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Exposure to vesicants, including sulfur mustard and nitrogen mustard, causes damage to the epithelia of the respiratory tract and the lung. With time, this progresses to chronic disease, most notably, pulmonary fibrosis. The pathogenic process involves persistent inflammation and the release of cytotoxic oxidants, cytokines, chemokines, and profibrotic growth factors, which leads to the collapse of lung architecture, with fibrotic involution of the lung parenchyma. At present, there are no effective treatments available to combat this pathological process. Recently, much interest has focused on nutraceuticals, substances derived from plants, herbs, and fruits, that exert pleiotropic effects on inflammatory cells and parenchymal cells that may be useful in reducing fibrogenesis. Some promising results have been obtained with nutraceuticals in experimental animal models of inflammation-driven fibrosis. This review summarizes the current knowledge on the putative preventive/therapeutic efficacy of nutraceuticals in progressive pulmonary fibrosis, with a focus on their activity against inflammatory reactions and profibrotic cell differentiation.

Wheat is the third most cultivated cereal in the world and represents the major contributor to human nutrition. Milling wheat by-products such as husks (17–20% of the total processing output weight), even if still containing high-value-added bioactive compounds, are often left untreated or unused, thus resulting in environmental and human health burdens. In these regards, the present study is aimed at evaluating in a multimethodological approach the nutraceutical properties of durum wheat husks belonging to the ancient cultivar “Senatore Cappelli”, thus assessing their potential as bioactive compound sources in terms of phytochemical, cytotoxic, and nutraceutical properties. By means of HPLC-FD analyses, wheat husk samples analyzed revealed a higher content of serotonin, amounting to 35% of the total BAs, and were confirmed to occur at biogenic amines quality index (BAQI) values

Neuropeptide Y (NPY) is an abundantly expressed peptide capable of modulating innate and adaptive immune responses and regulating chemotaxis and cytokine secretion by macrophages. Abnormal regulation of NPY is involved in the development of atherosclerosis. The inflammatory infiltrate within atherosclerotic plaque is characterized by accumulation of macrophages, which are subject to reprogram their phenotypes in response to environmental signals. Macrophage number and phenotype influence plaque fate. Here, we investigated the effect of NPY on the changes in phenotype and functions of human macrophages, from the pro-inflammatory phenotype M1 to the reparative M2, indicative of atherosclerosis regression or stabilization. Human monocytes were differentiated in vitro into macrophages with M-CSF (M0) and polarized towards an M1 phenotype with IFN-γ plus LPS M(IFN-γ/LPS) or M2 with IL-10 (M IL-10) and further challenged with NPY (10−7–10−9 M) for 8–36 h. Cell phenotype and functions were analyzed by immunofluorescence and immunochemical analyses. NPY affected macrophage surface markers and secretome profile expression, thus shifting macrophages toward an M2-like phenotype. NPY also prevented the impairment of endocytosis triggered by the oxysterol 7-keto-cholesterol (7KC) and prevented 7KC-induced foam cell formation by reducing the lipid droplet accumulation in M0 macrophages. NPY-treated M0 macrophages enhanced the autophagosome formation by upregulating the cell content of the autophagy markers LC3-II and p62-SQSTM1, increased activation of the anti-oxidative transcription factor NRF2 (NF-E2-related factor 2), and subsequently induced its target gene HMOX1 that encodes heme oxygenase-1. Our findings indicate that NPY has a cytoprotective effect with respect to the progression of the inflammatory pathway, both enhancing p62/SQSTM1-dependent autophagy and the NRF2–antioxidant signaling pathway in macrophages. NPY signaling may have a crucial role in tissue homeostasis in host inflammatory responses through the regulation of macrophage balance and functions within atherosclerosis.

Background: Hyperbaric exposure (HE) is proven to be a stressor to several mechanisms in living cells. Even if after homeostasis restoration, harmful effects are expected, in particular a presence of free radicals. These latter are the stimulus to negative phenomenon as inflammation or cancer. In Italy, with 7500 km of sea shores, a large quantity of workers is exposed to HE during occupational activities. A deep knowledge of HE and bodily effects is not well defined; hence a multidisciplinary assessment of risk is needed. To detect one or more indicators of HE a research group is organised, under the INAIL sponsorship. The research project focused on the oxidative stress (OxS) and this paper details on the possible protocol to estimate, with a large amount of techniques on several human liquids, the relationship between OxS and HE. Specific attention will be paid to identify confounding factors and their influence. Methods: Blood and urine will be sampled. Several lab techniques will be performed on samples, both targeted, to measure the level of well-known biomarkers, and untargeted. Regard the formers: products of oxidation of DNA and RNA in urine; inflammation and temperature cytokines and protein carbonyles in blood. Untargeted evaluation will be performed for a metabolomics analysis in urine. Confounding factors: temperature, body fat, fitness, allergies and dietary habits. These factors will be assessed, directly or indirectly, prior and after HE. The final scope of the project is to determine one or more indicators that relates to HE in hits twofold nature: depth and duration. Conclusion: The relationship between OxS and HE is not deeply investigated and literature proposes diverging results. The project aims to define the time dependence of biomarkers related to OxS, to rise knowledge in risk assessment in workers exposed to HE.

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer’s disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-β peptide (Aβ). The morphological evaluation showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aβ. Moreover, URB597 reduced both the increase of Rho protein activation in Aβ-treated BV-2 cells and the Aβ-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.

Background It is a common opinion that Primary Sjögren Syndrome (pSS) damages the exocrine glands and determines the reduction of secreted saliva, some studies show that there are qualitative anomalies of the mucins produced in saliva, including MUC7, MUC5B, MUC1. The purpose of this study is to trace all the information useful to establish whether there is a qualitative or quantitative defect of the mucins in the pSS. Material and Methods We reviewed the literature by looking for publications relevant to the topic in electronic databases. Sixteen articles met the search criteria. The studies were divided into two categories, those that studied the rheological characteristics of the saliva and those that studied the structural and / or metabolism modifications of the muciparous cells in the salivary glands. Results in Patients with pSS, xerostomia and the reduction of salivary spinnbarkeit are only partially related to the reduction of the unstimulated salivary flow. In pSS, pathological alterations of mucins’ chemical-physical properties prevail as a cause of the clinical characteristics. Moreover, in pSS there are structural and metabolism changes in salivary glands’ muciparous cells. Conclusions There is much evidence that supports the presence of qualitative alterations in the saliva’s rheological properties in Patients with pSS, and these are the main cause, more than the reduction of the unstimulated salivary flow, of the disease clinical characteristics - dry mouth and complications in the oral cavity. Therefore we propose to add to the classification criteria of pSS also a qualitative test of salivary glycoproteins. Key words:Primary Sjögren's syndrome, mucin, MUC7, MUC5B, MUC1, sulphate oligosaccharides.

Objectives The diagnosis of primary Sjogren’s syndrome (pSS) continues to be difficult and several patients keep symptomatic for years with different diagnoses before confirmation of pSS. Since the IL-23-IL-17 axis is involved in the etiopathogenesis of pSS we evaluated by immunohistochemistry and morphometric methods the presence of IL-17 as well as IL-23 within minor salivary glands (MSG) obtained from patients with uncertain diagnosis of pSS. Materials and Methods 42 patients, with symptoms attributable to pSS, and 8 patients used as a control, were enrolled for the study. Autoantibody detection, histological analysis for the presence of Germinal Centers (GC+), immunohistochemistry to detect IL-23 and IL-17 were performed. Results The detection of GC + anti-SSA and anti-SSB antibody in parallel with the detection of IL-17 and IL-23, displays only a diagnostic reinforcement value. Instead, the detection of a positive reaction for both IL-17 and IL-23 without GC + or autoantibody within minor salivary glands, as detected in 36 % of patients with uncertain diagnosis, may be hold as a sensitive and specific marker to identify those patients who are likely to evolve into pSS. Conclusion we suggest to use the IL-17/ IL-23 immunohistochemical detection to improve the identification of patients with a possible diagnosis in all cases which do not fully meet the American-European criteria for pSS, in particular when the GC + are not present at histopathological analysis and anti-SSA and anti-SSB antibody are undetectable in the serum.

Aim To establish a mouse model for the study of congenital defects, using exposure of pregnant females to the teratogen BMS-189453, a multiple retinoic acid competitive antagonist. Aim We found not less than 60% of fetuses had transposition of the great arteries and l5% had other congenital heart defects such as double outlet right ventricle, tetralogy of Fallot, truncus and right aortic arch. Newborns exposed in utero to BMS-189453 were affected by thymus aplasia or hypoplasia, and severe congenital anomalies of the central nervous system due to neural tube defects. An anterior rotation of the right lung was also frequently present in our model. We also report a case of murine congenital diaphragmatic hernia associated with thymic aplasia and transposition of the great arteries. Conclusion These findings support the hypothesis that the combination of diaphragmatic hernia and congenital heart defects may be related to an alteration of the retinoic acid signaling pathways.

To prospectively compare two different approaches for estimating the amount of intravenous contrast media (CM) needed for multiphasic MDCT of the liver in obese patients. This single-center, HIPAA-compliant prospective study was approved by our Institutional Review Board. Ninety-six patients (55 men, 41 women), with a total of 42 hypovascular liver lesions, underwent MDCT of the liver. The amount of contrast medium injected was computed according to the patient’s lean body weight which was estimated using either a bioimpedance device (Group A) or the James formula (Group B). The following variables were compared between the two groups: the amount of contrast medium injected (in grams of Iodine, gI), the contrast enhancement index (CEI) and the lesion-to-liver contrast-to-noise ratio. Protocols A and B yielded significant differences in the amount of CM injected (mean values 41.9 ± 4.41 gI in Group A vs. 35.9 ± 5.75 gI in Group B; P = 0.021). The mean CEI value and lesion-to-liver contrast-to-noise ratio measured on the portal phase were significantly higher with protocol A than with protocol B (P

Purpose of review This review summarises the most recent evidence regarding the effects of diet in preventing and reducing age-related cognitive decline and neurodegenerative diseases. Recent findings Recent evidence indicates that nutraceuticals and whole diet approaches may protect against the development of age-related cognitive decline and pathological neurodegeneration. The neuroprotective effects are diverse depending on the nutrient employed and may involve a reduction of neuroinflammation, an activation of the endogenous antioxidant defence system and a modulation of the gut microbiota structure and function. Summary This review summarises the existing evidence in favour of diet as a viable alternative approach to directly impact cognitive decline and neurodegenerative diseases. The single nutrient (polyphenols, B vitamins, long-chain polyunsaturated fatty acids) versus whole diet approach (Mediterranean diet, Dietary Approaches to Stop Hypertension, MIND, Nordic, ketogenic) is presented and discussed. Potential mechanisms of action underlying the beneficial effects of these diets are also described. Implementation of large-scale preventive interventions based on dietary patterns identified as being beneficial to brain health should be a research and public health priority, ideally in conjunction with other health-promoting lifestyle factors.

Alzheimer&rsquo, s disease (AD) is characterized by the abnormal accumulation of amyloid-&beta, (A&beta, ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of A&beta, across the blood&ndash, brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the A&beta, load in the AD brain, little information is available about the effect of KBs on BBB and their effect on A&beta, transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on A&beta, transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in A&beta, clearance. Finally, the combined use of KBs promotes A&beta, efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote A&beta, clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.

Extracellular vesicles (EVs) have emerged as key regulators of cell-cell communication during inflammatory responses to lung injury induced by diverse pulmonary toxicants including cigarette smoke, air pollutants, hyperoxia, acids, and endotoxin. Many lung cell types, including epithelial cells and endothelial cells, as well as infiltrating macrophages generate EVs. EVs appear to function by transporting cargo to recipient cells that, in most instances, promote their inflammatory activity. Biologically active cargo transported by EVs include miRNAs, cytokines/chemokines, damage-associated molecular patterns (DAMPs), tissue factor (TF)s, and caspases. Findings that EVs are taken up by target cells such as macrophages, and that this leads to increased proinflammatory functioning provide support for their role in the development of pathologies associated with toxicant exposure. Understanding the nature of EVs responding to toxic exposures and their cargo may lead to the development of novel therapeutic approaches to mitigating lung injury.

New civil wars and waves of terrorism are causing crucial social changes, with consequences in all fields, including health care. In particular, skin injuries are evolving as an epidemic issue. From a physiological standpoint, although wound repair takes place more rapidly in the skin than in other tissues, it is still a complex organ to reconstruct. Genetic and clinical variables, such as diabetes, smoking, and inflammatory/immunological pathologies, are also important risk factors limiting the regenerative potential of many therapeutic applications. Therefore, optimization of current clinical strategies is critical. Here, we summarize the current state of the field by focusing on stem cell therapy applications in wound healing, with an emphasis on current clinical approaches being developed. These involve protocols for the ex vivo expansion of adipose tissue-derived mesenchymal stem cells by means of a patented Good Manufacturing Practice-compliant platelet lysate. Combinations of multiple strategies, including genetic modifications and stem cells, biomimetic scaffolds, and novel vehicles, such as nanoparticles, are also discussed as future approaches.

Sulfur mustard (SM) and nitrogen mustard (NM) are cytotoxic alkylating agents that cause severe and progressive injury to the respiratory tract, resulting in significant morbidity and mortality. Evidence suggests that macrophages and the inflammatory mediators they release play roles in both acute and long-term pulmonary injuries caused by mustards. In this article, we review the pathogenic effects of SM and NM on the respiratory tract and potential inflammatory mechanisms contributing to this activity.

Pulmonary fibrosis is characterized by destruction and remodeling of the lung due to an accumulation of collagen and other extracellular matrix components in the tissue. This results in progressive irreversible decreases in lung capacity, impaired gas exchange and eventually, hypoxemia. A number of inhaled and systemic toxicants including bleomycin, silica, asbestos, nanoparticles, mustard vesicants, nitrofurantoin, amiodarone, and ionizing radiation have been identified. In this article, we review the role of innate and adaptive immune cells and mediators they release in the pathogenesis of fibrotic pathologies induced by pulmonary toxicants. A better understanding of the pathogenic mechanisms underlying fibrogenesis may lead to the development of new therapeutic approaches for patients with these debilitating and largely irreversible chronic diseases.

Microglia, the innate immune cells of the CNS, respond to brain injury by activating and modifying their morphology. Our study arises from the great interest that has been focused on blueberry (BB) for the antioxidant and pharmacological properties displayed by its components. We analyzed the influence of hydroalcoholic BB extract in resting or lipopolysaccharide (LPS)-stimulated microglia BV-2 cells. BB exerted a protective effect against LPS-induced cytotoxicity, as indicated by cell viability. BB was also able to influence the actin cytoskeleton organization, to recover the control phenotype after LPS insult, and also to reduce LPS-driven migration. We evaluated the activity of Rho and Rac1 GTPases, which regulate both actin cytoskeletal organization and migratory capacity. LPS caused an increase in Rac1 activity, which was counteracted by BB extract. Furthermore, we demonstrated that, in the presence of BB, mRNA expression of pro-inflammatory cytokines IL-1&beta, IL-6 and TNF-&alpha, decreased, as did the immunofluorescence signal of iNOS, whereas that of Arg-1 was increased. Taken together, our results show that, during the inflammatory response, BB extract shifts the M1 polarization towards the M2 phenotype through an actin cytoskeletal rearrangement. Based on that, we might consider BB as a nutraceutical with anti-inflammatory activities.

Neuropeptide Y (NPY), a powerful neurotransmitter of the central nervous system, is a key regulator of angiogenesis and biology of adipose depots. Intriguingly, its peripheral vascular and angiogenic powerful activity is strictly associated to platelets, which are source of clinical hemoderivates, such as platelet lysate (PL), routinely employed in several clinical applications as wound healing, and to preserve ex vivo the progenitor properties of the adipose stromal cells pool. So far, the presence of NPY in PL and its biological effects on the adipose stromal cell fraction (ASCs) have never been investigated. Here, we aimed to identify endogenous sources of NPY such as PL-based preparations and to investigate which biological properties PL-derived NPY is able to exert on ASCs. The results show that PL contains a high amount of NPY, which is in part also excreted by ASCs when stimulated with PL. The protein levels of the three main NPY subtype receptors (Y1, Y2, Y5) are unaltered by stimulation of ASCs with PL, but their inhibition through selective pharmacological antagonists, considerably enhances migration, and a parallel reduction of angiogenic features of ASCs including decrease in VEGF mRNA and intracellular calcium levels, both downstream targets of NPY. The expression of VEGF and NPY is enhanced within the sites of neovascularisation of difficult wounds in patients after treatment with leuco-platelet concentrates. Our data highlight the presence of NPY in PL preparations and its peripheral effects on adipose progenitors.

We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.

OBJECTIVE: Treatment of wounds difficult to heal concerns 50% of the elderly population in Italy and is therefore an extreme relevance social burden. The present study shows how the treatment with autologous leuco-platelets reduces the healing time of wounds improving the functional recovery. PATIENTS AND METHODS: One hundred patients with ulcers of the legs were divided in two groups: 1) 50 patients treated with conventional therapies; 2) 50 patients treated with autologous leuco-platelet concentrate (LPC) and HYAFF as a scaffold. RESULTS: After 2 months a 49% reduction of the wound area was observed in the second group and in a remarkable share (about 65%) wound reduction was obtained in 15 days (4 LPC dressings). On the contrary, patients treated only by simple medications, showed a longer healing time and a greater percentage of failures. Morphometric analysis of bioptic samples obtained from the edge as well as from the bottom of the lesions obtained from the LPC group, detected an abundant presence of neoformed capillaries, characterized by a cubic, "reactive endothelium", close to the site of leuco-platelet infiltration. CONCLUSIONS: These results suggest that healing was promoted not only by limiting bacterial infections but also by the release of chemiotatic and proangiogenetic factors from leukocytes and platelets, improving the neoformation of capillaries.

Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes' production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

Background: Most of the recent reports suggest that inflammatory mediators play a central role in the etiopathogenesis of Alzheimer's disease (AD) and that the conditions leading to a chronic low-grade inflammation, such as stress, depression, obesity and metabolic syndrome, increase the odds of developing Mild Cognitive Impairment (MCI) and AD. Microglia cells are the main actors in the AD process: stimuli from the microenvironment may induce microglia cells to switch to a classically activated inflammatory phenotype M1, or, on the contrary to an alternatively activated M2 phenotype characterized by the secretion of different types of cytokines. Many attempts are currently being made in order to delay the progression of AD by reducing inflammatory mechanisms underlying the disease. Several studies support a relationship among neuroinflammation and nutrients, foods or dietary patterns, taking into account the synergistic or antagonistic biochemical interactions among nutrients as well as the different food sources of the same nutrient. Natural antioxidant and anti-inflammatory compounds found in plant foods, such as fruits, particularly berries (such as strawberry, blueberry, blackcurrant, blackberry, blueberry and mulberry) have been shown to exert neuroprotective activity. It is still unclear whether the dietary bioactive compounds enter the Blood Brain Barrier (BBB) playing a direct antiinflammatory or pro-inflammatory effect on microglia and/or other Central Nervous System (CNS) cells. Another hypothesis is that they may trigger a peripheral reaction that induce indirectly a CNS' response. The subsequent synthesis of cytokines may drive microglia polarization by different ways. So, via an indirect route microglia detects and responds to immune-to-brain signaling. Conclusion: This review summarizes current evidence about the potential mechanisms of the interaction among diet, neuroinflammation and AD.

Macrophages’ phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs’ expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes’ mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes’ expression and PLC proteins’ presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

Atherosclerosis initiation and progression is controlled by inflammatory molecular and cellular mediators. Cells of innate immunity, stimulated by various endogenous molecules that have undergone a transformation following an oxidative stress or nonenzymatic glycation processes, activate cells of the adaptive immunity, found at the borders of atheromas. In this way, an immune response against endogenous modified antigens takes place and gives rise to chronic low-level inflammation leading to the slow development of complex atherosclerotic plaques. These lesions will occasionally ulcerate, thus ending with fatal clinical events. Plaque macrophages represent the majority of leukocytes in the atherosclerotic lesions, and their secretory activity, including proinflammatory cytokines and matrix-degrading proteases, may be related to the fragilization of the fibrous cap and then to the rupture of the plaque. A considerable amount of work is currently focused on the identification of locally released proinflammatory factors that influence the evolution of the plaque to an unstable phenotype. A better understanding of these molecular processes may contribute to new treatment strategies. Mediators released by the immune system and associated with the development of carotid atherosclerosis are discussed.

AimsThe signalling system of phosphoinositides (PIs) is involved in a number of cell and tissue functions including membrane trafficking, ion channel activity, cell cycle, apoptosis, differentiation and cell and tissue polarity. Recently, a role in cell migration was hypothesised for PI and related molecules including the phosphoinositide-specific phospholipases C (PI-PLCs), main players in PI signalling. The expression of PI-PLCs is tissue-specific and evidence suggests that it varies under different conditions such as tumour progression or cell activation. In order to obtain a complete picture, the expression of all PI-PLC isoforms was analysed in human endothelial cells (EC).MethodsUsing molecular biology methods (RT-PCR), the expression of PI-PLC isoforms was analysed in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for human EC.ResultsAll the PI-PLC isoforms except PI-PLC β1, PI-PLC ɛ and PI-PLC ζ were expressed in HUVEC.ConclusionsThe growing interest in the complex cascade of events occurring in angiogenesis will provide useful insights for therapeutic strategies. The expression of PI-PLC isoforms in HUVEC is a useful tool for further studies directed to understanding their role in angiogenesis. However, although HUVEC represent a widely used experimental model for human macrovascular EC, limitations remain in that they cannot fully represent the metabolic properties and interactions of the EC distributed in the entire organism.

The ketogenic diet, originally developed for the treatment of epilepsy in non-responder children, is spreading to be used in the treatment of many diseases, including Alzheimerrsquo;s disease. The main activity of the ketogenic diet has been related to improved mitochondrial function and decreased oxidative stress. B-Hydroxybutyrate, the most studied ketone body, has been shown to reduce the production of reactive oxygen species (ROS), improving mitochondrial respiration: it stimulates the cellular endogenous antioxidant system with the activation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2), it modulates the ratio between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) and it increases the efficiency of electron transport chain through the expression of uncoupling proteins. Furthermore, the ketogenic diet performs anti-inflammatory activity by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome as well as inhibiting histone deacetylases (HDACs), improving memory encoding. The underlying mechanisms and the perspectives for the treatment of Alzheimerrsquo;s disease are discussed.

The new civil wars and waves of terrorism are causing crucial social changes, with consequences in all fields, including health care. In particular, skin injuries are evolving as an epidemic issue. From a physiological standpoint, although wound repair takes place more rapidly in the skin than in other tissues, it is still a complex organ to reconstruct. Genetic and clinical variables, such as diabetes, smoking, and inflammatory/immunological pathologies, are also important risk factors limiting the regenerative potential of many therapeutic applications. Therefore, optimization of current clinical strategies is critical. Here we summarize the current state of the field by focusing on stem cell therapy applications in wound healing, with an emphasis on current clinical approaches being developed at Sapienza University. These involve protocols for the ex vivo expansion of adipose tissue–derived mesenchymal stem cells by means of a patented GMP-compliant platelet lysate, Mesengen™. A combination of multiple strategies, including genetic modifications of stem cells, biomimetic scaffolds, or novel vehicles such as nanoparticles, are also discussed as future approaches.

Studies aimed at elucidating the pathogenetic mechanisms underlying the initiation and progression of human atherosclerosis have emphasized the central role of inflammatory and immune cells. Atherosclerotic plaques are infiltrated by activated macrophages, T and B lymphocytes, plasma cells, and mast cells, releasing inflammatory molecules, which amplify the severity of the disease. Endothelial cells subjected to various stress conditions express increased amounts of heat shock proteins (HSPs), some of the most successfully conserved proteins throughout evolution. Many experimental observations reviewed in this article draw attention to several HSPs targeted by a specific cellular and humoral immune response in patients with atherosclerotic disease. The review also reports preliminary data obtained by our group on the possible role of HSP90 as a candidate autoantigen in carotid atherosclerosis. Our study deals with the presence of specific antibodies and T cells directed against HSP90 in patients with carotid atherosclerotic plaques. In 60% of these subjects' sera but in none of the sera from healthy controls immunoblotting (IB) detected the presence of specific antibodies. Moreover, 20% of peripheral blood mononuclear cells (PBMC) samples from patients but none from healthy subjects proliferated in response to human purified HSP90. In vitro experiments showed an upregulation of HSP90 expression in endothelial cells exposed to oxidative stress by treatment with H(2)O(2) and greater release of soluble HSP90 in culture supernatants from H(2)O(2)-treated cells than from untreated cells.

Globesity is referred to a global epidemic of obesity, affecting millions of individuals. Molecules released by the enlarged adipose tissue, most of which are pro-inflammatory, have been named adipokines. The present review deals with function, molecular targets and the potential clinical relevance of adipokines. Currently, more than 600 adipokines have been identified, many of them, including leptin, visfatin, resistin as well as Retinol Binding Protein4 may serve as informative markers for metabolic and cardiovascular diseases and play important roles in glucose homeostasis, insulin sensitivity as well as metabolic regulation of energy expenditure. Adiponectin on the contrary exerts anti-inflammatory and insulin sensitizing activity. Adiponectin has additional anti-atherogenic effects and low adiponectin serum concentrations are associated with increased risk for cardiovascular diseases. The understanding of the role of adipokines has provided a wealth of information that has opened great opportunities for new therapeutic advances. Adiponectin may be the most prominent example for the potential use of an adipokine in the treatment of obesity and obesity-associated metabolic diseases. In many studies, administration of recombinant adiponectin results in improved insulin sensitivity, increased insulin secretion and beneficial effects on body weight and hyperglycemia. Up-regulation of adiponectin/adiponectin receptors or enhancing adiponectin receptor function may be an interesting therapeutic strategy for obesity-linked insulin resistance. Moreover, the therapeutic use of combined amylin/leptin agonism (with pramlintide and metreleptin) demonstrated a significant weight-lowering effect in obese subjects. Therefore, adipokines may be clinically relevant either as therapeutic tools or as target in the treatment of obesity related diseases.

Neuropeptide Y (NPY), a major autonomic nervous system and stress mediator, is emerging as an important regulator of inflammation, implicated in autoimmunity, asthma, atherosclerosis, and cancer. Yet the role of NPY in regulating phenotype and functions of dendritic cells (DCs), the professional antigen-presenting cells, remains undefined. Here we investigated whether NPY could induce DCs to migrate, mature, and polarize naive T lymphocytes. We found that NPY induced a dose-dependent migration of human monocyte-derived immature DCs through the engagement of NPY Y1 receptor and the activation of ERK and p38 mitogen-activated protein kinases. NPY promoted DC adhesion to endothelial cells and transendothelial migration. It failed to induce phenotypic DC maturation, whereas it conferred a T helper 2 (Th2) polarizing profile to DCs through the up-regulation of interleukin (IL)-6 and IL-10 production. Thus, during an immune/inflammatory response NPY may exert proinflammatory effects through the recruitment of immature DCs, but it may exert antiinflammatory effects by promoting a Th2 polarization. Locally, at inflammatory sites, cell recruitment could be amplified in conditions of intense acute, chronic, or cold stress. Thus, altered or amplified signaling through the NPY-NPY-Y1 receptor-DC axis may have implications for the development of inflammatory conditions.-Buttari, B., Profumo, E., Domenici, G., Tagliani, A., Ippoliti, F., Bonini, S., Businaro, R., Elenkov, I., Rigano, R. Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a Th2 polarization.

alpha2-Macroglobulin (alpha2M) has been identified as a carrier protein for beta-amyloid (Abeta) decreasing fibril formation and affecting the neurotoxicity of this peptide. The alpha2-macroglobulin receptor/low density lipoprotein receptor related protein (LRP) is involved in the internalization and degradation of the alpha2M/Abeta complexes and its impairment has been reported to occur in Alzheimer's disease. Previous studies have shown alpha2M to determine an enhancement or a reduction of Abeta toxicity in different culture systems. In order to clarify the role of alpha2M in Abeta neurotoxicity, we challenged human neuroblastoma cell lines with activated alpha2M in combination with Abeta. Our results show that in neuroblastoma cells expressing high levels of LRP, the administration of activated alpha2M protects the cells from Abeta neurotoxicity. Conversely, when this receptor is not present alpha2M determines an increase in Abeta toxicity as evaluated by MTT and TUNEL assays. In LRP-negative cells transfected with the full-length human LRP, the addition of activated alpha2M resulted to be protective against Abeta-induced neurotoxicity. By means of recombinant proteins we ascribed the neurotoxic activity of alpha2M to its FP3 fragment which has been previously shown to bind and neutralize transforming growth factor-beta. These studies provide evidence for both a neuroprotective and neurotoxic role of alpha2M regulated by the expression of its receptor LRP.

The presence of the alpha2macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2Mr/LRP) and its ligands alpha2macroglobulin (alpha2M), apoliprotein E, and plasminogen activators was detected in senile plaques of Alzheimer's disease (AD). To explore a possible role of alpha2M in neurodegenerative processes occurring in AD, we analyzed the effect of alpha2M on Abeta 25-35-induced neurotoxicity. Treatment of LAN5 human neuroblastoma cells with 10 microM beta-amyloid peptide fragment 25-35 (Abeta 25-35) for 72 h resulted in a 50% decrease in cell viability as determined by MTT incorporation and cell counts. The addition of alpha2M to the culture medium of these cells did not determine any effect, but when the activated form alpha2M* was used a dose-dependent decrease in cell viability was observed, the maximum effect being reached at 140 and 280 nM. Moreover, treatment of LAN5 cells with alpha2M* in combination with Abeta 25-35 increased the neurotoxicity of the amyloid peptide by 25%. This neurotoxic effect of alpha2M* seems to be related to its capability to bind and inactivate TGFbeta in the culture medium, since it was mimicked by a TGFbeta neutralizing antibody. A possible involvement of receptor-mediated endocytosis was ruled out, since alpha2M receptor is not present on LAN5, as revealed by RT-PCR and Western blotting experiments. The presence of alpha2M* in amyloid deposits of Alzheimer's disease has been recently reported and a possible impairment of LRP internalization processes has been hypothesized. Our data suggest that the local accumulation of alpha2M* in AD plaques may increase Abeta 25-35-induced neurotoxicity by neutralizing TGFbeta-mediated neuroprotective mechanisms.

Macrophages consist of two main subsets: the proinflammatory M1 subset and the anti-inflammatory M2 one. 7-oxo-cholesterol, the most abundant cholesterol autoxidation product within atherosclerotic plaque, is able to skew the M1/M2 balance towards a proinflammatory profile. In the present study, we explored the ability of the polyphenolic compound resveratrol to counteract the 7-oxo-cholesterol-triggered proinflammatory signaling in macrophages. Resveratrol-pretreated human monocyte-derived M1 and M2 macrophages were challenged with 7-oxo-cholesterol and analyzed for phenotype and endocytic ability by flow cytometry, for metalloproteinase- (MMP-) 2 and MMP-9 by gelatin zymography, and for cytokine, chemokine, and growth factor secretome by a multiplex immunoassay. We also investigated the NF-κB signaling pathway. In the M1 subset, resveratrol prevented the downregulation of CD16 and the upregulation of MMP-2 in response to 7-oxo-cholesterol, whereas in M2 macrophages it prevented the upregulation of CD14, MMP-2, and MMP-9 and the downregulation of endocytosis. Resveratrol prevented the upregulation of several proinflammatory and proangiogenic molecules in both subsets. We identified modulation of NF-κB as a potential mechanism implicated in 7-oxo-cholesterol and resveratrol effects. Our results strengthen previous findings on the immunomodulatory ability of resveratrol and highlight its role as potential therapeutic or preventive compound, to counteract the proatherogenic oxysterol signaling within atherosclerotic plaque.

Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC γ1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3-6-h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.

Many research efforts were addressed to identify individuals at high risk for multifactorial diseases, such as cardiovascular alterations and related diseases. Great interest was paid to investigate the genetic liability in multifactorial illnesses. The prognosis of high-risk patients might be greatly ameliorated using genetic predisposition risk factors, such as the polymorphisms of neuropeptide Y (NPY) and Angiotensin converting enzyme (ACE) genes. Epidemiologic results suggest that selected polymorphisms of both NPY and ACE might be helpful to improve the evaluation of patients, offering a powerful prognostic tool and paving the way to novel molecular therapeutic strategies. We present a case report of a male, sudden-death from myocardial infarction, presenting with left ventricular hypertrophy. The patient carried polymorphisms of ACE and NPY genes, respectively ACE genotype ID and NPY genotype T-399C, actually considered as risk factors.

The low density lipoprotein receptor-related protein (LRP) has been localized in human brain at the level of neurons, astrocytes and along capillary membranes. It is a multifunctional receptor responsible for binding and internalization of lipoproteins enriched with apoliprotein E, lipoprotein lipase, protease-alpha 2 macroglobulin complexes and plasminogen activator-inhibitor complexes. LRP expression is observed in cells involved in Alzheimer's disease, neoplastic transformation and tissue repair. Moreover, its synthesis is modulated during brain development. In this study we used the SK-N-AS human neuroblastoma cell line as a model system to study LRP expression during cellular differentiation induced by phorbol esters, retinoic acid and interferon gamma. Since LRP plays a major role in the regulation of lipid metabolism, the decreased levels of LRP measured by immunofluorescence, western blot and PCR on differentiated neuroblastoma cells may be the consequence of the lower requirements of cholesterol and lipids of differentiated cells in relation to their reduced mitotic index.

Atherosclerosis is a chronic inflammatory process, lasting for several decades until the onset of its clinical manifestations. The progression of the atherosclerotic lesion to a stable fibrotic plaque, narrowing the vascular lumen, or to a vulnerable plaque leading to main vascular complications, is associated to the involvement of several cell subpopulations of the innate as well as of the adaptive immunity, and to the release of chemokines and pro-inflammatory cytokines. Emerging evidence outlines that the cardiovascular risk is dependent on stress-mediators influencing cell migration and vascular remodeling. The view that atherosclerosis is initiated by monocytes and lymphocytes adhering to dysfunctional endothelial cells is substantiated by experimental and clinical observations. Macrophages, dendritic cells, T and B lymphocytes, granulocytes accumulating into the subendothelial space secrete and are stimulated by soluble factors, including peptides, proteases and cytokines acting synergistically. The final step of the disease, leading to plaque destabilization and rupture, is induced by the release, at the level of the fibrous cap, of metalloproteinases and elastases by the activated leukocytes which accumulate locally. Recruitment of specific cell subpopulations as well as the progression of atherosclerotic lesions towards a stable or an unstable phenotype, are related to the unbalance between pro-atherogenic and anti-atherogenic factors. In this connection stress hormones deserve particular attention, since their role in vascular remodeling, via vascular smooth cell proliferation, as well as in neoangiogenesis, via stimulation of endothelial cell proliferation and migration, has been already established.