Isolamento e caratterizzazione di una linea cellulare umana da Non Small Cell Lung Cancer

Il carcinoma del polmone rappresenta ad oggi la prima causa di morte per patologia neoplastica. Nonostante siano stati identificati i principali fattori eziologici alla base della neoplasia, i meccanismi biologici sono ancora poco conosciuti e ciò spiega la scarsa efficacia delle terapie oggi utilizzate. Recenti studi hanno messo in evidenza che, in aggiunta alle neoplasie ematologiche, anche nelle neoplasie solide esiste una controparte neoplastica della cellula staminale normale, denominata Cancer Initiating Cell (CIC), che sembra avere un ruolo chiave nell’insorgenza, progressione e metastatizzazione del tumore; queste cellule rappresentano quindi un potenziale target terapeutico in grado di modificare la storia naturale dei tumori polmonari. L’obiettivo principale del progetto di ricerca denominato “Lung Cancer Initiating Cell” (LCIC) è l’isolamento e la caratterizzazione di fenotipi staminali di origine polmonare, a partire da lobi polmonari di pazienti affetti da neoplasia e sottoposti ad intervento chirurgico presso il Dipartimento di Scienze Chirurgiche, Unità Operativa di Chirurgia Toracica, dell’Università degli Studi di Parma. A partire da colture primarie, sono state purificate ed amplificate nel nostro laboratorio linee di cellule mesenchimali stromali e linee di cellule endoteliali ottenute sia dal nodulo neoplastico che dalla porzione di parenchima distale indenne da neoplasia. Da diversi casi analizzati siamo riusciti ad isolare diverse linee cellulari con morfologia e fenotipo caratteristici del compartimento epiteliale. Questo studio mostra i dati preliminari relativi alla caratterizzazione fenotipica e funzionale di una di queste linee epiteliali, denominata Tumor Epitelial Cell (TEpC), ottenuta da un paziente maschio di anni 58, ex fumatore, affetto da Adenocarcinoma polmonare (stadio G2). In particolare, la caratterizzazione fenotipica delle linea di TEpCs per la ricerca di marcatori caratteristici dell’epitelio polmonare o della linea mesenchimale è stata effettuata mediante analisi FACS, analisi immunoistochimica e un’analisi ultrastrutturale al microscopio elettronico a trasmissione (TEM). I dati ottenuti sono stati confrontati con una linea di controllo isolata da adenocarcinoma polmonare umano, denominata Calu-3, già ampiamente caratterizzata. La rivelazione immunocitochimica e l’analisi FACS hanno evidenziato la positività per i marcatori epiteliali tipici quali E-caderina, pan-citocheratina e CD44 sia per la linea di TEpC che per la linea di controllo delle Calu-3. L’analisi TEM ha inoltre dimostrato la presenza di tratti cellulari comuni in entrambe le linee analizzate. Per valutare le proprietà funzionali della popolazione epiteliale isolata e, in particolare, il loro potenziale tumorigenico, è stato effettuato uno studio in vivo in cui TEpCs e Calu-3 sono state inoculate in topi femmine BALB/c nudi. Dopo il sacrificio i noduli neoformati sono stati excisi ed analizzati per valutarne la composizione tissutale. Inoltre, una porzione non destinata all’analisi immunoistochimica è stata processata per isolare e riespandere in vitro le cellule tumorali inoculate. L’analisi morfometrica ha documentato una forte somiglianza tra il nodulo formato dall’iniezione delle TEpC e il tumore d’origine da cui sono state isolate. In conclusione, la possibilità di isolare, espandere e caratterizzare cellule epiteliali neoplastiche paziente e tumore-specifiche rappresenta il punto di partenza per l’identificazione di sottopopolazioni cellulari implicate nella genesi e sviluppo della neoplasia. In ambito clinico, si prospetta inoltre l’opportunità di sviluppare tests di sensibilità in vitro allo scopo di individuare terapie farmacologiche personalizzate

La suggestiva "Hedda Gabler" di Antonio Calenda

This article examines the very recent mise-en-scène made by the Teatro Stabile di Trieste, directed by Antonio Calenda (Manuela Mandracchia as the protagonist), and highlights the tied relationship that the director has intended to establish with the more provocative Ibsenian critics, according to which Hedda could be anything except the character of a proto-martyr of the Decadency ideology. The Ibsenian text – in Calenda’s staging – is haunted by an incestuous ghost between Hedda and her father, the General Gabler, which – in the past – stopped her love to Løvborg, and – in the present – stops her marriage with Tesman (Hedda accepts to marry Tesman, but she keeps her maiden name (Hedda Gabler, not Hedda Tesman).This article examines the very recent mise-en-scène made by the Teatro Stabile di Trieste, directed by Antonio Calenda (Manuela Mandracchia as the protagonist), and highlights the tied relationship that the director has intended to establish with the more provocative Ibsenian critics, according to which Hedda could be anything except the character of a proto-martyr of the Decadency ideology. The Ibsenian text – in Calenda’s staging – is haunted by an incestuous ghost between Hedda and her father, the General Gabler, which – in the past – stopped her love to Løvborg, and – in the present – stops her marriage with Tesman (Hedda accepts to marry Tesman, but she keeps her maiden name (Hedda Gabler, not Hedda Tesman)

Oxidant stress and platelet activation in hypercholesterolemia

Hypercholesterolemia is the dominant risk factor associated with atherothrombotic disorders in the western world. Consequently, much attention has been devoted to defining its role in the pathogenesis of atherosclerosis. It is currently recognized that hypercholesterolemia induces phenotypic changes in the microcirculation that are consistent with oxidative and nitrosative stresses. Superoxide is generated via several cellular systems and, once formed, participates in a number of reactions, yielding various free radicals, such as hydrogen peroxide, peroxynitrite, or oxidized low-density lipoproteins. Once oxidant stress is invoked, characteristic pathophysiologic features ensue, such as platelet activation and lipid peroxidation, which are both involved in the initiation and progression of the atherosclerotic lesions. Thus, therapeutic strategies that act to maintain the normal balance in the oxidant status of the vascular bed may prove effective in reducing the deleterious consequences of hypercholesterolemia

Molecular mechanisms of helicobacter pylori pathogenesis

Helicobacter pylori infects 50% of mankind. The vast majority of H. pylori infection occurs in the developing countries where up to 80% of the middle-aged adults may be infected. Bacterial infection causes an inflammatory response that proceeds through a series of intermediated stages of precancerous lesions (gastritis, atrophy, intestinal metaplasia, and dysplasia). Among infected individuals, approximately 10% develops severe gastric lesions such as peptic ulcer disease, 1-3% progresses to gastric cancer (GC) with a low 5-year survival rate, and 0.1% develops mucosa-associated lymphoid tissue (MALT). GC is one of the most common cancer and the third leading cause of cancer-related deaths worldwide. In this review, we have summarized the most recent papers about molecular mechanisms of H. pylori pathogenesis. The main important steps of H. pylori infection such as adhesion, entry in epithelial gastric cells, activation of intracellular pathways until epigenetic modifications have been described

Semirigid Ligands Enhance Different Coordination Behavior of Nd and Dy Relevant to Their Separation and Recovery in a Non-aqueous Environment

Rare-earth elements are widely used in high-end technologies, the production of permanent magnets (PMs) being one of the sectors with the greatest current demand and likely greater future demand. The combination of Nd and Dy in NdFeB PMs enhances their magnetic properties but makes their recycling more challenging. Due to the similar chemical properties of Nd and Dy, their separation is expensive and currently limited to the small scale. It is therefore crucially important to devise efficient and selective methods that can recover and then reuse those critical metals. To address these issues, a series of heptadentate Trensal-based ligands were used for the complexation of Dy3+ and Nd3+ ions, with the goal of indicating the role of coordination and solubility equilibria in the selective precipitation of Ln3+-metal complexes from multimetal non-water solutions. Specifically, for a 1:1 Nd/Dy mixture, a selective and fast precipitation of the Dy complex occurred in acetone with the Trensalp-OMe ligand at room temperature, with a concomitant enrichment of Nd in the solution phase. In acetone, complexes of Nd and Dy with Trensalp-OMe were characterized by very similar formation constants of 7.0(2) and 7.3(2), respectively. From the structural analysis of an array of Dy and Nd complexes with TrensalR ligands, we showed that Dy invariably provided complexes with coordination number (cn) of 7, whereas the larger Nd experienced an expansion of the coordination sphere by recruiting additional solvent molecules and giving a cn of >7. The significant structural differences have been identified as the main premises upon which a suitable separation strategy can be devised with these kind of ligands, as well as other preorganized polydentate ligands that can exploit the small differences in Ln3+ coordination requirements

Small leucine rich proteoglycans are differently distributed in normal and pathological endometrium

BACKGROUND: During the woman's fertile period, the non-pregnant uterus is subject to constant cyclic changes. The complex mechanisms that control the balance among proliferation, differentiation, cell death and the structural remodeling of the extracellular matrix can contribute to the benign or malignant endometrial pathological state. The small leucine-rich proteoglycans (SLRPs) are important components of cell surface and extracellular matrices. MATERIALS AND METHODS: Using immunohistochemistry, we showed that the distribution patterns of SLRPs were completely modified in the pathological compared to normal endometrium. RESULTS: The expression of SLRPs was low/absent in all endometrial pathologies examined compared to normal endometrium. We observed an increase of lumican from proliferative to secretory phase of the endometrium and a decrease of fibromodulin, biglycan and decorin. In menopause endometrial tissue, the level of expression of fibromodulin, biglycan, decorin and lumican dramatically decreased. CONCLUSION: The results revealed the prominence and importance of proteoglycans in the tissue architecture and extracellular matrix organization

Trimethylamine-N-Oxide (TMAO)-Induced Impairment of Cardiomyocyte Function and the Protective Role of Urolithin B-Glucuronide

One of the most recently proposed candidates as a potential trigger for cardiovascular diseases is trimethylamine-N-oxide (TMAO). Possible direct effects of TMAO on myocardial tissue, independent of vascular damage, have been only partially explored so far. In the present study, we assessed the detrimental direct effects of TMAO on cardiomyocyte contractility and intracellular calcium dynamics, and the ability of urolithin B-glucuronide (Uro B-gluc) in counteracting TMAO-induced cell damage. Cell mechanics and calcium transients were measured, and ultrastructural analysis was performed in ventricular cardiomyocytes isolated from the heart of normal adult rats. Cells were either untreated, exposed to TMAO, or to TMAO and Uro B-gluc. TMAO exposure worsened cardiomyocyte mechanics and intracellular calcium handling, as documented by the decrease in the fraction of shortening (FS) and the maximal rate of shortening and re-lengthening, associated with reduced efficiency in the intracellular calcium removal. Ultrastructurally, TMAO-treated cardiomyocytes also exhibited glycogen accumulation, a higher number of mitochondria and lipofuscin-like pigment deposition, suggesting an altered cellular energetic metabolism and a higher rate of protein oxidative damage, respectively. Uro B-gluc led to a complete recovery of cellular contractility and calcium dynamics, and morphologically to a reduced glycogen accumulation. We demonstrated for the first time a direct negative role of TMAO on cardiomyocyte functional properties and the ability of Uro B-gluc in counteracting these detrimental effects

Circulating tumor cells detection has independent prognostic impact in high-risk non-muscle invasive bladder cancer

High-risk non-muscle invasive bladder cancer (NMIBC) progresses to metastatic disease in 10–15% of cases, suggesting that micrometastases may be present at first diagnosis. The prediction of risks of progression relies upon EORTC scoring systems, based on clinical and pathological parameters, which do not accurately identify which patients will progress. Aim of the study was to investigate whether the presence of CTC may improve prognostication in a large population of patients with Stage I bladder cancer who were all candidate to conservative surgery. A prospective single center trial was designed to correlate the presence of CTC to local recurrence and progression of disease in high-risk T1G3 bladder cancer. One hundred two patients were found eligible, all candidate to transurethral resection of the tumor followed by endovesical adjuvant immunotherapy with BCG. Median follow-up was 24.3 months (minimum–maximum: 4–36). The FDA-approved CellSearch System was used to enumerate CTC. Kaplan–Meier methods, log-rank test and multivariable Cox proportional hazard analysis was applied to establish the association of circulating tumor cells with time to first recurrence (TFR) and progression-free survival. CTC were detected in 20% of patients and predicted both decreased TFR (log-rank p < 0.001; multivariable adjusted hazard ratio [HR] 2.92 [95% confidence interval: 1.38–6.18], p 5 0.005), and time to progression (log-rank p < 0.001; HR 7.17 [1.89–27.21], p 5 0.004). The present findings provide evidence that CTC analyses can identify patients with Stage I bladder cancer who have already a systemic disease at diagnosis and might, therefore, potentially benefit from systemic treatment

Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus: a New Polyextremophilic Marine Fungus from Barents Sea

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillusprotuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 μg/mL. © 2018, Springer Science+Business Media, LLC, part of Springer Nature

Localization and distribution of wolframin in human female reproductive tract

Wolframin, a transmembrane glycoprotein of endoplasmic reticulum consisting of 890 amino acids, is encoded by the WFS1 gene, mutated in the Wolfram syndrome. This pathology, also called DIDMOAD, is an autosomal recessive disorder defined by the association of diabetes mellitus, optic atrophy, and further organ abnormalities. To gain further insight into the pathogenesis of diseases associated with WFS1 mutations, we conducted an immunohistochemical study to investigate its pattern of expression in human female reproductive tract in physiologic and pathologic conditions. For this purpose, samples of physiologic and pathologic endometrium, samples of placenta throughout pregnancy in normal and diabetic pregnant women, were used. In physiologic endometrium, we observed a light increase of wolframin from proliferative to secretory phase where wolframin was localized in the glands, stroma and cells lining blood vessels. In menopause, wolframin expression increased with a glandular and stromal localization. In pathologic endometrium, we observed an increase of wolframin expression from hyperplasia to polyps until a higher expression in carcinoma tissues. In normal placenta there was a modulation of wolframin throughout pregnancy with a strong level of expression during the first trimester and a moderate level in the third trimester. In diabetic women, the wolframin expression was strongly reduced in the third trimester of gestation. In human endometrium, wolframin seems to have a role in differentiation program. Deregulation of these functions may induce the onset of several endometrial pathologies. Moreover, in normal placenta wolframin may be required to sustain normal rates of cytotrophoblast cell proliferation during the first trimester of gestation. The decrease of wolframin expression in diabetic placentae may hypothesize that this protein is directly regulated by insulin concentration also in the placenta, suggesting that this protein physiologically maintain the glucose homeostasis in this organ