Tania Di Raimo

Adipokines and their Involvement as a Target of New Drugs

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.

IL-1β and IL-23 in amniotic fluids of ultrasound-detected aortic intima/media thickness and growth retardation

Intrauterine growth restriction (IUGR) and/or neonatal low birth weight are often associated with increased intima/media thickness of the abdominal aortic wall (aIMT). Several studies in children suggested that aIMT might be related to inflammation, probably indicating an early stage of adulthood diseases, such as atherosclerosis. Our previous study performed on the abdominal aortic wall of a stillbirth presenting with IUGR and aIMT suggested an association among IUGR, aIMT, and inflammation, also highlighting the presence of fibroblastoid cells, which are thought to represent peculiar elements of the pre-atherosclerotic lesions. These observations led us to analyze two cytokines involved in the inflammation cascade, IL-1β and IL-23, in amniotic fluid samples of IUGR fetuses and small-for-gestational-age newborns presenting with aIMT and in normal controls. Our results indicate that IL-23, but not IL-1β, concentrations differed in the groups analyzed. Therefore, IL-23, a regulatory element that bridges the innate and adaptive arms of the immune system, might be involved in the inflammatory process observed in fetal aIMT.

Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages

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.

Multidisciplinary approaches to stimulate wound healing.

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.

Interleukin-18 modulation in autism spectrum disorders

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disease which affects 1 in 88 children. Its etiology remains basically unknown, but it is apparent that neuroinflammation is involved in disease development. Great attention has been focused on pro-inflammatory cytokines, and several studies have reported their dysfunction unbalance in serum as well as in the brain. The present work aimed at evaluating putative dysregulation of interleukin-18 (IL-18), a pro-inflammatory cytokine of the IL-1 family in the sera of patients with ASD of different grades, compared to healthy controls, as well as in postmortem brain samples obtained from patients with tuberous sclerosis as well as acute inflammatory diseases. Moreover, quantitative analysis of IL-18 was performed in the sera and brain obtained from Reeler mice, an experimental model of autism.MethodsSerum IL-18 levels were measured by ELISA. IL-18 was localized by immunohistochemical analysis in brain sections obtained from tuberous sclerosis and encephalitis patients, as well as from gender- and age-matched controls, and in the brain sections of both Reeler and wild-type mice. IL-18 was also quantified by Western blots in homogenates of Reeler and wild-type mice brains. IL-18 binding protein (IL-18BP) was evaluated in Reeler and wild-type mice plasma as well as in their brains (sections and homogenates).ResultsIL-18 content decreased in the sera of patients with autism compared to healthy subjects and in Reeler sera compared to wild-type controls. IL-18 was detected within glial cells and neurons in the brain of subjects affected by tuberous sclerosis and encephalitis whereas in healthy subjects, only a weak IL-18 positivity was detected at the level of glial cells. Western blot identified higher amounts of IL-18 in Reeler brain homogenates compared to wild-type littermates. IL-18BP was expressed in higher amounts in Reeler brain compared to the brain of wild-type mice, whereas no significant difference was detected comparing IL-18BP plasma levels.ConclusionsIL-18 is dysregulated in ASD patients. Further studies seemed necessary to clarify the molecular details behind IL-18 increase in the brain and IL-18 decrease in the sera of patients. An increase in the size of the patient cohort seems necessary to ascertain whether decreased IL-18 content in the sera can become a predictive biomarker of ASD and whether its measure, in combination with other markers (e.g., increased levels of brain-derived neurotrophic factor (BDNF)), may be included in a diagnostic panel.

Cognitive disability in alzheimer's disease and its management

Cognitive disability linked to neurodegenerative diseases and in particular to Alzheimers disease, remains an increasing cause for concern through a dramatic prevalence increment and associated socio-economic burdens. Initially Alzheimers disease develops asymptomatically with primary clinical signs, such as memory impairment, decline of spatial and perceptual abilities, occurring at a later stage. This delay implies the possibility of promoting early interventions during the pre-symptomatic stage of the disease. Different strategies have been applied in order to prevent/delay onset of Alzheimers disease or at least to improve quality of life and health conditions of Alzheimers disease patients and their caregivers, especially in the absence of current viable therapies. Multidomain interventions, aimed at affecting several risk factors simultaneously, offer a versatility that may attain improved outcomes in comparison with single-domain prevention trials. These multidomain interventions involve diet, physical exercise, cognitive training and social activities, while music therapy, improving self-consciousness and reducing neurofibrils, may contribute to deceleration/delay onset of Alzheimers disease progression. Information and Communication Technology (ICT) provides broad applications to improve quality of life and well-being of Alzheimers disease patients and caregivers, suffering from psychological distress, as well as reducing additional public health costs.

LPS, Oleuropein and Blueberry extracts affect the survival, morphology and Phosphoinositide signalling in stimulated human endothelial cells

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes’ expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.

Expression and localization of Phosphoinositide-specific Phospholipase C enzymes in polarized macrophages

The phenotypic and functional diversity of macrophages depends on differentiating programs being developed during the cells’ lives. Great interest was addressed to identify the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. Enzymes belonging to the PLC family are strictly tissue specific and the expression panel, as well as the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), M1 and M2 macrophages to list the isoforms expressed in the polarized macrophages and their subcellular localization. Our results confirmed that macrophages express a wide number of PLC isoforms. 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. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

Macrophage polarization by the microenvironment of atherosclerotic plaques

Macrophages are key cellular mediators of innate immunity: they are positionally and transcriptionally programmed to respond to pathogens and environmental challenges. When activated by inflammatory signals in their microenvironment they develop into functionally and phenotypically distinct polarized subpopulations: classically activated macrophages, M1, characterized by cytotoxix/proinflammatory activity; alternatively activated macrophages, M2, characterized by anti-inflammatory/wound repair activity. M1 pro-inflammatory macrophages drive atherosclerotic plaques progression towards instability, cap fragilization and rupture. Our study provide new informations about the role exherted by IL-23 and its receptor in human carotid atherosclerotic plaque progression. We show the presence of IL-23 immunoreactivity, mRNA and protein in macrophages infiltrating human carotid atherosclerotic plaques. Our immunohistochemical analysis demonstrated a strong IL-23 immunoreactivity within the inflammatory infiltrate at the shoulder of the plaques, and at the level of cells lining the fibrous cap. FISH analysis confirmed the expression of IL-23 detected by immunohistochemistry. Immunofluorescence, followed by FISH analysis, showed that cells positive for IL-23 mRNA bind anti-CD68 mAb, thus indicating that these cells belong to the macrophage components of the inflammatory infiltrate. This result was further confirmed by double labelling experiments. IL-23 immunoreactivity was detected within the fibrous layer and co-localized with cells belonging to the monocyte-macrophage lineage as shown by their strong CD68- and CD14-related reaction. Clusters of double-positive cells were found at the border of the plaque, as well as in the subendothelial space. Immunohistochemistry and immunofluorescence showed a strong immunoreactivity for IL-23R at the level of inflammatory mononuclear cells accumulated within the plaque. In vitro, only M1 pro-inflammatory, but not M2 anti-inflammatory macrophages produced IL-23, upon stimulation with zymosan or bacterial lipopolysaccharide. Our results suggests that a hyperactive and highly pathogenic IL-23-IL-23R system drives chronic inflammation in atherosclerosis, while the presence of IL-23 proximal to the fibrous cap may contribute to the atherosclerotic plaque instability.

Ketogenic diet: a nutritional protocol for a non-pharmacological treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is a progressively worsening disease that affects specific brain areas involved in spatial memory, short-term memory and has a negative impact on the individual, limiting his ability of independent life. AD remains asymptomatic for a considerable time before cognitive decline becomes clinically evident. For this reason, the establishment of appropriate nutritional protocols, in the early stage, may be more effective than any drug treatments in the fight against this disease. Ketogenic Diet (KD) is a diet high in fat, adequate-proteins and low carbohydrates producing ketone bodies (KBs) (alternative energy source to glucose for the brain). Nowadays, relations between KBs and cerebral Aβ accumulation are not clear. Most studies focus on the neuronal component and forget the vascular component of AD represented by the blood-brain barrier (BBB), located at the cerebral microvessels level. Consequently, it seems essential to focus on the BBB physiology, and in particular on the BBB’s functions of the receptors/transporters and enzymes involved in Aβ peptide transport and metabolism, to better understand the influence of a KD on the onset and the evolution of this disease. For 4 weeks, Wild type mice (129SV) were maintained on KD and Control Diet (CD). Glucose and Beta-hydroxybutyrate (BHB) levels were assessed in blood sample. Microvessel fractions were isolated from total brain and qPCR analyses were performed to study expression of transporters, receptors and enzymes involved in amyloid transport and metabolism at the BBB level. KD fed animals showed increased levels of BHB which was accompanied by an increased expression of Monocarboxylate Transporters 1 (MCT1) and Glucose transporter 1 (GLUT1) at the BBB level. There were not changes in the level of Glucose and body weight in these mice. In addition we observed modifications in the expression of some transporters involved in Aβ exchanges such as Low density lipoprotein receptor-related protein 1 (LRP1) and Multidrug resistance-associated protein 1 (MRP1). The expression of Aβ synthesis enzymes remains unchanged, instead an increase for the Aβ degradation enzyme Endothelin Converting Enzyme 1 (ECE1) was observed. These preliminary results show that dietary factors and in particular KD can modulate the expression of some actors implicated in brain Aβ metabolism at the BBB level.