Alessandro Pitruzzella

Nitric oxide modulation of the basal ganglia circuitry: therapeutic implication for Parkinson's disease and other motor disorders.

Several recent studies have emphasized a crucial role for the interactions between serotonergic and dopaminergic systems in movement control and the pathophysiology of basal ganglia. These observations are supported by anatomical evidence demonstrating large serotonergic innervation of all the basal ganglia nuclei. In fact, serotonergic terminals have been reported to make synaptic contacts with both substantia nigra dopamine-containing neurons and their terminal areas such as the striatum, the globus pallidus and the subthalamus. These brain areas contain a high concentration of serotonin (5-HT), with the substantia nigra pars reticulata receiving the greatest input. In this chapter, the distribution of different 5-HT receptor subtypes in the basal ganglia nuclei will be described. Furthermore, evidence demonstrating the serotonergic control of basal ganglia activity will be reviewed and the contribution of the different 5-HT receptor subtypes examined. The new avenues that the increasing knowledge of 5-HT in motor control has opened for exploring the pathophysiology and pharmacology of Parkinsons disease and other movement disorders will be discussed. It is clear that these avenues will be fruitful, despite the disappointing results so far obtained by clinical studies with selective 5-HT ligands. Nevertheless, these studies have led to a great increase in the attention given to the neurotransmitters of the basal ganglia and their connections.

Hsp60 and Hsp10 increase in colon mucosa of Crohn’s disease and ulcerative colitis

The purpose of this work was to determine in colon mucosa of Crohn’s disease (CD) and ulcerative colitis (UC) in relapse: a) the levels of the chaperonins Hsp60 and Hsp10; b) the quantity of inflammatory cells; and c) if the levels of chaperonins parallel those of inflammation cells. Twenty cases of CD and UC and twenty normal controls (NC) were studied using immunohistochemistry, Western blotting and immunofluorescence. Immunohistochemically, Hsp60 and Hsp10 were increased in both inflammatory bowel diseases (IBD) compared to NC. These results were confirmed by Western blotting. Hsp60 and Hsp10 occurred in the cytoplasm of epithelial cells in CD and UC but not in NC. Hsp60 and Hsp10 co-localised to epithelial cells of mucosal glands but not always in connective tissue cells of lamina propria, where only Hsp60 or, less often, Hsp10 was found. Cells typical of inflammation were significantly more abundant in CD and UC than in NC. Since chaperonins are key factors in the activation of the immune system leading to inflammation, we propose that they play a central role in the pathogenesis of the two diseases, which, consequently, ought to be studied as chaperonopathies.

Heat Shock Protein 60 Levels in Tissue and Circulating Exosomes in Human Large Bowel Cancer Before and After Ablative Surgery

Heat shock protein 60 (Hsp60) is a chaperonin involved in tumorigenesis, but its participation in tumor development and progression is not well understood and its value as a tumor biomarker has not been fully elucidated. In the current study, the authors presented evidence supporting the theory that Hsp60 has potential as a biomarker as well as a therapeutic target in patients with large bowel cancer.

Changes in immunohistochemical levels and subcellular localization after therapy and correlation and colocalization with CD68 suggest a pathogenetic role of Hsp60 in ulcerative colitis.

In an earlier work, the role of heat shock protein (Hsp60) in the pathogenesis of ulcerative colitis (UC) was suggested by its significant increase in the pathological mucosa parallel with an increase in inflammatory cells. More data in this direction are reported in this work. We analyzed by immunohistochemistry biopsies of colon tissue from 2 groups of patients with UC and treated with either 5-aminosalicylic acid (5-ASA) alone or in combination with a probiotic. We looked for inflammatory markers and Hsp60. Both the treatments were effective in reducing symptoms but the group treated with both 5-ASA and probiotics showed better clinical results. Amelioration of symptoms was associated with reduction of both inflammation and Hsp60, a reduction that was most marked in the group treated with 5-ASA and probiotics. The levels of Hsp60 positively correlated with those of CD68-positive cells, and double immunofluorescence showed a high index of colocalization of the chaperonin and CD68 in lamina propria. Immunoelectron microscopy showed that Hsp60—classically a mitochondrial protein—was abundantly also present in cytosol in biopsies taken at the time of diagnosis, but not after the treatment. Our data suggest that Hsp60 is an active player in pathogenesis of UC and it can be hypothesized that the chaperonin is responsible, at least in part, for initiation and maintenance of disease.

Circulating miR-130a, miR-27b, and miR-210 in Patients With Peripheral Artery Disease and Their Potential Relationship With Oxidative Stress A Pilot Study

Some emerging risk factors such as oxidative stress biomarkers and microRNAs (miRs) may add additional value to the established risk factors for peripheral artery disease (PAD). We enrolled 27 patients with PAD and 27 age-matched controls. We examined the levels of a series of miRs (miR-130a, miR-27b, and miR-210) in serum samples. The level of well-established oxidative stress biomarkers, such as lipid hydroperoxides, isoprostanes, hemeoxygenase-1 (HO-1) and reduced glutathione, was also measured in plasma and their relationship with the miRs was determined. Levels of miR-130a, miR-27b, and miR-210 were significantly increased in patients with PAD when compared to the controls. The level of miR-130 was positively correlated with body mass index, whereas miR-210 was inversely associated with pain-free walking distance (PfWD). None of the evaluated miRs was associated with lowered PfWD of patients with PAD (stage IIa > 250 m, IIb < 250 m) or oxidative stress parameters. In conclusion, our findings suggest the need for more research to assess if miRs can serve as useful markers for the early diagnosis and monitoring of PAD.

Oxidative stress markers at birth: Analyses of a neonatal population

In order to further understand neonatal stress and, thus, control it efficaciously, there is a need for more information on the manifestations of stress at the molecular level in the newborn, with particular regard to oxidants, and anti-oxidant and anti-stress mechanisms, including mitochondrial heat shock protein-chaperones such as Hsp60. We investigated patterns of anti-oxidants, biomarkers of oxidative stress, and Hsp60 levels in sera from newborns and found significant associations between glutathione (GSH) levels and gestational age, delivery modality, and lipid hydroperoxydes (LOOH) level. LOOH levels and spontaneous (vaginal) delivery were independently associated with increased GSH levels when these were above the median. Hsp60 and LOOH levels were positively correlated whereas Hsp60 and GSH levels were inversely correlated in spontaneously delivered newborns; in contrast, Hsp60 and GSH levels were positively correlated in newborns delivered by cesarea. Our results point to new directions in the search for definite patterns of GSH, LOOH, and Hsp60 in the newborns serum that might have functional and diagnostic significance and that could help in the monitoring of newborn health during and after delivery. In addition, the data provide a starting basis for investigating the precise roles and interplay of GSH and Hsp60 in the maintenance of an optimal redox balance at birth to cope with the stress inherent to delivery, and also for investigating the predictive value of any given pattern of GSH, LOOH, and Hsp60 at birth with regard to health status and risk of disease in adult life.

Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells.

Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin‐releasing hormone, luteinizing hormone, and follicle‐stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real‐time PCR. The results obtained show that testosterone levels increase at a 3.9 μM concentration of nandrolone and return to the basal level a 15.6 μM dose of nandrolone. Nandrolone‐induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a‐hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down‐regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects. J. Cell. Physiol. 231: 1385–1391, 2016.

Nandrolone decanoate interferes with testosterone biosynthesis altering blood–testis barrier components

The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography–mass spectrometry. Western blot analysis and quantitative real‐time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood–testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein‐1 (TJP1). ND stimulation deregulated metalloproteinase‐9, metalloproteinase‐2 (MMP‐2) and the tissue inhibitor of MMP‐2. Moreover, ND administration resulted in a mislocalization of mucin‐1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP‐2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone.

Mild Aerobic Exercise Training Hardly Affects the Diaphragm of mdx Mice

In the mdx mice model of Duchenne Muscular Dystrophy (DMD), mild endurance exercise training positively affected limb skeletal muscles, whereas few and controversial data exist on the effects of training on the diaphragm. The diaphragm was examined in mdx (C57BL/10ScSn‐Dmdmdx) and wild‐type (WT, C57BL/10ScSc) mice under sedentary conditions (mdx‐SD, WT‐SD) and during mild exercise training (mdx‐EX, WT‐EX). At baseline, and after 30 and 45 days (training: 5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonins Hsp60 and Hsp70 and the p65 subunit of nuclear factor‐kB (NF‐kB) were measured in diaphragm, gastrocnemius, and quadriceps in each experimental group at all time points. Although morphological analysis showed unchanged total area of necrosis/regeneration in the diaphragm after training, there was a trend for larger areas of regeneration than necrosis in the diaphragm of mdx‐EX compared to mdx‐SD mice. However, the levels of Cx39, a protein associated with active regeneration in damaged muscle, were similar in the diaphragm of mdx‐EX and mdx‐SD mice. Hsp60 significantly decreased at 45 days in the diaphragm, but not in limb muscles, in both trained and sedentary mdx compared to WT mice. In limb muscles, but not in the diaphragm, Hsp70 and NF‐kB p65 levels were increased in mdx mice irrespective of training at 30 and 45 days. Therefore, the diaphragm of mdx mice showed little inflammatory and stress responses over time, and appeared hardly affected by mild endurance training. J. Cell. Physiol. 232: 2044–2052, 2017.

Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

Tissue-engineered oral mucosal equivalents have been developed for in vitro studies for a few years now. However, the usefulness of currently available models is still limited by many factors, mainly the lack of a physiological extracellular matrix (ECM) and the use of cell populations that do not reflect the properly differentiated cytotypes of the mucosa of the oral cavity. For this reason, we have developed a novel three-dimensional culture model reflecting the normal architecture of the human oral mucosa, with the main aim of creating a better in vitro model where to test cellular responses to drugs administration. This novel 3D cell culture model (3D outgrowth) was set up using an artificial extracellular matrix (Matrigel™ ), allowing the interactions required for proper differentiation of the various citotypes which form the mucosal layer. Biopsies of human oral mucosa, in fragments of about 0.5 mm3, were placed onto 6.5mm Transwells, covered with Matrigel™ and grown in a specific culture medium. A gradual formation of an architectural structure similar to that of the in vivo oral mucosa was observed. Transmission electron and confocal microscopy were employed to characterize the newly developed model: the cell components (keratinocytes and fibroblasts) differentiated properly within the outgrowth and reconstituted, in vitro, the physiological structure of the human oral mucosa, including a stratified non-keratinized squamous layer composed of four different layers, a proper basal membrane and a lamina propria where fibroblasts produce ECM. Moreover, keratinocytes expressed CK5, CK13, CK19 and E-cadherin, whereas fibroblasts expressed collagen type I and IV, laminin and fibronectin. 3D outgrowths could be considered a valid alternative to animal models, and provide useful information for researchers interested in studying the responses of the human oral mucosa to locally delivered drugs or other exogenous treatments.