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Background Social distancing and quarantine imposed by the authority during the COVID-19 pandemic caused restrictions, which had a negative impact on eating behavior, especially among adolescents. We proposed a retrospective study aimed to evaluate the effect of the COVID-19 pandemic on eating disorders risk and symptoms. Methods In this study, a group of 127 pediatric patients (117 females and 10 males) with eating disorders admitted to the Bambino Gesù Children’s Hospital of Rome (Italy), in the period between August 2019 and April 2021, was analyzed. All patient data were collected from patients’ electronic medical records. Results We found that 80.3% of patients were at the onset of eating disorders and that 26% of patients had familiarity for psychotic disorders. Often these patients had comorbidities and alterations in blood parameters such as leukocytopenia, neutropenia, hypovitaminosis and hormonal problems that could affect their future. Conclusions Our findings could provide a framework for developing clinical and educational interventions to mitigate the short- and long-term negative impact of the pandemic on adolescent future health.

Red blood cells (RBCs) are recognized to be important pathogenetic determinants in several human cardiovascular diseases (CVD). Undergoing to functional alterations when submitted to risk factors, RBCs modify their own intracellular signaling and the redox balance, shift their status from antioxidant defense to pro-oxidant agents, become a potent atherogenic stimulus playing a key role in the dysregulation of the vascular homeostasis favoring the developing and progression of CVD. Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with a significantly increased risk of cardiovascular mortality with a prevalence from two to five more likely in woman, mainly attributed to accelerated atherosclerosis. The purpose of this study was to correlate the RA disease activity and the RBCs functional characteristics. Thirty-two women (aged more than 18 years) with RA, and 25 age-matched healthy women were included in this study. The disease activity, measured as the number of swollen and painful joints (DAS-28), was correlated with 1) the expression of RBCs estrogen receptors, which modulate the RBC intracellular signaling, 2) the activation of the estrogen-linked kinase ERK½, which is a key regulator of RBC adhesion and survival, and 3) the levels of inflammatory- and oxidative stress-related biomarkers, such as the acute-phase reactants, the antioxidant capacity of plasma, the reactive oxidizing species formation and 3-nitrotyrosine. All the biomarkers were evaluated in RA patients at baseline and 6 months after treatment with disease-modifying anti-rheumatic drugs (DMARDs). We found, for the first times, that in RA patients 1) the DAS-28 correlated with RBC ER-α expression, and did not correlate with total antioxidant capacity of plasma; 2) the RBC ER-α expression correlated with systemic inflammatory biomarkers and oxidative stress parameters, as well as ERK½ phosphorylation; and 3) the DMARDs treatments improved the clinical condition measured by DAS-28 score decrease, although the RBCs appeared to be more prone to pro-oxidant status associated to the expression of survival molecules. These findings represent an important advance in the study of RA determinants favoring the developing of CVD, because strongly suggest that RBCs could also participate in the vascular homeostasis through fine modulation of an intracellular signal linked to the ER-α.

Background. Social distancing and quarantine imposed by the authority during the COVID-19 pandemic caused restrictions, which had a negative impact on eating behavior, especially among adolescents. We proposed a retrospective study aimed to identify the main indicators/predictors of eating disorders occurring in this pandemic period. Methods.In this study, a group of 127 pediatric patients (117 females and 10 males) with eating disorders admitted to the Bambino Gesù Children’s Hospital of Rome (Italy), in the period between August 2019 and April 2021, was analyzed. Results. We found that 80.3% of patients were at the onset of eating disorders and that 26% of patients had familiarity for psychotic disorders. Often these patients had comorbidities and alterations in blood parameters such as leukocytopenia, neutropenia, hypovitaminosis and hormonal problems that could affect their future. Conclusions. Based on this study, we suggest that the therapeutic path of patients with eating disorders should guarantee not only psychological treatment, but also that of comorbidities.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H

Aging is a process characterised by a general decline in physiological functions. The high bioavailability of reactive oxygen species (ROS) plays an important role in the aging rate. Due to the close relationship between aging and oxidative stress (OS), functional foods rich in flavonoids are excellent candidates to counteract age-related changes. This study aimed to verify the protective role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. Markers of OS, including ROS production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, as well as the anion exchange capability through Band 3 protein (B3p) and glycated haemoglobin (A1c) have been analysed in erythrocytes treated with d-Gal for 24 h, with or without pre-incubation for 1 h with 0.5-10 µg/mL Açaì extract. Our results show that the extract avoided the formation of acanthocytes and leptocytes observed after exposure to 50 and 100 mM d-Gal, respectively, prevented d-Gal-induced OS damage, and restored alterations in the distribution of B3p and CD47 proteins. Interestingly, d-Gal exposure was associated with an acceleration of the rate constant of SO

Metabolic syndrome (MS) represents worldwide public health issue characterized by a set of cardiovascular risk factors including obesity, diabetes, dyslipidemia, hypertension, and impaired glucose tolerance. The link between the MS and the associated diseases is represented by oxidative stress (OS) and by the intracellular redox imbalance, both caused by the persistence of chronic inflammatory conditions that characterize MS. The increase in oxidizing species formation in MS has been accepted as a major underlying mechanism for mitochondrial dysfunction, accumulation of protein and lipid oxidation products, and impairment of the antioxidant systems. These oxidative modifications are recognized as relevant OS biomarkers potentially able to (i) clarify the role of reactive oxygen and nitrogen species in the etiology of the MS, (ii) contribute to the diagnosis/evaluation of the disease’s severity, and (iii) evaluate the utility of possible therapeutic strategies based on natural antioxidants. The antioxidant therapies indeed could be able to (i) counteract systemic as well as mitochondrial-derived OS, (ii) enhance the endogenous antioxidant defenses, (iii) alleviate MS symptoms, and (iv) prevent the complications linked to MS-derived cardiovascular diseases. The focus of this review is to summarize the current knowledge about the role of OS in the development of metabolic alterations characterizing MS, with particular regard to the occurrence of OS-correlated biomarkers, as well as to the use of therapeutic strategies based on natural antioxidants.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 μM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42− uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 μM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Aging is a process characterised by a general decline in physiological functions. The high bioavailability of reactive oxygen species (ROS) plays an important role in the aging rate. Due to the close relationship between aging and oxidative stress (OS), functional foods rich in flavonoids are excellent candidates to counteract age-related changes. This study aimed to verify the protective role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. Markers of OS, including ROS production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, as well as the anion exchange capability through Band 3 protein (B3p) and glycated haemoglobin (A1c) have been analysed in erythrocytes treated with d-Gal for 24 h, with or without pre-incubation for 1 h with 0.5–10 µg/mL Açaì extract. Our results show that the extract avoided the formation of acanthocytes and leptocytes observed after exposure to 50 and 100 mM d-Gal, respectively, prevented d-Gal-induced OS damage, and restored alterations in the distribution of B3p and CD47 proteins. Interestingly, d-Gal exposure was associated with an acceleration of the rate constant of SO42− uptake through B3p, as well as A1c formation. Both alterations have been attenuated by pre-treatment with the Açaì extract. These findings contribute to clarify the aging mechanisms in human erythrocytes and propose functional foods rich in flavonoids as natural antioxidants for the treatment and prevention of OS-related disease conditions.

SARS-CoV-2 is a highly contagious respiratory virus that in December 2019 emerged in Wuhan (Hubei Province, China) and spread rapidly worldwide. Despite the high number of people affected, data on clinical features and prognostic factors in children and adolescents are limited. Children become less ill than adults and most of them contract the infection through close contact with their parents or family members. In this short communication, we commented data of our study on an Italian pediatric population with COVID-19, published on the Italian Journal of Pediatrics.

Background: Since December 2019 coronavirus disease (COVID-19) emerged in Wuhan and spread rapidly worldwide. Despite the high number of people affected, data on clinical features and prognostic factors in children and adolescents are limited. We propose a retrospective study aimed to identify sex differences in a pediatric population with COVI-19.Methods: A pediatric population admitted with COVID-19 to Bambino Gesù Children's Hospital of Rome (Italy) in the period from March to May 2020 has been studied taking into account sex differences. Medical history, comorbidities, symptoms and laboratory findings were obtained from patients' electronic medical records. Results: In 37 patients (19 males and 18 females) we found that: i) fever and cough were the dominant symptoms, while gastrointestinal symptoms were rare; and ii) all ages of childhood were susceptible to COVID-19. Moreover, we found that females with COVID-19 were older than males (p < 0.01); required more days of hospitalization (p < 0.04); needed of treatment with multiple drugs; and had higher serum lactate dehydrogenase values (p < 0.04) than males. Conversely, males had, although not significant, higher values of C reactive protein and erythrocyte sedimentation rate than females.Conclusions: Based on the data listed above sex differences were detected in an Italian pediatric population. Compared to the adults we found that COVID-19 infection in children is a non-severe inflammatory disease in both males and females. In any case, many detailed studies should be conducted.

Background Since December 2019 coronavirus disease (COVID-19) emerged in Wuhan and spread rapidly worldwide. Despite the high number of people affected, data on clinical features and prognostic factors in children and adolescents are limited. We propose a retrospective study aimed to evaluate clinical characteristics of children infected with SARS-CoV-2 in Italy. Methods A pediatric population admitted with COVID-19 to Bambino Gesù Children’s Hospital of Rome (Italy) in the period from the end of February to July 2020 has been studied. Medical history, comorbidities, symptoms and laboratory findings were obtained from patients’ electronic medical records. Results In 66 patients (35 males and 31 females) we found that: i) fever and cough were the dominant symptoms, while vomit and convulsions were rare symptoms; and ii) all ages of childhood were susceptible to COVID-19. Furthermore, we found that, compared to females, males with COVID-19, although not significantly, had higher values of inflammatory markers such as C-reactive protein (CRP) and ESR. Conversely, we found that COVID-19 positive females were older than males and required more days of hospitalization. Both males and females COVID-19 positives had procalcitonin values within the normal range and D-Dimer values slightly higher than the normal range. With regard to this latter marker, the value measured in females, although not significant, was higher than that measured in males. Interestingly, the presence of leukopenia was found in both sexes. Conclusions Compared to the adults we found that COVID-19 infection in children is a non-severe inflammatory disease in both males and females. In any case, many detailed studies should be conducted.

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.

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.

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.

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.

Adult cardiac progenitor cells (CPCs), isolated as cardiosphere-derived cells (CDCs), represent promising candidates for cardiac regenerative therapy. CDCs can be expanded in vitro manyfolds without losing their differentiation potential, reaching numbers that are appropriate for clinical applications. Since mechanisms of successful CDC survival and engraftment in the damaged myocardium are still critical and unresolved issues, we aimed at deciphering possible key factors capable of bolstering CDC function. In particular, the response and the phenotype of CDCs exposed to low concentrations of the multifunctional cytokine tumor necrosis factor α (TNF-α), known to be capable of activating cell survival pathways, have been investigated. Furthermore, differential biological responses of CDCs from male and female donors, in terms of cell cycle progression and cell spreading, have also been assessed. The results obtained indicate that (i) the intracellular signaling activated in our experimental conditions is most likely due to the prosurvival and proliferative signaling of TNF-α receptor 2 and that (ii) cells from female patients appear more responsive to TNF-α treatment in terms of cell cycle progression and migration ability. In conclusion, the present report highlights the hypothesis that TNF-stimulated CDCs isolated from females may represent a promising candidate for cardiac regenerative therapy applications.

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

Aim Although constitutive autophagy is linked to redox state and participates in cell homeostasis, it is scarcely known if redox state, autophagy, and lysosomal function depend on sex, a factor that largely influences health and diseases. Therefore, we evaluated the existence of sex differences in redox state and constitutive autophagy in rat tissues. Main methods 7 week old Sprague–Dawley rats were used to obtain organs. Malondialdehyde (MDA), and carbonylated proteins were measured by spectrophotometric methods for redox state assessment. The autophagy biomarkers Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3), the mammalian target of rapamycin (mTOR; checkpoint in autophagic process), and the lysosomal associated membrane protein (LAMP-1; biomarker of lysosomes) were evaluated by Western blotting. Immunofluorescence analysis was also performed for LC3 and LAMP-1 colocalization. Key findings In the heart, Beclin-1, and LC3-II/LC3-I were higher in males than in females suggesting that the male heart has a major constitutive autophagy and this was linked with higher levels of carbonyl groups, indicating that protein oxidation could play a role. In the liver, it was found that LAMP-1 was higher in males and greatly colocalized with LC3 indicating a larger number of autophagolysosomes. None of the above parameters was significantly different in the kidneys of both sexes with the exception of MDA, which was significantly higher in females. Significance The above results suggest that sex differences exist in redox state and autophagy and they occur in an organ-specific way. Importantly, it seems that the protein oxidation is more linked with constitutive autophagy, at least in cardiac ventricles, in comparison with lipid peroxidation.

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.This review briefly describes our current knowledge on gender medicine and on the Italian legislation in risk management. 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. The Istituto Superiore di Sanità (ISS) is beginning a project funded by INAIL, to monitor the hepatitis B, measles, rubella, mumps, pertussis, chickenpox, tetanus vaccines immune coverage in health care workers in Italy.The analysis of the collected data will be disaggregated for men and women and statistically analyzed with respect to the time intervals from vaccination.The results could provide useful elements to implement prevention and health surveillance programs in occupational medicine.La medicina di genere valuta l’impatto del genere sulla fisiologia umana, sulla fisiopatologia, e sulle caratteristiche cliniche delle malattie, integrando aspetti biologici, sociologici, etnici e culturali. Il decreto legge 81/2008, ha introdotto il genere nella valutazione dei rischi sul luogo di lavoro, tuttavia non esiste ancora una normativa generespecifica riguardante il personale delle professioni sanitarie e delle istituzioni scientifiche, che è soggetto ad agenti biologici, fisici e chimici, oltre che a problemi psicosociali. L’Istituto Superiore di Sanità, ha avviato un progetto insieme a INAIL per la raccolta dei dati di copertura immunologica vaccinale nei/lle lavoratori/trici del comparto sanitario, nei riguardi dei vaccini antivirali (anti morbillo-parotite-rosolia, pertosse, epatite B, varicella, tetano). I dati saranno raccolti in una banca dati, ed analizzati in modo disaggregato per sesso. I risultati forniranno elementi per implementare i sistemi di sorveglianza sanitaria e prevenzione della salute dei lavoratori, riguardo all’immunoprofilassi primaria.

Despite considerable advances in the treatment of human inflammatory diseases, such as cardiovascular and neurological disorders, they remain the leading cause of death in developed countries. From a clinical perspective, an active area of investigation focuses on the identification of diagnostic and prognostic biomarkers, because preventing events in those at risk of chronic inflammatory disease is likely to have a substantial impact on the overall public-health burden. The sex difference has not been considered previously as important in the evaluation of biomarkers of human diseases, notwithstanding it is now ascertained that the severity of these disorders is correlated with sex hormones which modulate the inflammatory response. The aim of the present brief review is to report and comment the state of art regarding the sex-related biomarkers in cardiovascular and neurodegenerative disorders, focusing on those compounds showing potential prognostic- and diagnostic values, and/or acting as indicators of the therapeutic treatment efficacy.

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.

A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100_ and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved

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.

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.

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.

Changes in the mitochondrial membrane potential play a key role in determining cell fate. Mitochondria membrane hyperpolarization has been found to occur after cell activation, e.g. in lymphocytes, whereas depolarization is associated with apoptosis. The aim of this study was to investigate the effects of an immunological stimulus, i.e. opsonized zymosan A, on human platelet mitochondria by means of flow and static cytometry analyses as well as biochemical methods. We found that opsonized zymosan induced significant changes of platelet morphology at early time points (90 min). This was associated with increased production of reactive oxygen species, and, intriguingly, mitochondrial membrane hyperpolarization. At a later time point (24 h), opsonized zymosan was found to induce increased expression of CD47 adhesion molecule, platelet aggregation, mitochondrial membrane depolarization and phosphatidylserine externalization. Although these late events usually represent signs of apoptosis in nucleated cells, in opsonized zymosan-treated platelets they were not associated with membrane integrity loss, changes in Bcl-2 family protein expression or caspase activation. In addition, pre-treatment with low doses of the 'mitochondriotropic' protonophore carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone counteracted mitochondrial membrane potential alterations, production of reactive oxygen species and phosphatidylserine externalization induced by opsonized zymosan. Our data suggest that mitochondrial hyperpolarization represents a key event in platelet activation and remodeling under opsonized zymosan immunological stimulation, and opsonized zymosan immunological stimulation may represent a useful tool for understanding of the pathogenetic role of platelet alterations associated with vascular complications occurring in metabolic and autoimmune diseases.

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis and is endowed to modulate several immune responses. We previously demonstrated that both immature and mature human dendritic cells (DCs) express a functional leptin receptor, and we found that leptin activates DCs, licenses them for Th1 priming, and promotes DC survival. Moreover, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Here we monitor the effects of leptin on DC migratory capacities, focusing on the intracellular signaling driving cytoskeleton rearrangement. We found that leptin increases immature DC migratory performance both by favoring cytoskeleton dynamics and by up-regulating CCR7 surface expression, thus favoring chemotactic responsiveness. We found that in immature DCs, leptin activates cofilin, favoring the turnover of actin microfilaments, and, by triggering Vav phosphorylation, promotes Rac1 activation. Finally, we found that in immature DCs, leptin up-regulates interleukin-12p70 production on CD40 stimulation and, more importantly, increases their capacity to stimulate activation of autologous CD8(+) T cells. Taken altogether, the findings herein highlight the potential use of leptin as an adjuvant tool in vaccination protocols employing ex vivo-generated autologous DCs.

Several experimental models have so far been developed to improve our knowledge of the pathogenetic mechanisms of type 2 diabetes mellitus (T2D), to determine the possible pharmacological targets of this disease and to better evaluate diabetes-associated complications, e.g. the cardiovascular disease. In particular, the study of T2D gained the attention of several groups working with different animal species: rodents, cats or pigs, as well as other non-human primate species. Each of these species provided useful and different clues. However, T2D has to be considered as a gender-associated disease: sex differences play in fact a key role in the onset as well as in the progression of the disease and a higher mortality for cardiovascular diseases is detected in diabetic women with respect to men. The results obtained from all the available animal models appear to only partially address this issue so that the search for more precise information in this respect appears to be mandatory. In this review we summarize these concepts and literature in the field and propose a reappraisal of the various animal models for a study of T2D that would take into consideration a gender perspective.

Reactive oxygen and nitrogen species have been implicated in diverse subcellular activities, including cell proliferation,differentiation and, in some instances, cell injury and death. The implications of reactive species inhuman pathology have also been studied in detail. However, although the role of free radicals in the pathogenesis of human diseases has been extensively analyzed in different systems (i.e., in vitro, ex vivo, and in vivo),it is still far from elucidated. In particular, the possible role of gender 4 differences in human pathophysiology associated with reactive species is a promising new field of investigation. Although the complex scenario this presents is still incomplete, important gender-associated "redox features" of cells have already been described in the literature. Here we summarize the different aspects of redox-associated molecules and enzymes in regard to gender differences in terms of the intracellular production and biochemical activity of reactive species. These are often associated with the pathogenetic mechanisms underlying several human morbidities(e.g., degenerative diseases) and can represent a specific target for new pharmacologic strategies. Gender differences may thus pose an important challenge for future studies aimed at the clinical management of diseases characterized by a redox imbalance.

Carboxyfullerenes (CF) act as free radical scavengers in many cell settings and prevent apoptosis in vitro and in vivo. CF protect normal human keratinocytes from UVB-induced apoptosis, although the mechanisms underlying this effect remain to be clarified. Double-staining confocal laser microscopy revealed that CF penetrate the cell and colocalize with cytokeratin-18 within cytoplasm. This localization was confirmed by transmission electron microscopy that showed CF intermingled with keratin filaments. Moreover, double-staining with the mitochondrial marker anti-F1-ATPase antibody demonstrated that CF are expressed in mitochondria. Transmission electron microscopy confirmed that CF actually localize within mitochondria. Then, normal human keratinocytes were UVB-irradiated in the presence or absence of CF at different doses. CF protected keratinocytes from apoptosis induced by reactive oxygen species. CF scavenging effect is associated with a partial blockade of the UVB-induced intrinsic apoptotic pathway by down-modulating caspase-9 activation and cytochrome c release, and by inhibiting the down-regulation of the inhibitor of apoptosis proteins (IAP) survivin, livin, IAP-1 and IAP-2. Finally, CF prevented the cleavage of Bid, up-regulation of Bad and down-regulation of Mcl-1 induced by UVB. Taken together, these results indicate that CF penetrate human keratinocytes, localize within mitochondria where they act both by scavenging free radicals and by protecting cells from apoptosis.

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies. Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides. These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with UV-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell–cell and cell–substrate contacts, cell detachment and cell death by apoptosis. The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with α-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as α-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity in Western countries. The increased oxidative stress, caused by the release of reactive oxygen and nitrogen species (ROS/RNS) from inflammatory airways cells, contributes to the pathogenesis of the disease. The aim of the present study was to evaluate (a) whether the oxidative imbalance can lead to specific alterations of red blood cells (RBCs) from stable COPD patients; (b) whether treatment with N-acetyl-cysteine (NAC), in widespread use as mucolytic agent in clinical practice, can counteract these effects; and (c) whether an in vitro model represented by the exposure of RBC to ROS/RNS could mimic the in vivo situation. The results obtained clearly indicated that the RBC integrity and function are similarly altered in COPD patients and in ROS/RNS in vitro-treated samples and that NAC administration was capable of counteracting RBC oxidative modifications both in vivo, as detected by clinical and laboratory evaluations, and in vitro. Altogether these results point to RBC oxidative modifications as valuable bioindicators in the clinical management of COPD and indicate that in vitro RBC exposure to ROS/RNS as a useful tool in experimental studies aimed at the comprehension of the pathogenic mechanisms of the redox-associated diseases.

The multidrug resistance protein 1 (MRP1) is a drug transporter that protects cells from oxidative stress, which increases HIV-1 replication. The aim of this study was to characterize the expression, function, and role of lymphocyte MRP1 in HIV-1 infection and its modulation by antiretroviral drugs such as the protease inhibitors (PIs). Peripheral blood mononuclear cells (PBMCs) from HIV-positive individuals do not show significant alterations of MRP1 expression despite highly active antiretroviral therapy and HIV plasma viral load levels; however, they exhibit different intracellular MRP1 expression as compared with healthy subjects. By contrast, MRP efflux function is increased in subjects with primary HIV infection and becomes defective in later stages of the infection. PI- and probenecid (PBCD)-mediated inhibition of MRP lowers the in vitro stress-induced response of lymphoid cells by reducing the level of the specific reactive oxygen species superoxide anion and hydrogen peroxide. Finally, the blockade of MRP by PBCD and PIs down-modulates HIV-1 replication by a mechanism independent of inhibition of the HIV-1 protease. Our results are consistent with a model wherein HIV replication is favored by the MRP1-related oxidative stress and inhibition of MRP1 may contribute to the antiviral activity of PIs.

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis. Leptin is capable of modulating several immune responses. However, the effect of leptin on dendritic cells (DCs) has not yet been recognized. Because DCs are instrumental in the development of immune responses, in this study, we evaluated the impact of leptin on DC activation. We demonstrated the presence of leptin receptor in human immature and mature DCs both at mRNA and protein level and its capacity to transduce leptin signaling leading to STAT-3 phosphorylation. We found no consistent modulation of DC surface molecules known to be critical for their APC function in response to leptin. In contrast, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. At a functional level, leptin up-regulates the IL-1β, IL-6, IL-12, TNF-α, and MIP-1α production. Coincident with this, leptin-treated DCs stimulate stronger heterologous T cell responses. Furthermore, we found that leptin down-regulates IL-10 production by DCs and drives naive T cell polarization toward Th1 phenotype. Finally, we found that leptin partly protects DCs from spontaneous and UVB-induced apoptosis. Consistent with the antiapoptotic effect of leptin, we observed the activation of NF-κB and a parallel up-regulation of bcl-2 and bcl-xL gene expression. These results provide new insights on the immunoregulatory function of leptin demonstrating its ability to improve DC functions and to promote DC survival. This is of relevance considering a potential application of leptin in immunotherapeutic approaches and its possible use as adjuvant in vaccination protocols.

Changes in the oxidative status of erythrocytes can reduce cell lifetime, oxygen transport, and delivery capacity to peripheral tissues and have been associated with a plethora of human diseases. Among reactive oxygen and nitrogen species of importance in red blood cell (RBC) homeostasis, superoxide and nitric oxide radicals play a key role. In the present work, we evaluated subcellular effects induced by peroxynitrite, the product of the fast reaction between superoxide and nitric oxide. Peroxynitrite induced 1) oxidation of oxyhemoglobin to methemoglobin, 2) cytoskeleton rearrangement, 3) ultrastructural alterations, and 4) altered expression of band-3 and decreased expression of glycophorin A. With respect to control cells, this occurred in a significantly higher percentage of human RBC (approximately 40%). The presence of antioxidants inhibited these modifications. Furthermore, besides these senescence-associated changes, other important modifications, absent in control RBC and usually associated with apoptotic cell death, were detected in a small but significant subset of peroxynitrite-exposed RBC (approximately 7%). Active protease cathepsin E and mu-calpain increased; activation of caspase 2 and caspase 3 was detected; and phosphatidylserine externalization, an early marker of apoptosis, was observed. Conversely, inhibition of cathepsin E, mu-calpain, as well as caspase 2 and 3 by specific inhibitors resulted in a significant impairment of erythrocyte "apoptosis" Altogether, these results indicate that peroxynitrite, a milestone of redox-mediated damage in human pathology, can hijack human RBC toward senescence and apoptosis by a mechanism involving both cysteinyl and aspartyl proteases.

Galectins are a family of beta-galactoside binding molecules involved in cell--extracellular matrix adhesion processes. Specifically, Galectin-3 (Gal-3), one of the members of this family of molecules plays a role in cell adhesion processes as well as in cell survival or apoptosis. Gal-3 was also hypothesized to represent a useful tool in tumor characterization, for example, in thyroid tumors. We report herein the results obtained by evaluating Gal-3 expression of colon cells from human adenomas and adenocarcinomas with two different methodologies: immunohistochemistry and flow cytometry of living dispersed cells. We found that (1) the expression of Gal-3 was significantly increased on the surface of cells from adenomas with respect to normal mucosa from the same patient; (2) Gal-3 ligand, 90k molecule, was increased in the blood plasma from patients with both adenomatous and adenocarcinomatous lesions; and (3) Gal-3 overexpression was not related with the presence of K-ras mutation. Altogether these results clearly indicate that the evaluation of Gal-3 expression (and of its ligand, 90k) can be of interest in the characterization of nonmalignant and malignant colon cancers.

The accessory HIV-1 Nef protein plays a key role in AIDS pathogenesis. We recently demonstrated that exogenous Nef triggers phenotypic and functional differentiation of immature dendritic cells (DCs). Here we investigated whether the Nef-induced DC differentiation occurs with morphological remodeling and have focused on the interference of Nef in the signaling pathways that regulates DC maturation. We found that exogenous Nef enters immature DCs, promoting their functional and morphological differentiation. Specifically, Nef promotes interleukin (IL) -12 release, which closely fits with nuclear factor (NF) -kappaB activation. Nef induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Moreover, Nef increases the capacity of DCs to form clusters with allogeneic CD4+ T cells, improving immunological synapse formation. Searching for molecules involved in Nef-triggered signaling pathways driving the DC maturation, we found that Nef targets Vav and promotes its tyrosine phosphorylation, associated with its nucleus-to-cytoplasm redistribution. This has a direct effect on Vav guanine nucleotide exchange factor activity for the small GTPase Rac1. We hypothesize that targeting Vav, Nef modulates both early signaling events (such as cytoskeletal rearrangement) and delayed responses (such as transcriptional regulation), promoting DC differentiation. Our results highlight how Nef may enhance T lymphocyte activation, thus fostering virus dissemination, manipulating the DC arm of the immune response.

It has been reported that the incidence of thrombotic events can display a gender disparity. In particular, a lower thrombotic risk has been described in female gender. The mechanisms underlying this disparity are still poorly understood. Of great interest is the hypothesis that hormones, estrogen in particular, could play a key role. In fact, the possibility that some hormonal factors could protect women from thrombotic events appears well documented in literature. For instance, several studies aimed at the analysis of the impact of estrogen and estrogen receptors in thrombogenesis claim for the implication of these hormones either in megakaryocyte differentiation or, more intriguingly, directly affecting platelet integrity and function. In consideration of the absence of the nucleus, platelet susceptibility appears quite striking and probably due to the non-nuclear estrogen receptor function. In this review we briefly summarize our knowledge as concerns the role of estrogen and estrogen receptors in determining megakaryocyte/platelet functions and thrombogenicity.

Our previous article reported that retroviral transduction of human type I consensus interferon-coding sequence into two human melanoma cells increased their susceptibility to cisplatin-induced apoptosis. Importantly, primary melanoma cells were significantly more sensitive to cisplatin-induced apoptosis with respect to metastatic melanoma cells. The aim of this study was to elucidate the subcellular mechanisms involved in this interferon-induced apoptotic proneness. Our results indicate that 1) cisplatin-induced apoptosis can be referred to as the type II apoptosis, ie, to the mitochondrially driven cascade; 2) treatment of interferon-producing melanoma cells with other type II apoptotic stimuli, such as radiation or staurosporine, also resulted in massive apoptosis, whereas type I stimuli, ie, anti-Fas, were ineffective; 3) interferon sensitization involved the caspase cascade in primary melanoma cells and the alternative pathway represented by cathepsin-mediated apoptosis in metastatic melanoma cells; 4) interferon production sensitizes cells to apoptosis by inducing, as the earliest event, mitochondrial membrane hyperpolarization. These results suggest that constitutive production of type I interferon by melanoma cells can act as an intracellular booster capable of increasing cell proneness to apoptosis by specifically modifying mitochondrial homeostasis and independently from the apoptotic cascade involved.

Summary This study investigates the in vitro effects of oxidized low-density lipoproteins (ox-LDL), ‘physiological’ pro-oxidants, N-acetylcysteine (NAC), a free radical scavenger and glutathione precursor, and their combination on human peripheral blood mononuclear cell functions. We found that treatment with ox-LDL induced a significant down-regulation of proliferative response to mitogens, antigens and interleukin-2. Lipid extracts from ox-LDL were able to reproduce the same effect as the lipoprotein. On the other hand, NAC exposure induced a significant up-regulation of proliferative responses to all the stimuli used. Moreover, we showed that natural killer (NK) cell-mediated cytotoxic activity was significantly down-regulated by ox-LDL while treatment with NAC induced a significant up-regulation of NK-cell activity. Finally, we found that ox-LDL and NAC exerted opposite effects on the cytokine network, interfering both at the protein secretion level and the messenger RNA synthesis level. More importantly, when NAC was used in combination with ox-LDL the proliferative responses, NK-cell-mediated cytotoxic activity and cytokine production were restored to values comparable to controls. These data indicate that ox-LDL and NAC modulate immune functions, exerting opposite effects reflecting their pro-oxidant and antioxidant behaviours. Our results add new insights to the key role played by redox imbalance as a modulator of immune system homeostasis and suggest that an antioxidant drug such as NAC could be useful against pathologies associated with an increase in lipid peroxidation.

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma-glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells.

In mice, the Fas/Fas ligand (FasL) system has been shown to be involved in germ cell apoptosis. In the present study we evaluated the expression of Fas and Fas ligand (FasL) in fetal and adult human testis. Semiquantitative RT-PCR demonstrated the expression of Fas and FasL messenger ribonucleic acids in adult testis, but not in fetal testis (20-22 weeks gestation). In situ RT-PCR and immunohistochemistry experiments on adult human testis demonstrated the expression of FasL messenger ribonucleic acid and protein in Sertoli and Leydig cells, whereas the expression of Fas was confined to the Leydig cells and sporadic degenerating spermatocytes. The number of Fas-positive germ cells per 100 Sertoli cell nuclei was increased in 10 biopsies with postmeiotic germ cell arrest compared to 10 normal testis biopsies (mean, 3.82 +/- 0.45 vs. 2.02 +/- 0.29; P = 0.0001), but not in 10 biopsies with meiotic germ cell arrest (mean, 1.56 +/- 1.07). Fas and FasL proteins were not expressed in cases of idiopathic hypogonadotropic hypogonadism. Together, these findings may suggest that Fas/FasL expression in the human testis is developmentally regulated and under gonadotropin control. The increased germ cell expression of Fas in patients with postmeiotic germ cell arrest suggests that the Fas/FasL system may be involved in the quality control mechanism of the produced gametes.

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3-fullero-tris-methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration-dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4-induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.

Anoikia is a type of apoptotic cell death that occurs in cells that are substrate-restricted in their growth. Buckminster-fullerenes represent a new class of chemical compounds with wide potential pharmacological antioxidant activity. In this report we provide the first demonstration that a water-soluble fullerene derivative,C3-fullero-tris-methanodicar☐ylic acid, synthesized in our laboratories, is capable of inducing anoikia resistance in epithelial cells by a mechanism involving a ‘trophic’ effect on cell spreading-associated cytoskeletal components, i.e. on actin microfilaments.

Several lines of evidence have demonstrated that IFNs could be relevant in the treatment of certain neoplastic diseases such as carcinomas. In particular, IFN-α, in addition to the anti-proliferative and cytostatic effects, was demonstrated to be capable of inducing cell death by apoptosis both in vivo and in vitro. Numerous protocols have also been proposed which consider the association of IFN-α with other drugs. Among these are retinoids, a class of compounds capable of inducing inhibition of cell growth and differentiation. We address the question here by analyzing the role of cell adhesion in susceptibility to IFN-α, RA and their combination of a human cell line derived from a squamous carcinoma of the cervix, the Bcl-2-negative SiHa cell line. In this context, cytoskeleton components and several surface molecules playing a role in cell substrate and cell-to-cell relationships have been evaluated. We found that RA treatment is capable of improving stress fiber formation, decreasing cell detachment and increasing cell-adhesion capability. However, no variations in the ability to adhere to specific extracellular-matrix molecules were found in RA-treated cells. No quantitative changes were detected in integrins involved as receptors for extracellular matrix molecules (VLA1-VLA5) or in other cell-adhesion-associated molecules (e.g., CD44). By contrast, 2 important molecules involved in cell-adhesion processes appeared to be up-regulated by RA exposure: focal adhesion kinase and E-cadherin, involved in adhesion plaque formation and cell-to-cell contacts, respectively. Keeping in mind the importance of adhesion properties in the cell-growth pathway, our findings could be of interest in the study of carcinoma-cell proliferation and metastatic potential. Int. J. Cancer 76:531–540, 1998.© 1998 Wiley-Liss, Inc.