Gut acellular matrix for the in vitro study of Enteric Nervous System cells

Enteric nervous system (ENS) cells respond to the intestinal extracellular matrix (ECM) signals changing their proliferation rate, migration and differentiation. In this study, we explored in vitro the adaptive response of primary ENS cell cultures to the stimulation of gut acellular matrix (AM) defining the gene expression profile of neuronal functionality markers. Scanning electron microscopy was used to detect the acquisition of specific morphological features. Intestinal AM was prepared using an enzyme-detergent treatment. Primary rat enteric cells were isolated from the myenteric plexus of postnatal rats using an enzymatic method and seeded on intestinal AM in the presence of exogenous neurotrophic factors. The morphological properties and the expression of specific differentiation markers were evaluated by Scanning Electron Microscopy (SEM) and wholemount fluorescent staining. In order to verify the synergic activity of soluble factors and AM, the gene expression of neurotransmitter receptors was evaluated by qPCR in ENS cells cultured in SM conditions in the presence or not of AM. The development of interconnected ganglion-like structures and the expression of neurotransmitter receptors suggested that gut matrix engineered with ENS cells could be useful for medical applications of regenerative medicine or for the in vitro assessment of tridimensional culture system of ENS

Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

Background:: Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages. Methods:: In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage. Results:: After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair. Conclusion:: Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy

Infrapatellar Fat Pad Stem Cells Responsiveness to Microenvironment in Osteoarthritis: From Morphology to Function

Recently, infrapatellar fat pad (IFP) has been considered as a source of stem cells for cartilage regeneration in osteoarthritis (OA) due to their ability for differentiation into chondrocytes. However, stressful conditions, like that related to OA, may induce a pathogenic reprograming. The aim of this study was to characterize the structural and functional properties of a new population of stem cells isolated from osteoarthritic infrapatellar fat pad (OA-IFP). Nine OA patients undergoing total knee arthroplasty (TKA) were enrolled in this study [median age = 74 years, interquartile range (IQR) = 78.25-67.7; median body mass index = 29.4 Kg/m2, IQR = 31.7-27.4]. OA-IFP stem cells were isolated and characterized for morphology, stemness, metabolic profile and multi-differentiative potential by transmission electron microscopy, flow cytometric analysis, gene expression study and cytochemistry. OA-IFP stem cells displayed a spindle-like morphology, self-renewal potential and responsiveness (CD44, CD105, VEGFR2, FGFR2, IL1R, and IL6R) to microenvironmental stimuli. Characterized by high grade of stemness (STAT3, NOTCH1, c-Myc, OCT-4, KLF4, and NANOG), the cells showed peculiar immunophenotypic properties (CD73+/CD39+/CD90+/CD105+/CD44\u2013/+/CD45\u2013). The expression of HLA-DR, CD34, Fas and FasL was indicative of a possible phenotypic reprograming induced by inflammation. Moreover, the response to mechanical stimuli together with high expression level of COL1A1 gene, suggested their possible protective response against in vivo mechanical overloading. Conversely, the low expression of CD38/NADase was indicative of their inability to counteract NAD+-mediated OA inflammation. Based on the ultrastructural, immunophenotypic and functional characterization, OA-IFP stem cells were hypothesized to be primed by the pathological environment and to exert incomplete protective activity from OA inflammation

Inflammatory profile of neurotrophins, IL-6, IL1-β, TNF-α, VEGF, ICAM-1 and TGF-β in the Human Waldeyer’s ring

The palatine tonsils, nasopharyngeal tonsil (adenoid) and lingual tonsil constitute the major part of Waldeyer’s ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components. The lymphoid tissue of Waldeyer’s ring is located at the gateway of the respiratory and alimentary tract and belongs to the mucosa-associated lymphoid tissue (MALT). The lymphatic tissue is known to interact with the nervous system and several organs implicated in the host response to a wide range of stressors (Otten et al., 1995; Kaneko et al., 2012; Ogasawara et al., 2011). This study focusses on the expression of some neurotrophins (NTs), their high- and low-affinity receptors in human adenoid tissues, lingual and palatine tonsils via immunohistochemical analysis, as well as on the expression of some inflammatory cytokines and other tissue growth factors (IL-6, IL1-β, TNF-α, VEGF, ICAM-1 and TGF-β)). Light microscopy immunohistochemistry performed on human samples showed to be generally positive for all the NTs investigated (NGF, BDNF, NT-3) and their receptors (TrKA, TrKB and TrKC) as well as the other cytokines and growth factors studied with some different expression levels. Real time PCR analysis is in progress to quantitate these data. Our data corroborate previous studies, suggesting that neurotrophins and inflammatory cytokines may mediate functional signals in lymphoid aggregates (Yusuf-Makagiansar et al., 2002; Ruoco et al., 1990)

S100B protein as a therapeutic target in multiple sclerosis: The S100B inhibitor arundic acid protects from chronic experimental autoimmune encephalomyelitis

S100B is an astrocytic protein behaving at high concentration as a damage-associated molecular pattern molecule. A direct correlation between the increased amount of S100B and inflammatory processes has been demonstrated, and in particular, the inhibitor of S100B activity pentamidine has been shown to ameliorate clinical scores and neuropathologic-biomolecular parameters in the relapsing-remitting experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. This study investigates the effect of arundic acid (AA), a known inhibitor of astrocytic S100B synthesis, in the chronic experimental autoimmune encephalomyelitis, which is another mouse model of multiple sclerosis usually studied. By the daily evaluation of clinical scores and neuropathologic-molecular analysis performed in the spinal cord, we observed that the AA-treated group showed lower severity compared to the vehicle-treated mice, particularly in the early phase of disease onset. We also observed a significant reduction of astrocytosis, demyelination, immune infiltrates, proinflammatory cytokines expression and enzymatic oxidative reactivity in the AA-treated group. Overall, our results reinforce the involvement of S100B in the development of animal models of multiple sclerosis and propose AA targeting the S100B protein as a focused potential drug to be considered for multiple sclerosis treatment

Umbilical cord mesenchymal stem cells modulate dextran sulphate sodium induced acute colitis in immunodeficient mice.

Inflammatory bowel diseases (IBD) are complex multi-factorial diseases with increasing incidence worldwide but their treatment is far from satisfactory. Unconventional strategies have consequently been investigated, proposing the use of stem cells as an effective alternative approach to IBD. In the present study we examined the protective potential of exogenously administered human umbilical cord derived mesenchymal stem cells (UCMSCs) against Dextran Sulphate Sodium (DSS) induced acute colitis in immunodeficient NOD.CB17-Prkdc scid/J mice with particular attention to endoplasmic reticulum (ER) stress. METHODS: UCMSCs were injected in NOD.CB17-Prkdc scid/J via the tail vein at day 1 and 4 after DSS administration. To verify attenuation of DSS induced damage by UCMSCs, Disease Activity Index (DAI) and body weight changes was monitored daily. Moreover, colon length, histological changes, myeloperoxidase and catalase activities, metalloproteinase (MMP) 2 and 9 expression and endoplasmic reticulum (ER) stress related proteins were evaluated on day 7. RESULTS: UCMSCs administration to immunodeficient NOD.CB17-Prkdc scid/J mice after DSS damage significantly reduced DAI (1.45\u2009\ub1\u20090.16 vs 2.08\u2009\ub1\u20090.18, p\u20093-fold), which were significantly reduced in mice receiving UCMSCs. Moreover, positive modulation in ER stress related proteins was observed after UCMSC administration. CONCLUSIONS: Our results demonstrated that UCMSCs are able to prevent DSS-induced colitis in immunodeficient mice. Using these mice we demonstrated that our UCMSCs have a direct preventive effect other than the T-cell immunomodulatory properties which are already known. Moreover we demonstrated a key function of MMPs and ER stress in the establishment of colitis suggesting them to be potential therapeutic targets in IBD treatment

Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation

La chiesa di San Giacomo, poi di San Pasquale, a Lecce

La chiesa leccese di San Giacomo, poi dedicata a San Pasquale, ha intrecciato nel tempo uno stretto rapporto con il potere feudale e politico: dalla fondazione di Raimondo Orsini del Balzo, alla ricostruzione del figlio Giovanni Antonio, alla vicinanza alla Torre del Parco – residenza orsiniana extra moenia, ‘luogo di delizie’ e sede della Zecca – e al sito dove si teneva la Fiera di san Giacomo. La Torre fu residenza dei Viceré provinciali e dei Presidi della Provincia, rappresentanti del potere iberico, e nel parco si tenevano feste e giostre all’uso spagnolo il 25 luglio, giorno della festa di san Giacomo. Nonostante il cambio di dedicazione, l’immagine del Santo pellegrino è presente in punti fondamentali dell’edificio sacro, come la facciata e l’ancona dell’altare maggiore, anche dopo il passaggio della chiesa agli Alcantarini nel 1671, e sulla colonna nel chiostro superiore restituita dall’incisione ottocentesca del Wenzel

"Histological Study on Bone Retrived Massive Allografts: Preliminary Observations"

Histological Study on Bone Retrived Massive Allografts: Preliminary Observation