First Exploration of the Altered Microbial Gut-Lung Axis in the Pathogenesis of Human Refractory Chronic Cough.

PURPOSE: Cough represents a natural mechanism that plays an important defensive role in the respiratory tract, but in some conditions, it may become persistent, nonproductive, and harmful. In general, refractory chronic cough (RCC) occurs in about 20% of individuals; hence, we aimed to assess the presence of altered gut-lung communication in RCC patients through a compositional and functional characterization of both gut (GM) and oral microbiota (OM). METHODS: 16S rRNA sequencing was used to characterize both GM and OM composition of RCC patients and healthy controls (HC). PICRUST2 assessed functional changes in microbial communities while gas chromatography was used to evaluate fecal short-chain fatty acid levels and serum-free fatty acid (FFA) abundances. RESULTS: In comparison with HC, RCC patients reported increased saliva alpha-diversity and statistically significant beta-diversity in both GM and OM. Also, a, respectively, significant increased or reduced Firmicutes/Bacteroidota ratio in stool and saliva samples of RCC patients has been shown, in addition to a modification of the abundances of several taxa in both GM and OM. Moreover, a potential fecal over-expression of lipopolysaccharide biosynthesis and lipoic acid metabolism pathways and several differences in serum FFA levels have been reported in RCC patients than in HC. CONCLUSION: Since differences in both GM and OM of RCC patients have been documented, these findings could provide new information about RCC pathogenesis and also pave the way for the development of novel nutritional or pharmacological interventions for the management of RCC through the restoration of eubiotic gut-lung communication.

Technical Evaluation of a New Medical Device Based on Rigenase in the Treatment of Chronic Skin Lesions.

Chronic wound is characterized by slow healing time, persistence, and abnormal healing progress. Therefore, serious complications can lead at worst to the tissue removal. In this scenario, there is an urgent need for an ideal dressing capable of high absorbency, moisture retention and antimicrobial properties. Herein we investigate the technical properties of a novel advanced non-woven triple layer gauze imbibed with a cream containing Rigenase, an aqueous extract of Triticum vulgare used for the treatment of skin injuries. To assess the applicability of this system we analyzed the dressing properties by wettability, dehydration, absorbency, Water Vapor Transmission Rate (WVTR), lateral diffusion and microbiological tests. The dressing showed an exudate absorption up to 50%. It created a most environment allowing a proper gaseous exchange as attested by the WVTR and a controlled dehydration rate. The results candidate the new dressing as an ideal medical device for the treatment of the chronic wound repairing process. It acts as a mechanical barrier providing a good management of the bacterial load and proper absorption of abundant wound exudate. Finally, its vertical transmission minimizes horizontal diffusion and side effects on perilesional skin as maceration and bacterial infection.

Ethanol-induced AMPA alterations are mediated by mGLU5 receptors through miRNA upregulation in hippocampal slices.

Prenatal alcohol exposure (PAE) affects neuronal networks and brain development causing a range of physical, cognitive and behavioural disorders in newborns that persist into adulthood. The array of consequences associated with PAE can be grouped under the umbrella-term 'fetal alcohol spectrum disorders' (FASD). Unfortunately, there is no cure for FASD as the molecular mechanisms underlying this pathology are still unknown. We have recently demonstrated that chronic EtOH exposure, followed by withdrawal, induces a significant decrease in AMPA receptor (AMPAR) expression and function in developing hippocampus in vitro. Here, we explored the EtOH-dependent pathways leading to hippocampal AMPAR suppression. Organotypic hippocampal slices (2 days in cultures) were exposed to EtOH (150 mM) for 7 days followed by 24 h EtOH withdrawal. Then, the slices were analysed by means of RT-PCR for miRNA content, western blotting for AMPA and NMDA related-synaptic proteins expression in postsynaptic compartment and electrophysiology to record electrical properties from CA1 pyramidal neurons. We observed that EtOH induces a significant downregulation of postsynaptic AMPA and NMDA subunits and relative scaffolding protein expression and, accordingly, a decrease of AMPA-mediated neurotransmission. Simultaneously, we found that chronic EtOH induced-upregulation of miRNA 137 and 501-3p and decreased AMPA-mediated neurotransmission are prevented by application of the selective mGlu5 antagonist MPEP during EtOH withdrawal. Our data indicate mGlu5 via miRNA137 and 501-3p expression as key factors in the regulation of AMPAergic neurotransmission that may contribute, at least in part, to the pathogenesis of FASD.

Tisochrysis lutea F&M-M36 Mitigates Risk Factors of Metabolic Syndrome and Promotes Visceral Fat Browning through ß3-Adrenergic Receptor/UCP1 Signaling.

Pre-metabolic syndrome (pre-MetS) may represent the best transition phase to start treatments aimed at reducing cardiometabolic risk factors of MetS. In this study, we investigated the effects of the marine microalga Tisochrysis lutea F&M-M36 (T. lutea) on cardiometabolic components of pre-MetS and its underlying mechanisms. Rats were fed a standard (5% fat) or a high-fat diet (20% fat) supplemented or not with 5% of T. lutea or fenofibrate (100 mg/Kg) for 3 months. Like fenofibrate, T. lutea decreased blood triglycerides (p < 0.01) and glucose levels (p < 0.01), increased fecal lipid excretion (p < 0.05) and adiponectin (p < 0.001) without affecting weight gain. Unlike fenofibrate, T. lutea did not increase liver weight and steatosis, reduced renal fat (p < 0.05), diastolic (p < 0.05) and mean arterial pressure (p < 0.05). In visceral adipose tissue (VAT), T. lutea, but not fenofibrate, increased the ß3-adrenergic receptor (ß3ADR) (p < 0.05) and Uncoupling protein 1 (UCP-1) (p < 0.001) while both induced glucagon-like peptide-1 receptor (GLP1R) protein expression (p < 0.001) and decreased interleukin (IL)-6 and IL-1ß gene expression (p < 0.05). Pathway analysis on VAT whole-gene expression profiles showed that T. lutea up-regulated energy-metabolism-related genes and down-regulated inflammatory and autophagy pathways. The multitarget activity of T. lutea suggests that this microalga could be useful in mitigating risk factors of MetS.

Impact of Preoperative Immunonutrition on Oxidative Stress and Gut Barrier Function in Surgical Patients with Crohn's Disease.

Several international guidelines recommend a peri-operative immunonutrition (IN) support for patients care in elective colorectal surgery, to reduce postoperative complications, particularly infections. In Crohn's patients, is also used to mitigate the severity of the disease. We performed a pilot study on 16 Crohn's patients undergoing intestinal surgery for active disease, not responsive to pharmacological treatment; half of them received an oral nutritional supplement enriched with immunonutrients (IN patients) for 7 days prior to surgery, in addition to normal food intake. Markers of oxidative stress (Advanced Glycated End-products (AGEs) and Advanced Oxidation Protein Products (AOPPs) were measured both in plasma and tissue samples wherein the Receptor for Advanced Glycation End products (RAGE) and Tight Junction Protein 1 (TJP1) gene expression were also determined. Plasma AGEs were significantly and positively correlated with tissue levels of AGEs (p = 0.0354) and AOPPs (p = 0.0043) while they were negatively correlated with TJP1 expression (p = 0.0159). The expression of RAGE was also negatively correlated with that of TJP1 gene (p = 0.0146). IN patients exhibited significantly lower AGEs plasma levels (p = 0.0321) and a higher mucosal TJP1 expression (p = 0.0182). No patient had postoperative complications and the length of hospital stay was similar in the two groups, but IN patients, showed a significantly shorter time to resume fluid and solid diet. These preliminary data suggest that IN might support patient's recovery by improving intestinal mucosa barrier function through the regulation of AGEs/RAGE signaling.

Niosomes Functionalized with a Synthetic Carbohydrate Binding Agent for Mannose-Targeted Doxorubicin Delivery.

Niosomes are a potential tool for the development of active targeted drug delivery systems (DDS) for cancer therapy because of their excellent behaviour in encapsulating antitumorals and the possibility to easily functionalise their surface with targeting agents. Recently, some of us developed a synthetic carbohydrate binding agent (CBA) able to target the mannosidic residues of high-mannose-type glycans overexpressed on the surface of several cancer cell lines, promoting their apoptosis. In this article, we modified the structure of this mannose receptor to obtain an amphiphilic analogue suitable for the functionalization of doxorubicin-based niosomes. Several niosomal formulations and preparation methods were investigated deeply to finally obtain functionalized niosomes suitable for parental administration, which were stable for over six months and able to encapsulate up to 85% of doxorubicin (DOXO). In vitro studies, carried out towards triple-negative cancer cells (MDA-MB231), overexpressing high-mannose-type glycans, showed a cytotoxic activity comparable to that of DOXO but with an appreciable increment in apoptosis given by the CBA. Moreover, niosomal formulation was observed to reduce doxorubicin-induced cytotoxicity towards normal cell lines of rat cardiomyocytes (H9C2). This study is propaedeutic to further in vivo investigations that can aim to shed light on the antitumoral activity and pharmacokinetics of the developed active targeted DDS.

Cyclic Acetals as Novel Long-Lasting Mosquito Repellents.

The use of skin repellents against hematophagous mosquitoes is an important personal protection practice wherever these insects are abundant and where they are vectors of diseases. DEET and Icaridin are the major synthetic insect repellents in commercial formulations and are considered the most effective. Here, we tested against the mosquito Aedes albopictus several cyclic hydroxyacetals synthesized by acetalization of commercially available aliphatic carbonyl compounds (ranging from C3 to C15) with either glycerol, 1,1,1-trismethyloletane, or 1,1,1-trismethylolpropane and compared their efficacy with commercial repellents. We found that several hydroxyacetals were comparable with DEET and Icaridin both in terms of the required dose and repellence duration, while a few performed better. For those most active, toxicity was investigated, finding that a few of them were less cytotoxic than DEET and less prone to permeate through cell layers. Therefore, such results indicate that novel safe mosquito repellents could be developed among cyclic hydroxyacetals.

Inadequate food literacy is related to the worst health status and limitations in daily life in subjects with inflammatory bowel disease.

PURPOSE: Diet affects Inflammatory Bowel Disease (IBD) patients' quality of life. An adequate Food Literacy (FL) level enables adequate food choices. Currently, there is a lack of knowledge on the FL degree in patients with IBD. To deepen this item, we measured for the first time the degree of FL in IBD patients and then we analyzed its correlation with health and socio-demographic variables. METHODS: This is an observational prospective study includingsubjects with IBD belonging to A.M.I.C.I. ONLUS association. We first measured the degree of FL through Literacy Survey (FLS-IT) questionnaire (containing the Newest Vital Sign (NVS)). In addition, we analyzed the relationship between FL individual level and health and sociodemographic variables, evaluating which of these aspects have a positive or negative correlation with FL level. In detail, continuous variables were analyzed by Mann Whitney test. Differences among proportions were assessed using the chi-square test. A Spearman's rank correlation analysis was performed to correlate the sociodemographic and health status variable with FL index and NVS. A stepwise linear regression analysis with backward selection was performed to identify possible predictor of good food literary skills in IBS patients. RESULTS: Overall, 450 IBD subjects completed the FLS-IT questionnaire. Among them, 69.78% (n = 314) showed an inadequate FL level. Concerning NVS test, 14.22% showed an insufficient ability to interpret food labels. In addition, higher FL level was associated to better subjective health conditions (r = 0.1513; p = 0.0013), less limitations in daily living activities (r = 0.1430; p = 0.0026), higher physical activity (r = 1200; p = 0.0110) and no alcohol consumption (p = 0.0020). Finally, higher NVS scores were shown by women (r = 0.1408; p = 0.0028) and by younger subjects (r = -0.1686; p = 0.0003). CONCLUSIONS: We showed that an inadequate level of FL, related to the worst health status and the presence of several limitations in daily life, is widespread among our Italian cohort of IBD patients, and health and social status influence the ability to make adequate food choices. For this reason, it is necessary to investigate the problem and identify effective intervention strategies that will improve the patient's nutritional awareness.

A Comparison between Open and Minimally Invasive Techniques for the Resection of Colorectal Liver Metastasis.

The liver is the most common site of colorectal cancer metastasis. Liver surgery is a cornerstone in treatment, with progressive expansion of minimally invasive surgery (MIS). This study aims to compare short- and long-term outcomes of open surgery and MIS for the treatment of colorectal adenocarcinoma liver metastasis during the first three years of increasing caseload and implementation of MIS use in liver surgery. All patients treated between November 2018 and August 2021 at Careggi Teaching Hospital in Florence, Italy, were prospectively entered into a database and retrospectively reviewed. Fifty-one patients were resected (41 open, 10 MIS). Considering that patients with a significantly higher number of lesions underwent open surgery and operative results were similar, postoperative morbidity rate and length of hospital stay were significantly higher in the open group. No differences were found in the pathological specimen. The postoperative mortality rate was 2%. Mean overall survival and disease-free survival were 46 months (95% CI 42-50) and 22 months (95% CI 15.6-29), respectively. The use of minimally invasive techniques in liver surgery is safe and feasible if surgeons have adequate expertise. MIS and parenchymal sparing resections should be preferred whenever technically feasible.

Trophoblast microRNAs (miRNAs), preeclampsia and intrauterine growth restriction.

MicroRNAs (miRNAs) are small single-stranded non-coding RNA molecules that play a role in regulating gene expression in a tissue-specific manner. Placental miRNAs expression pattern dynamically changes during pregnancy influencing cell proliferation, differentiation and apoptosis. Changes of specific miRNA levels have been described in pregnancies complicated by hypertensive disorders or gestational diabetes and a growing interest in understanding miRNA role on placental development and placental disorders is currently going on. The present review evaluates the possible roles of miRNAs in trophoblastic invasion and placental development as well as their potential role as biomarkers for the prediction of placental disorders focusing the attention on intrauterine growth restriction.

Crohn's disease recurrence updates: first surgery vs. surgical relapse patients display different profiles of ileal microbiota and systemic microbial-associated inflammatory factors.

Background and aims: Crohn's disease (CD) pathogenesis is still unclear. Remodeling in mucosal microbiota and systemic immunoregulation may represent an important component in tissue injury. Here, we aim to characterize the ileal microbiota in both pathological and healthy settings and to evaluate the correlated systemic microbial-associated inflammatory markers comparing first-time surgery and relapse clinical conditions. Methods: We enrolled 28 CD patients at surgery; we collected inflamed and non-inflamed mucosa tissues and blood samples from each patient. Bacterial wall adherence was observed histologically, while its composition was assessed through amplicon sequencing of the 16S rRNA gene. In addition, we evaluated the systemic microRNA (miRNA) using quantitative real-time PCR amplification and free fatty acids (FFAs) using gas chromatography-mass spectroscopy. Results: The total number of mucosal adherent microbiota was enriched in healthy compared to inflamed mucosa. In contrast, the phylum Tenericutes, the family Ruminococcaceae, and the genera Mesoplasma and Mycoplasma were significantly enriched in the pathological setting. Significant microbiota differences were observed between the relapse and first surgery patients regarding the families Bacillaceae 2 and Brucellaceae and the genera Escherichia/Shigella, Finegoldia, Antrobacter, Gemmatimonas, Moraxella, Anoxibacillus, and Proteus. At the systemic level, we observed a significant downregulation of circulating miR-155 and miR-223, as well as 2-methyl butyric, isobutyric, and hexanoic (caproic) acids in recurrence compared to the first surgery patients. In addition, the level of hexanoic acid seems to act as a predictor of recurrence risk in CD patients (OR 18; 95% confidence interval 1.24-261.81; p = 0.006). Conclusions: We describe a dissimilarity of ileal microbiota composition comparing CD and healthy settings, as well as systemic microbial-associated inflammatory factors between first surgery and surgical relapse. We suggest that patterns of microbiota, associated with healthy ileal tissue, could be involved in triggering CD recurrence. Our findings may provide insight into the dynamics of the gut microbiota-immunity axis in CD surgical recurrence, paving the way for new diagnostics and therapeutics aimed not only at reducing inflammation but also at maintaining a general state of eubiosis in healthy tissue.

Low levels of miR-34c in nasal washings as a candidate marker of aggressive disease in wood and leather exposed workers with sinonasal intestinal-type adenocarcinomas (ITACs).

INTRODUCTION: Sinonasal intestinal-type adenocarcinomas (ITACs) are rare and aggressive tumors, closely related to professional exposure to wood dusts or leather. Here we explored the role of non-coding RNAs controlling MUC2 in liquid biopsies and tumors from ITAC patients with the aim of identifying biomarkers and molecular mechanisms to improve early diagnosis, prognosis, and therapeutic approaches for this rare cancer. METHODS: MiR-34c-3p, lncRNA AF147447 and MUC2 were measured in tumors and normal mucosa, in nasal washings (NW) from the affected and non-affected nostril and in plasma from 17 ITAC patients. The Apparent Diffusion Coefficient (ADC) was also evaluated by Magnetic Resonance Imaging. RESULTS: MiR-34c was higher in ITACs compared to the corresponding normal mucosa (p = 0.021). Differentiated tumors exhibited higher miR-34c levels (p = 0.025) and lower ADC values (p<0.001) compared to mucinous ones and these parameters were also inversely correlated (r = 0.87; p = 0.001). High MUC2 tumor expression was associated with orbital extension (p = 0.010). Low miR-34c levels in NW were associated with orbital (p = 0.009) and intracranial (p = 0.031) extension and with advanced TNM stage (p = 0.054). Functional analysis identified Wnt, Focal adhesion, MAPK and inflammatory signalings among the pathways most enriched in mir-34c targets. DISCUSSION: Our results suggest measuring miR-34c in NW as a biomarker for early diagnosis and monitoring of ITAC patients and for the surveillance of wood and leather exposed workers. Further research on the involvement of miR-34c regulated pathways in ITAC tumorigenesis may also allow the development of new therapeutic approaches for this rare cancer.

Is the age at surgery in Crohn's disease clinically relevant? Differences and peculiarities: a wide single centre experience after long-term follow-up.

PURPOSE: The Montreal classification for Crohn's disease includes "age at diagnosis" as a parameter but few is reported about the age at surgery. The aim of this study is to evaluate the short- and long-term differences in the postoperative surgical outcome and disease behaviour, according to the age at the first surgery. METHODS: Patients consecutively operated for abdominal Crohn's disease during the period 1986-2012 at our centre were systematically analysed according to their age at first surgery. In our retrospective cohort, the age at first surgery ranged from 13 to 83 years, and patients were arbitrarily divided into four groups: ≤ 19 (G1), 20-39 (G2), 40-59 (G3) and ≥ 60 (G4) years old. RESULTS: In total, 1051 patients were included with a median follow-up time of 232 months. The four groups exhibited statistically significant differences in age at diagnosis, smoke habit, time between diagnosis and surgery, disease location and behaviour, history of perianal fistula or abscess, severe malnutrition requiring total parental nutrition before surgery, type of surgery, total length of resected bowel, median duration of hospitalization, incidence of abdominal recurrences and number of surgical recurrences. G1 displays an inverse linear trend with time in the severity of clinical characteristics when compared to G4 groups. On the contrary, the incidence of short-term complications, types of abdominal recurrence and presence of concomitant perianal disease did not vary among groups. In addition, at multivariate analysis, the age at surgery and the disease location were the only independent risk factors for abdominal surgical recurrence. CONCLUSION: Despite first surgery is extremely more frequent between 20 and 59 years, patients from G1 and G4 groups showed clinical differences and peculiarities when compared to the other age groups. The most indolent CD behaviour and occurrence of surgical recurrence was observed in patients having their first abdominal surgery in the elderly, while patients operated before the age of 19 experienced a more aggressive disease course.

Dexamethasone Promotes a Stem-Like Phenotype in Human Melanoma Cells via Tryptophan 2,3 Dioxygenase.

In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers (CD133, CD44, ganglioside GD2) was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect.

Involvement of MIR-126 and MMP9 in the Pathogenesis of Intra-Abdominal Fistulizing Crohn's Disease: A Brief Research Report.

Background: Intra-abdominal fistulas are complications that affect a significant proportion of Crohn's disease patients, often requiring surgery. The aim of the present work was to correlate the occurrence of intestinal fistulization to the clinico-pathological features of these patients and to the plasma levels of MMP9, a gelatinase involved in the pathophysiology of fistula formation, and of miR-126, appearing to modulate MMP9 expression. Methods: In a series of 31 consecutive Crohn's patients admitted to surgery due to therapeutic failure and/or complicated disease, we identified nine cases of abdominal fistulas, mainly entero-enteric fistulas. MMP9 protein was determined in plasma and at the intestinal level using immunometric assays. Circulating miR-126 was also measured in all plasma samples by real-time PCR. Results: Comparing patients with and without intra-abdominal fistulas, we did not observe differences in terms of age, gender, disease location and duration, number of previous surgeries and pre-biologic medications. However, cases with intra-abdominal fistulas had a significantly higher CDAI (p < 0.0001) and a significantly lower circulating miR-126 (p < 0.05). Patients with intra-abdominal fistulas had also a significantly higher amount of circulating MMP9 (p < 0.0001) and this data was correlated with an increased expression of MMP9 protein in the mucosa and with reduced levels of circulating miR-126. Receiver operating characteristic (ROC) analysis pointed out the ability of circulating MMP9 to discriminate patients with and without intra-abdominal fistulas. Conclusions: These data confirm that circulating MMP9 can be used for the identification of cases with intra-abdominal fistulas and suggest that miR-126 may be also involved in the pathogenesis of this complication and that it may be further investigated as a new therapeutic strategy or for monitoring therapeutic response in these patients.

Role of Macrophages and Mast Cells as Key Players in the Maintenance of Gastrointestinal Smooth Muscle Homeostasis and Disease.

The gut contains the largest macrophage pool in the body, with populations of macrophages residing in the mucosa and muscularis propria of the gastrointestinal (GI) tract. Muscularis macrophages (MMs), which are located within the muscularis propria, interact with cells essential for GI function, such as interstitial cells of Cajal, enteric neurons, smooth muscle cells, enteric glia, and fibroblast-like cells, suggesting that these immune cells contribute to several aspects of GI function. This review focuses on the latest insights on the factors contributing to MM heterogeneity and the functional interaction of MMs with other cell types essential for GI function. This review integrates the latest findings on macrophages in other organs with increasing knowledge of MMs to better understand their role in a healthy and diseased gut. We describe the factors that contribute to (muscularis macrophage) MM heterogeneity, and the nature of MM interactions with cells regulating GI function. Finally, we also describe the increasing evidence suggesting a critical role of another immune cell type, the mast cell, in normal and diseased GI physiology.

Evaluation of the increase of the thymoquinone permeability formulated in polymeric micelles: In vitro test and in vivo toxicity assessment in Zebrafish embryos.

Thymoquinone (TQ) is a natural compound present in the essential oil and in the fixed oil of Nigella sativa L. Like many natural substances, it is characterized by poor aqueous solubility and low stability which limit its bioavailability. Soluplus®-Solutol® HS15 polymeric micelles (TQ-MP) were developed to increase the permeability of TQ with particular attention to overcoming intestinal barrier and the blood brain barrier, for possible oral and parenteral administration. The optimized micelles have dimensions < 100 nm and PdI < 0.2 indicating that the formulation was homogeneous as confirmed also by TEM experiments. EE% was 92.4 ± 0.3%. Stability studies showed a stable formulation following subsequent dilutions and in the gastric-intestinal media. In vitro studies have revealed that the carrier enhances the permeability of TQ in the intestine and in the blood-brain barrier using Parallel Artificial Membrane Permeability Assay (PAMPA) assay and cellular tests with Caco-2 cells and hCMEC/D3 monolayer cells. Up-take study, cell viability and cytotoxicity studies were also conducted. Fluorescent micelles (FITC-MP), were also optimized to perform in vitro up-take study in Caco-2 cells and to study their toxicity in Zebrafish model. The toxicity was evaluated on three lines of Zebrafish: wild type, transgenic line Tg(Myl7:EGFP) in which cardiomyocytes are marked with green fluorescence protein and Tg(flk1-GFP) line which expresses GFP under the control of the vascular endothelial growth factor receptor 2 (vegfr2) promoter.

Colon fibroblasts from Pirc rats (F344/NTac-Apcam1137 ) exhibit a proliferative and inflammatory phenotype that could support early stages of colon carcinogenesis.

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc+/- colon fibroblasts have been previously characterized: however, little is known about their behavior at very early-stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc+/- ) of Pirc rats (F344/NTac-Apcam1137 ) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age-matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P < .001) and delayed replicative senescence onset (P < .05) compared to WCF, along with a lower level of oxidative DNA damage (P < .05). Furthermore, a constitutively higher expression of COX-2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P < .05), accompanied by higher invasive capability (P < .05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P < .05). However, they neither expressed CAFs markers (α-SMA, IL-6) nor responded to CAFs activating stimuli (TGF-ß). Accordingly, CAFs markers and activating stimuli resulted down-regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc+/- colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients.

Extracellular vesicles miR-210 as a potential biomarker for diagnosis and survival prediction of oral squamous cell carcinoma patients.

BACKGROUND: The identification of early diagnostic and prognostic biomarkers in oral squamous cell carcinoma (OSCC) is an unmet clinical need. We hypothesized that extracellular vesicles miR-210 expression (EV-miR-210) could be a potential biomarker for OSCC diagnosis and follow-up. METHODS: The expression of EV-miR-210 was measured in the plasma of OSCC patients (n = 30) and compared to that of controls (n = 14). RESULTS: The median EV-miR-210 expression was significantly higher in OSCC patients compared to controls who had often, undetectable levels (p < 0.0001). We performed receiver operating characteristic (ROC) analysis for discriminating OSCC cases from controls. EV-miR-210 yielded an area under the curve (AUC) of 0.9513 with sensitivity 92.3% and specificity 86.6%. Kaplan-Meier curves indicated that high EV-miR-210 expression was associated with worse 3 years' survival (p < 0.05). Cox regression hazard model indicated that high EV-miR-210, G2, and G3 grading and pathological nodal status (pN)>1 were independent predictors of worse survival in OSCC patients. CONCLUSION: These preliminary data suggest that EV-mir-210 may be a novel diagnostic and prognostic biomarker in OSCC.