Dynamic markers based on blood perfusion fluctuations for selecting skin melanocytic lesions for biopsy

Skin malignant melanoma is a highly angiogenic cancer, necessitating early diagnosis for positive prognosis. The current diagnostic standard of biopsy and histological examination inevitably leads to many unnecessary invasive excisions. Here, we propose a non-invasive method of identification of melanoma based on blood flow dynamics. We consider a wide frequency range from 0.005 – 2 Hz associated with both local vascular regulation and effects of cardiac pulsation. Combining uniquely the power of oscillations associated with individual physiological processes we obtain a marker which distinguishes between melanoma and atypical nevi with sensitivity of 100% and specificity of 90.9%. The method reveals valuable functional information about the melanoma microenvironment. It also provides the means for simple, accurate, in vivo distinction between malignant melanoma and atypical nevi, and may lead to a substantial reduction in the number of biopsies currently undertaken

miRNAs as candidate biomarker for the accurate detection of atypical endometrial hyperplasia/endometrial intraepithelial neoplasia

Endometrial cancer is the most common gynecologic malignancy in developed countries. Estrogen-dependent tumors (type I, endometrioid) account for 80% of cases and non-estrogen-dependent (type II, non-endometrioid) account for the rest. Endometrial cancer type I is generally thought to develop via precursor lesions along with the increasing accumulation of molecular genetic alterations. Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia is the least common type of hyperplasia but it is the type most likely to progress to type I cancer, whereas endometrial hyperplasia without atypia rarely progresses to carcinoma. MicroRNAs are a class of small, non-coding, single-stranded RNAs that negatively regulate gene expression mainly binding to 3'-untranslated region of target mRNAs. In the current study, we identified a microRNAs signature (miR-205, miR-146a, miR-1260b) able to discriminate between atypical and typical endometrial hyperplasia in two independent cohorts of patients. The identification of molecular markers that can distinguish between these two distinct pathological conditions is considered to be highly useful for the clinical management of patients because hyperplasia with an atypical change is associated with a higher risk of developing cancer. We show that the combination of miR-205, -146a, and -1260b has the best predictive power in discriminating these two conditions (>90%). With the aim to find a biological role for these three microRNAs, we focused our attention on a common putative target involved in endometrial carcinogenesis: the oncosuppressor gene SMAD4. We showed that miRs-146a, -205, and-1260b directly target SMAD4 and their enforced expression induced proliferation and migration of Endometrioid Cancer derived cell lines, Hec1 a cells. These data suggest that microRNAs-mediated impairment of the TGF-beta pathway, due to inhibition of its effector molecule SMAD4, is a relevant molecular alteration in endometrial carcinoma development. Our findings show a potential diagnostic role of this microRNAs signature for the accurate diagnosis of Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia and improve the understanding of their pivotal role in SMAD4 regulation

New miRNA Signature Heralds Human NK Cell Subsets at Different Maturation Steps: Involvement of miR-146a-5p in the Regulation of KIR Expression

Natural killer cells are cytotoxic innate lymphoid cells that play an important role for early host defenses against infectious pathogens and surveillance against tumor. In humans, NK cells may be divided in various subsets on the basis of the relative CD56 expression and of the low-affinity FcγRIIIA CD16. In particular, the two main NK cell subsets are represented by the CD56bright/CD16−/dim and the CD56dim/CD16bright NK cells. Experimental evidences indicate that CD56bright and CD56dim NK cells represent different maturative stages of the NK cell developmental pathway. We identified multiple miRNAs differentially expressed in CD56bright/CD16− and CD56dim/CD16bright NK cells using both univariate and multivariate analyses. Among these, we found a few miRNAs with a consistent differential expression in the two NK cell subsets, and with an intermediate expression in the CD56bright/CD16dim NK cell subset, representing a transitional step of maturation of NK cells. These analyses allowed us to establish the existence of a miRNA signature able to efficiently discriminate the two main NK cell subsets regardless of their surface phenotype. In addition, by analyzing the putative targets of representative miRNAs we show that hsa-miR-146a-5p, may be involved in the regulation of killer Ig-like receptor (KIR) expression. These results contribute to a better understanding of the physiologic significance of miRNAs in the regulation of the development/function of human NK cells. Moreover, our results suggest that hsa-miR-146a-5p targeting, resulting in KIR down-regulation, may be exploited to generate/increment the effect of NK KIR-mismatching against HLA-class I+ tumor cells and thus improve the NK-mediated anti-tumor activity

Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p < 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

IL RUOLO DELLA EZRINA E DELLA FASCINA, PROTEINE ASSOCIATE AL CITOSCHELETRO, E DELLA GLUTATIONE S-TRANSFERASI OMEGA NELLA PROGRESSIONE NEOPLASTICA DA ESOFAGO DI BARRETT AD ADENOCARCINOMA ESOFAGEO.

RIASSUNTO L’ Esofago di Barrett è una condizione metaplastica nella quale il normale epitelio squamoso di rivestimento dell’esofago distale viene sostituito da un epitelio colonnare di un tipo intestinale specializzato. E’ noto ormai che la metaplasia di Barrett sia la causa di un aumentato rischio di sviluppo di adenocarcinoma esofageo e che la presenza di displasia aumenta notevolmente il rischio di progressione neoplastica. Sebbene alcuni studi abbiano suggerito che la displasia o, come suggerito dall’O.M.S Non-invasive Neoplasia (NiN), di alto grado nell’Esofago di Barrett (HG-NiN) sia predittiva di una trasformazione maligna incipiente o addirittura di carcinoma in situ. I rischi associati ad un basso grado di displasia nell’Esofago di Barrett (LG-NiN) sono ancora meno definiti. Pertanto una migliore comprensione degli eventi molecolari coinvolti nel processo di modificazione displastica e di progressione verso l’adenocarcinoma può certamente essere d’aiuto nell’identificare i pazienti a più alto rischio anche in considerazione del fatto che la progressione metaplasia-displasia-neoplasia è un processo multi-step che può richiedere anche molti anni. La conversione di cellule epiteliali normali verso un fenotipo maligno è associato a cambiamenti nell’espressione e nella funzione dei sistemi di reciproca adesione tra cellule le quali assumono caratteristiche migratorie che conferiscono al tumore la capacità di invadere i tessuti e di dare metastasi. Queste ultime caratteristiche sono spesso il risultato di un’acquisizione sequenziale di una maggiore motilità che le rende in grado di prevaricare l’interazione cellula-cellula o cellula-matrice e di invadere i tessuti circostanti. Spesso le cellule tumorali invasive presentano una specifica morfologia caratterizzata da protrusioni della membrana citoplasmatica, perdita di comunicazioni giunzionali a causa di un riarrangiamento dei microfilamenti di actina. E’ proprio per questo motivo che si è ritenuto interessante valutare un possibile ruolo delle proteine del citoscheletro nella progressione verso un fenotipo maligno delle cellule esofagee affette da Barrett. L’Ezrina è una proteina che modula l’associazione tra la membrana plasmatica ed i filamenti di actina che compongono il citoscheletro. E’ stato dimostrato che l’Ezrina è coinvolta in un’ampia varietà di processi cellulari quali la sopravvivenza, l’adesione e la motilità cellulare nonché la trasduzione di segnali. Durante il processo di cancerogenesi, l’Ezrina mostra la tendenza a traslocare dalla membrana plasmatica, concentrandosi maggiormente al livello citoplasmatico e questa ridistribuzione subcellulare è significativamente correlata con la progressione neoplastica. La Fascina è una proteina globulare coinvolta nel riarrangiamento del citoscheletro attraverso il legame con l’actina e capace di regolare i meccanismi di protrusioni cellulari che coinvolgono processi di adesione cellulare. Nell’uomo, la Fascina è generalmente poco espressa, mentre i suoi livelli aumentano in diversi tipi di tumore (ovaie, mammella, pancreas, colon, polmone e melanomi). Inoltre, recentemente è stata riscontrata una correlazione tra alti livelli di espressione della Fascina e la presenza di metastasi linfonodali e prognosi infausta in diversi tumori. Ad esempio, nel carcinoma esofageo a cellule squamose (ESCC), l’espressione della Fascina sembra aumentare gradualmente nella progressione da epitelio normale a semplice iperplasia, displasia fino al carcinoma in situ. La Glutatione S-Transferasi Omega (GSTO), scoperta presso il Dipartimento di Patologia dell’Università di Pisa dal Prof. Casini negli anni ‘80, appartiene alla superfamiglia di enzimi detossificanti di fase II che, utilizzando il glutatione come cofattore, contribuiscono alla biotrasformazione di numerosi composti di natura esogena ed endogena, come agenti cancerogeni, farmaci e prodotti dello stress ossidativi. Nell’uomo la famiglia delle GSTO include due membri: GSTO1 e GSTO2. Mentre il gene della GSTO1 è abbondantemente espresso nella maggior parte dei tessuti sia al livello citoplasmatico sia nucleare, la GSTO2 è espressa in alti livelli solo nei testicoli e in pochi altri tessuti. Studi recenti hanno mostrato come il polimorfismo della GST-O sia associato al rischio di carcinoma epatocellulare, colangiocarcinoma, cancro del seno e cancro delle ovaie. Recentemente, presso l’Università di Pisa, è stato dimostrato che nei pazienti affetti metaplasia di Barrett la GSTO potrebbe giocare un ruolo importante nella progressione neoplastica verso adenocarcinoma. Molti studi sono già stati condotti con l’intento di valutare se marcatori precoci di progressione neoplastica (ad esempio p16, ciclina D1, p53, E-caderina) potessero risultare utili nel monitoraggio dei pazienti EB. Lo scopo del presente lavoro di tesi è stato quello di osservare la localizzazione e il grado di espressione al livello cellulare della Fascina, dell’Ezrina e della GSTO in preparati bioptici di pazienti affetti da Barrett semplice, in pazienti Barrett con basso grado di displasia (LG-NiN) e in pazienti Barrett con alto grado di displasia (HG-NiN), al fine di valutare la potenzialità del loro utilizzo come marker precoci nei pazienti affetti da EB esposti ad alto rischio di adenocarcinoma esofageo

From Materials to Technique: A Complete Non-Invasive Investigation of a Group of Six Ukiyo-E Japanese Woodblock Prints of the Oriental Art Museum E. Chiossone (Genoa, Italy)

In the present work, a complete non-invasive scientific investigation of six Utagawa Kunisada’s woodblock prints (nishiki-e) belonging to the Oriental Art Museum “E. Chiossone” (Genoa, Italy), was performed in situ. The campaign started with high resolution multiband imaging (visible, multiband fluorescence, near infrared) followed by reflectance transformation imaging (RTI) to characterize and highlight the peculiar printing techniques and the condition of the support. Then fiber optics reflectance spectroscopy (FORS), spectrofluorimetry, Raman and reflectance Fourier-transform infrared (FTIR) spectroscopies were successfully applied in synergy for the investigation of the printing materials (pigments, binders, support). The results obtained represent a set of very important information for these never-before-studied works of art, useful to the different professionals involved: historians, conservators and curators. The materials identified were completely in agreement with those traditionally used in the Edo period in the 19th century, while the computational imaging technique RTI gave an additional amount of information in terms of surface characterization that could not be overlooked when studying these works of art. RTI data were further processed to enhance the texture visualization

Spatiotemporal Correlation Spectroscopy Reveals a Protective Effect of Peptide-Based GLP-1 Receptor Agonism against Lipotoxicity on Insulin Granule Dynamics in Primary Human β-Cells

Glucagon-like peptide-1 receptor (GLP-1R) agonists are being used for the treatment of type 2 diabetes (T2D) and may have beneficial effects on the pancreatic β-cells. Here, we evaluated the effects of GLP-1R agonism on insulin secretory granule (ISG) dynamics in primary β-cells isolated from human islets exposed to palmitate-induced lipotoxic stress. Islets cells were exposed for 48 h to 0.5 mM palmitate (hereafter, ‘Palm’) with or without the addition of a GLP-1 agonist, namely 10 nM exendin-4 (hereafter, ‘Ex-4’). Dissociated cells were first transfected with syncollin-EGFP in order to fluorescently mark the ISGs. Then, by applying a recently established spatiotemporal correlation spectroscopy technique, the average structural (i.e., size) and dynamic (i.e., the local diffusivity and mode of motion) properties of ISGs are extracted from a calculated imaging-derived Mean Square Displacement (iMSD) trace. Besides defining the structural/dynamic fingerprint of ISGs in human cells for the first time, iMSD analysis allowed to probe fingerprint variations under selected conditions: namely, it was shown that Palm affects ISGs dynamics in response to acute glucose stimulation by abolishing the ISGs mobilization typically imparted by glucose and, concomitantly, by reducing the extent of ISGs active/directed intracellular movement. By contrast, co-treatment with Ex-4 normalizes ISG dynamics, i.e., re-establish ISG mobilization and ability to perform active transport in response to glucose stimulation. These observations were correlated with standard glucose-stimulated insulin secretion (GSIS), which resulted in being reduced in cells exposed to Palm but preserved in cells concomitantly exposed to 10 nM Ex-4. Our data support the idea that GLP-1R agonism may exert its beneficial effect on human β-cells under metabolic stress by maintaining ISGs’ proper intracellular dynamics

Activated innate immunity and the involvement of CX3CR1-fractalkine in promoting hematuria in patients with IgA nephropathy

A hallmark of immunoglobulin A nephropathy (IgAN) is episodes of gross hematuria coinciding with mucosal infections that can represent the disease-triggering event. Here we performed a whole genomic screen of IgAN patients during gross hematuria to clarify the link between mucosal antigens and glomerular hematuria. Modulated genes showed a clear involvement of the intracellular interferon signaling, antigen-presenting pathway, and the immunoproteasome. The mRNA and protein level of the chemokine receptor characterizing cytotoxic effector lymphocytes, CX3CR1, was upregulated. In vitro antigenic stimulation of peripheral blood mononuclear cells from IgAN patients, healthy blood donors, and other nephropathies with microscopic hematuria showed that only in IgAN patients was CX3CR1 enhanced in a dose-dependent manner. A significantly higher amount of glomerular and urinary fractalkine, the only ligand of CX3CR1, was also found in IgAN patients with recurrent episodes of gross hematuria compared with other patients with microscopic or no hematuria. This suggests a predisposition for cytotoxic cell extravasation only in patients with recurrent gross hematuria. Thus, we found a defect in antigen handling in peripheral blood mononuclear cells of IgAN patients with a specific increase of CX3CR1. This constitutive upregulation of glomerular and urinary fractalkine suggests an involvement of the CX3CR1-fractalkine axis in the exacerbation of gross hematuria