Histological and Immunohistochemical Evaluation of Autologous Cultured Bone Marrow Mesenchymal Stem Cells and Bone Marrow Mononucleated Cells in Collagenase-Induced Tendinitis of Equine Superficial Digital Flexor Tendon

The aim of this study was to compare treatment with cultured bone marrow stromal cells (cBMSCs), bone marrow Mononucleated Cells (BMMNCs), and placebo to repair collagenase-induced tendinitis in horses. In six adult Standardbred horses, 4000 IU of collagenase were injected in the superficial digital flexor tendon (SDFT). Three weeks after collagenase treatment, an average of either 5.5 × 106 cBMSCs or 1.2 × 108 BMMNCs, fibrin glue, and saline solution was injected intralesionally in random order. In cBMSC- and BMMNCS-treated tendons, a high expression of cartilage oligomeric matrix protein (COMP) and type I collagen, but low levels of type III collagen were revealed by immunohistochemistry, with a normal longitudinally oriented fiber pattern. Placebo-treated tendons expressed very low quantities of COMP and type I collagen but large numbers of randomly oriented type III collagen fibers. Both cBMSC and BMMNCS grafts resulted in a qualitatively similar heling improvement of tendon extracellular matrix, in terms of the type I/III collagen ratio, fiber orientation, and COMP expression

Erythrocyte's aging in microgravity highlights how environmental stimuli shape metabolism and morphology

The determination of the function of cells in zero-gravity conditions is a subject of interest in many different research fields. Due to their metabolic unicity, the characterization of the behaviour of erythrocytes maintained in prolonged microgravity conditions is of particular importance. Here, we used a 3D-clinostat to assess the microgravity-induced modifications of the structure and function of these cells, by investigating how they translate these peculiar mechanical stimuli into modifications, with potential clinical interest, of the biochemical pathways and the aging processes. We compared the erythrocyte's structural parameters and selected metabolic indicators that are characteristic of the aging in microgravity and standard static incubation conditions. The results suggest that, at first, human erythrocytes react to external stimuli by adapting their metabolic patterns and the rate of consumption of the cell resources. On longer timeframes, the cells translate even small differences in the environment mechanical solicitations into structural and morphologic features, leading to distinctive morphological patterns of agin

Anti-Inflammatory activity of a polyphenolic extract from Arabidopsis thaliana in in vitro and in vivo models of Alzheimer's Disease

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the primary form of dementia in the elderly. One of the main features of AD is the increase in amyloid-beta (Aβ) peptide production and aggregation, leading to oxidative stress, neuroinflammation and neurodegeneration. Polyphenols are well known for their antioxidant, anti-inflammatory and neuroprotective effects and have been proposed as possible therapeutic agents against AD. Here, we investigated the effects of a polyphenolic extract of Arabidopsis thaliana (a plant belonging to the Brassicaceae family) on inflammatory response induced by Aβ. BV2 murine microglia cells treated with both Aβ25⁻35 peptide and extract showed a lower pro-inflammatory (IL-6, IL-1β, TNF-α) and a higher anti-inflammatory (IL-4, IL-10, IL-13) cytokine production compared to cells treated with Aβ only. The activation of the Nrf2-antioxidant response element signaling pathway in treated cells resulted in the upregulation of heme oxygenase-1 mRNA and in an increase of NAD(P)H:quinone oxidoreductase 1 activity. To establish whether the extract is also effective against Aβ-induced neurotoxicity in vivo, we evaluated its effect on the impaired climbing ability of AD Drosophila flies expressing human Aβ1⁻42. Arabidopsis extract significantly restored the locomotor activity of these flies, thus confirming its neuroprotective effects also in vivo. These results point to a protective effect of the Arabidopsis extract in AD, and prompt its use as a model in studying the impact of complex mixtures derived from plant-based food on neurodegenerative diseases

Ultrastructural study of cultured ovine bone marrow-derived mesenchymal stromal cells.

Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify “in situ” the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected

One- and Two-Electron Oxidations of β-Amyloid_25-35 by Carbonate Radical Anion (CO₃•-) and Peroxymonocarbonate (HCO₄-):Role of Sulfur in Radical Reactions and Peptide Aggregation

The β-amyloid (Aβ) peptide plays a key role in the pathogenesis of Alzheimer’s disease. The methionine (Met) residue at position 35 in Aβ C-terminal domain is critical for neurotoxicity, aggregation, and free radical formation initiated by the peptide. The role of Met in modulating toxicological properties of Aβ most likely involves an oxidative event at the sulfur atom. We therefore investigated the one- or two-electron oxidation of the Met residue of Aβ25-35 fragment and the effect of such oxidation on the behavior of the peptide. Bicarbonate promotes two-electron oxidations mediated by hydrogen peroxide after generation of peroxymonocarbonate (HCO4−, PMC). The bicarbonate/carbon dioxide pair stimulates one-electron oxidations mediated by carbonate radical anion (CO3•−). PMC efficiently oxidizes thioether sulfur of the Met residue to sulfoxide. Interestingly, such oxidation hampers the tendency of Aβ to aggregate. Conversely, CO3•− causes the one-electron oxidation of methionine residue to sulfur radical cation (MetS•+). The formation of this transient reactive intermediate during Aβ oxidation may play an important role in the process underlying amyloid neurotoxicity and free radical generation

ERp57 chaperon protein protects neuronal cells from Aβ-induced toxicity

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid-β peptide (Aβ) in the form of senile plaques. Aβ can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aβ peptides, suggesting that it may be a carrier protein which prevents aggregation of Aβ. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aβ with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aβ25-35 fragment in vitro with high affinity via two in silico-predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short-term Aβ25-35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aβ25-35 and restores cellular viability, by preventing Aβ25-35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aβ toxicity and highlights it as a possible therapeutic tool in the treatment of AD

Genetic determinants in a critical domain of ns5a correlate with hepatocellular carcinoma in cirrhotic patients infected with hcv genotype 1b

HCV is an important cause of hepatocellular carcinoma (HCC). HCV NS5A domain‐1 interacts with cellular proteins inducing pro‐oncogenic pathways. Thus, we explore genetic variations in NS5A domain‐1 and their association with HCC, by analyzing 188 NS5A sequences from HCV genotype‐1b infected DAA‐naïve cirrhotic patients: 34 with HCC and 154 without HCC. Specific NS5A mutations significantly correlate with HCC: S3T (8.8% vs. 1.3%, p = 0.01), T122M (8.8% vs. 0.0%, p < 0.001), M133I (20.6% vs. 3.9%, p < 0.001), and Q181E (11.8% vs. 0.6%, p < 0.001). By multivariable analysis, the presence of >1 of them independently correlates with HCC (OR (95%CI): 21.8 (5.7–82.3); p < 0.001). Focusing on HCC‐group, the presence of these mutations correlates with higher viremia (median (IQR): 5.7 (5.4–6.2) log IU/mL vs. 5.3 (4.4–5.6) log IU/mL, p = 0.02) and lower ALT (35 (30–71) vs. 83 (48–108) U/L, p = 0.004), suggesting a role in enhancing viral fitness without affecting necroinflammation. Notably, these mutations reside in NS5A regions known to interact with cellular proteins crucial for cell‐cycle regulation (p53, p85‐PIK3, and β‐ catenin), and introduce additional phosphorylation sites, a phenomenon known to ameliorate NS5A interaction with cellular proteins. Overall, these results provide a focus for further investigations on molecular bases of HCV‐mediated oncogenesis. The role of these NS5A domain‐1 mutations in triggering pro‐oncogenic stimuli that can persist also despite achievement of sustained virological response deserves further investigation

Isolation, Determination and Analysis of Bioactive Natural Sulfur Compounds

Sulfur-containing products contribute significantly to natural chemical diversity and allow fundamental biological functions that no other compounds allow [...

Resveratrol: an ethylene bridge between Chemistry and Biochemistry

La ricerca scientifica del Dr. Francioso si è focalizzata sullo studio di molecole antiossidanti di origine naturale. In particolar modo si è concentrata su una molecola polifenolica, il Resveratrolo, che sin dalla sua scoperta ha attratto l'interesse della comunità scientifica per le sue molteplici attività biologiche e farmacologiche. Il Resveratrolo infatti oltre ad avere notevoli proprietà antiossidanti possiede marcata attività antitumorale, antinfiammatoria, antivirale, cardioprotettiva, di inibizione dell’aggregazione piastrinica e di modulazione dell’apoptosi. Tuttavia, nonostante i suoi molteplici effetti positivi sulla salute umana, l’uso del Resveratrolo come farmaco è fortemente limitato dalla sua scarsa solubilità in ambiente acquoso e bassa biodisponibilità, oltre che a causa della sua limitata stabilità e tendenza ad auto-ossidazione. Recentemente il Dr. Francioso, in collaborazione con l’azienda farmaceutica NOOS Srl, ha messo a punto una nuova formulazione liquida biocompatibile in cui il Resveratrolo è complessato con il Carbossimetil(1,3/1,6)-β-D-Glucano, derivato carbossimetilico del β-glucano, polimero di origine naturale con note attività farmaceutiche e biologiche. Questa associazione ha reso il Resveratrolo più solubile e chimicamente stabile in ambiente acquoso come dimostrato dai risultati di solubilità e stabilità a lungo termine. Per evidenziarne l’efficacia terapeutica è stata testata per la prima volta l’attività antivirale contro Rhinovirus in sistemi ex-vivo, dimostrando la capacità di questa associazione di ridurre significativamente la replicazione virale e la produzione di citochine pro-infiammatorie associate all’infezione virale. Un altro progetto che il Dr. Francioso ha perseguito nell’ambito del dottorato di ricerca in Biochimica è stato quello di verificare la stabilità fotochimica del Resveratrolo che è noto essere altamente fotosensibile anche dopo brevi esposizioni ai raggi UV, e di studiare i meccanismi di reazione fotochimici a cui va incontro la molecola in seguito a irradiazione. Attraverso metodi cromatografici e spettrometrici si è riusciti per la prima volta a portare a termine la completa caratterizzazione e quindi l’identificazione inequivocabile di una delle specie chimiche che si generano dopo irradiazione ultravioletta, il 2,4,6-triidrossifenantrene derivato dall’isomerizzazione e successiva elettrociclizzazione-ossidazione foto-indotta del Resveratrolo. Data la tossicità delle sostanze chimiche a struttura idrocarburica policiclica aromatica si è deciso, in collaborazione con l’ Università de “La Habana”(Cuba), di studiare gli effetti tossici del fotoprodotto isolato e purificato, ponendo l’attenzione in particolar modo sugli aspetti genotossici che questo composto potrebbe esercitare. I dati ottenuti dal Dr. Francioso presso il Dipartimento di Biologia vegetale della Università de “La Habana”, dimostrano che la molecola esercita un forte effetto citotossico e genotossico anche a concentrazioni sub-micromolari, ponendo quindi una nuova prospettiva sull’uso di prodotti a base di Resveratrolo potenzialmente esposti alla luce ultravioletta. Il Dr. Francioso ha quindi condotto ulteriori studi per verificare se il 2,4,6-triidrossifenantrene sia prodotto a seguito esposizione alla luce ultravioletta anche in campioni alimentari come vino e uva e in matrici biologiche e naturali complesse come oli e integratori alimentari. I risultati ottenuti dimostrano che il 2,4,6-triidrossifenantrene viene generato anche dopo periodi relativamente brevi di esposizione alla luce ultravioletta, rimarcando ancora come sia importante tenere in considerazione questo aspetto per quanto concerne l’uso o il consumo di prodotti contenenti Resveratrolo