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Purpose: To evaluate client satisfaction with prosthetic and orthotic services in Sweden, determine if satisfaction differs between clients using different devices and identify factors which influence client satisfaction., Materials and Methods: A cross-sectional design was used to survey 7318 clients. The survey included items related to demographics, quality of life, device comfort, device use, the extent to which clients' needs were met and satisfaction with services. Ethics approval was provided by the Swedish Ethical Review Authority., Results: A total of 2925 surveys were returned reflecting a response rate of 41%. Mean OPUS-CSS point score was 61.9(SD 16.8) with differences observed between device categories ( p  < 0.001). Factors that were identified as most positively influencing client satisfaction were, being a limb prosthesis user and being under 65 years. When analysing scores for individual OPUS items breast prosthesis users scored higher than users of other devices. Clients were most satisfied with the level of respect they were shown by staff (mean = 2.72/3) and less satisfied with coordination of services with other therapists/doctors(mean = 1.88/3)., Conclusions: Prosthetic and orthotic users are reasonably satisfied with the services they receive. Attention should be directed towards understanding why prosthetic users are more satisfied than orthotic users and why clients under 65 years report higher satisfaction scores.

Background: Cancer-associated fibroblasts (CAFs), mainly responsible for the desmoplastic reaction hallmark of intrahepatic Cholangiocarcinoma (iCCA), likely have a role in tumor aggressiveness and resistance to therapy, although the molecular mechanisms involved are unknown. Aim of the study is to investigate how targeting hCAF/iCCA cross-talk with a Notch1 inhibitor, namely Crenigacestat, may affect cancer progression., Methods: We used different in vitro models in 2D and established new 3D hetero-spheroids with iCCA cells and human (h)CAFs. The results were confirmed in a xenograft model, and explanted tumoral tissues underwent transcriptomic and bioinformatic analysis., Results: hCAFs/iCCA cross-talk sustains increased migration of both KKU-M213 and KKU-M156 cells, while Crenigacestat significantly inhibits only the cross-talk stimulated migration. Hetero-spheroids grew larger than homo-spheroids, formed by only iCCA cells. Crenigacestat significantly reduced the invasion and growth of hetero- but not of homo-spheroids. In xenograft models, hCAFs/KKU-M213 tumors grew significantly larger than KKU-M213 tumors, but were significantly reduced in volume by Crenigacestat treatment, which also significantly decreased the fibrotic reaction. Ingenuity pathway analysis revealed that genes of hCAFs/KKU-M213 but not of KKU-M213 tumors increased tumor lesions, and that Crenigacestat treatment inhibited the modulated canonical pathways. Cell cycle checkpoints were the most notably modulated pathway and Crenigacestat reduced CCNE2 gene expression, consequently inducing cell cycle arrest. In hetero-spheroids, the number of cells increased in the G2/M cell cycle phase, while Crenigacestat significantly decreased cell numbers in the G2/M phase in hetero but not in homo-spheroids., Conclusions: The hCAFs/iCCA cross-talk is a new target for reducing cancer progression with drugs such as Crenigacestat., (© 2024. The Author(s).)

Background: Intrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness, poor prognosis, and scarce therapeutic possibilities. Therefore, new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types, we investigated its pathogenetic and therapeutic relevance in iCCA., Methods: Levels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition, the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines, human iCCA cancer-associated fibroblasts (hCAFs), and patient-derived organoids., Results: Human preinvasive, invasive, and metastatic iCCAs displayed widespread HSF1 upregulation, which was associated with a dismal prognosis of the patients. In addition, hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn), which suppresses HSF1 activity, significantly delayed cholangiocarcinogenesis in AKT/NICD, AKT/YAP, and AKT/TAZ mice. In iCCA cell lines, iCCA hCAFs, and patient-derived organoids, administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore, KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells., Conclusions: The present data underscore the critical pathogenetic, prognostic, and therapeutic role of HSF1 in cholangiocarcinogenesis., (© 2024. The Author(s).)

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation., (© 2024. The Author(s).)

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial-mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression.

Background and Aims: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis., Approach and Results: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells., Conclusions: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Background: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA stimulates cell proliferation and migration and promotes wound repair following tissue damage. ATX levels are directly correlated with stage and grade in several human cancers. Several small molecule ATX inhibitors have been developed in recent years. IOA-289 is a potent ATX inhibitor, developed to treat cancers containing fibrosis. In this study, we tested IOA-289 treatment on different gastrointestinal tract tumor cell lines, in order to evaluate its effects on viability and motility., Methods: To determine the effects on cell viability and proliferation of treatment with increasing concentrations of IOA-289, we used the crystal violet assay, a clonogenic assay in matrigel, and we evaluated the inhibitor's effect on formation of 3D spheroids in an in vitro model. The effect of IOA-289 on cell cycle phases was analysed with a redox dye reagent. Cell migration capacity was evaluated by wound healing assay and transwell migration assay. To evaluate the pro-apoptotic effect of the inhibitor, cells were stained with Annexin V and immunofluorescence and flow cytometry analysis were performed. An antibody array was also used, to discriminate, in various samples, the differential expression of 43 proteins involved in the apoptosis pathway., Results: We found that IOA-289 is able to inhibit both growth and migration of gastrointestinal tract tumor cell lines, both in 2D (crystal violet assay) and 3D in vitro models (spheroid formation and clonogenic assay in matrigel). This effect is dose-dependent, and the drug is most effective when administered in FBS-free culture medium. The inhibitory effect on cell growth is due to a pro-apoptotic effect of IOA-289. Staining with FITC-conjugated Annexin V showed that IOA-289 induced a dose-dependent increase in fluorescence following incubation for 24 h, and apoptotic cells were also distinguished in flow cytometry using Annexin/PI staining. The antibody array shows that treatment with IOA-289 causes the increased expression of several pro-apoptotic proteins in all tested cell lines., Conclusions: These results indicate that IOA-289 may be an effective drug for the treatment of tumors of the gastrointestinal tract, particularly those characterized by a high degree of fibrosis., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Aims: This study aimed to assess (1) the use of different offloading interventions in Sweden for the healing of diabetes-related plantar neuropathic forefoot ulcers, (2) factors influencing the offloading intervention choice, and (3) the awareness of current gold standard offloading devices., Methods: An online questionnaire was distributed via SurveyMonkey to 51 prosthetic and orthotic clinics in Sweden., Results: Thirty-five (69%) practitioners responded to the questionnaire. Eighty-six percent of the practitioners provided modified off-the-shelf footwear combined with insoles to treat diabetes-related plantar neuropathic forefoot ulcers. A total contact cast (TCC) was provided by 20% of the practitioners, and a nonremovable knee-high walker was provided by 0%. Multiple practitioner-, patient-, intervention-, and wound-related factors were considered when practitioners provided offloading interventions to patients with this type of ulcer. The majority of the practitioners did not or were unsure whether they considered TCC or a nonremovable knee-high walker to be the gold standard treatment., Conclusions: Practitioners mainly provided the offloading intervention that the International Working Group on the Diabetic Foot strongly recommends not be provided, namely, modified off-the-shelf footwear with insoles. In contrast, TCC and nonremovable knee-high walkers, as the gold standards, were vastly underutilised. Therefore, the pattern of providing offloading interventions was almost exactly opposite to the recommendations of evidence-based guidelines. Different factors were considered when providing offloading interventions to patients with diabetes-related plantar neuropathic forefoot ulcers. The practitioners' lack of awareness regarding gold standard devices may have contributed to the underutilisation of TCC and nonremovable knee-high walkers., (© 2023 The Authors. Diabetes/Metabolism Research and Reviews published by John Wiley & Sons Ltd.)

Grapes represent a significant source of phenolic compounds known for their health-promoting properties, such as antioxidant capacity on normal cells and prooxidant activity on tumor cells. The genotype highly affects the polyphenolic composition in grapes and, consequently, the nutritional quality of berries. This work aimed to characterize the phenolic composition, the antioxidant, and anticancer activity of grape skin extracts (GSEs) of nine new table grape genotypes selected from a breeding program to obtain new cultivars of seedless table grapes, well adapted to the climatic change and with higher nutraceutical properties. The grape polyphenolic profile was characterized by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry analysis. GSE antioxidant activity was determined by the ABTS, DPPH, and ORAC assays; GSE cell growth inhibition test was carried out in the Caco2 human cancer cell line. The nine GSEs showed different flavonoid and non-flavonoid profiles, and all possessed antioxidant activity, with the 'Aika N.', 'Turese N.', and 'Egnatia N.' the most active. As anticancer activity against the tested cancer cell line, 'Daunia N.' and 'Apenestae N.' showed the EC50 after 24 h of 35.60 µg/mL and 150.91 µg/mL, respectively. The relationship between polyphenolic profile and the antioxidant and anticancer activity of GSE was also investigated. Interestingly, among the different classes of polyphenolics, flavan-3-ols e proanthocyanidins showed the highest positive correlation with the anticancer activity of extracts. These findings can be helpful for the preparation of new extracts for the pharmaceutical and nutraceutical industry and geneticists working in vine breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Milella, De Rosso, Gasparro, Gigante, Debiase, Forleo, Marsico, Perniola, Tutino, Notarnicola, Velasco and Flamini.)

Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-β1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA., Methods: We investigated Crenigacestat's effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA., Results: In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted "liver fibrosis" as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p < 0.001) reduced peritumoral liver fibrosis. Similar results were obtained in a hydrodynamic model of iCCA. Bioinformatic prediction of the upstream regulators related to liver fibrosis in the iCCA PDX treated with Crenigacestat revealed the involvement of the TGF-β1 pathway as a master regulator gene showing a robust connection between TGF-β1 and Notch pathways. Consistently, drug treatment significantly (p < 0.05) reduced TGF-β1 mRNA and protein levels in tumoral tissue. In PDX tissues, Crenigacestat remarkably inhibited TGF-β signaling and extracellular matrix protein gene expression and reduced α-SMA expression. Furthermore, Crenigacestat synergistically increased Gemcitabine effectiveness in the iCCA PDX model. In 31 iCCA patients, TGF-β1 and α-SMA were upregulated in the tumoral compared with peritumoral tissues. In freshly isolated CAFs from patients with iCCA, Crenigacestat significantly (p < 0.001) inhibited Notch signaling, TGF-β1 secretion, and Smad-2 activation. Consequently, Crenigacestat also inactivated CAFs reducing (p < 0.001) α-SMA expression. Finally, CAFs treated with Crenigacestat produced less (p < 005) ECM components such as fibronectin, collagen 1A1, and collagen 1A2., Conclusions: Notch signaling inhibition reduces the peritumoral desmoplastic reaction in iCCA, blocking the TGF-β1 canonical pathway., (© 2022. The Author(s).)

Grape pomace (GP)-the major by-product of winemaking processes-still contains bioactive molecules with known beneficial properties for human health, such as an antiradical scavenging activity or an antiproliferative activity of tumors. In vitro studies have demonstrated that GP polyphenols specifically influence colon cancer cell proliferation. In addition to previously published work, we tested the phenolic compounds of Aglianico GP following an in vitro simulated gastrointestinal digestion on colorectal cancer cell lines at different degrees of differentiation. Our experiments, using HT29 and SW480 cells, confirmed the anti-proliferative effect of GP gastrointestinal digested extract and provided intriguing insights on the way it influences the cancer cell features (i.e., viability, proliferation, and apoptosis). We observed that Aglianico GP extract showed a great ability to affect cell proliferation and apoptosis. Interestingly, both HT29 and SW480 cells produced a significant increase in Bax, and a significant increase in the Bax/Bcl-2 ratio and caspase-3. The gastrointestinal digested GP extract was previously characterized both for antioxidant activity and phenolic composition. As a result, the TPC and the antioxidant activity reached high values in the Aglianico GP digested extract, and the main compounds assessed by UHPLC-DAD were anthocyanins, phenolic acids, and flavonoids. This work shed light on the use of digested GP extract as a dietary ingredient, a very sustainable source of nutritional compounds with potential health benefits for colon cancer cell proliferation.

In recent years, nutraceuticals have gained great popularity, owing to their physiological and potential health effects, such as anti-inflammatory, anti-cancer, antioxidant, and prebiotic effects, and their regulation of lipid metabolism. Since the Mediterranean diet is a nutritionally recommended dietary pattern including high-level consumption of nutraceuticals, this review aimed to summarize the main results obtained by our in vitro and in vivo studies on the effects of the major constituents of the Mediterranean diet (i.e., extra virgin olive oil compounds, polyunsaturated fatty acids, and fruit components). Based on experimental studies, the therapeutic purpose of nutraceuticals depends on their bioavailability, solubility, toxicity, and delivery system. This review provides more in-depth knowledge on the effects linked to nutraceuticals administration on human health, focusing the gastrointestinal tract and suggesting specific dietary components for personalized adjuvant therapies.

Background: Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell-cell and cell-matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway., Methods: THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively., Results: CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival., Conclusions: iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA., (© 2022. The Author(s).)

Scope: The study aims to investigate the effects of fresh table grape consumption in healthy subjects on circulating levels of the most common human microRNAs (miRNAs). The regulatory network governed by these modulated miRNAs is also investigated., Methods and Results: Autumn Royal table grape, used in this study, is chosen for its high polyphenolic content and antioxidant properties. The study is a randomized controlled trial, in which 40 consecutive subjects are recruited on a voluntary basis and randomly assigned to two groups of the study, the control group, receiving only dietary recommendations and a grape group receiving a daily dose of 5 g of fresh table grape per kg of body weight for 21 days. All analyses are performed at baseline and after 21 days of dietary treatment. Circulating miRNAs levels are detected by Real-Time quantitative PCR (RT-qPCR) followed by bioinformatic functional analysis. The study identifies 20 circulating miRNAs differentially expressed in healthy subjects after grape intake, and in particular, 18 of 20 are down-regulated and 2 are up-regulated., Conclusion: The dietary intake of table grape affects circulating miRNAs levels in healthy subjects, particularly the miRNAs related to pathways involved in counteracting cancer development, including gastrointestinal cancers., (© 2021 Wiley-VCH GmbH.)

Functional alterations in irritable bowel syndrome have been associated with defects in bioenergetics and the mitochondrial network. Effects of high fat, adequate-protein, low carbohydrate ketogenic diet (KD) involve oxidative stress, inflammation, mitochondrial function, and biogenesis. The aim was to evaluate the KD efficacy in reducing the effects of stress on gut mitochondria. Newborn Wistar rats were exposed to maternal deprivation to induce IBS in adulthood. Intestinal inflammation (COX-2 and TRL-4); cellular redox status (SOD 1, SOD 2, PrxIII, mtDNA oxidatively modified purines); mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, mtDNA content); and autophagy (Beclin-1, LC3 II) were evaluated in the colon of exposed rats fed with KD (IBD-KD) or standard diet (IBS-Std), and in unexposed controls (Ctrl). IBS-Std rats showed dysfunctional mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, and mtDNA contents lower than in Ctrl) associated with inflammation and increased oxidative stress (higher levels of COX-2 and TLR-4, SOD 1, SOD 2, PrxIII, and oxidatively modified purines than in Ctrl). Loss of autophagy efficacy appeared from reduced levels of Beclin-1 and LC3 II. Feeding of animals with KD elicited compensatory mechanisms able to reduce inflammation, oxidative stress, restore mitochondrial function, and baseline autophagy, possibly via the upregulation of the PPAR-γ/PGC-1α axis.

The administration of a ketogenic diet (KD) has been considered therapeutic in subjects with irritable bowel syndrome (IBS). This study aimed to investigate the molecular mechanisms by which a low-carbohydrate diet, such as KD, can improve gastrointestinal symptoms and functions in an animal model of IBS by evaluating possible changes in intestinal tissue expression of endocannabinoid receptors. In rats fed a KD, we detected a significant restoration of cell damage to the intestinal crypt base, a histological feature of IBS condition, and upregulation of CB1 and CB2 receptors. The diet also affected glucose metabolism and intestinal membrane permeability, with an overexpression of the glucose transporter GLUT1 and tight junction proteins in treated rats. The present data suggest that CB receptors represent one of the molecular pathways through which the KD works and support possible cannabinoid-mediated protection at the intestinal level in the IBS rats after dietary treatment.

Dendritic cells are the most important antigen-presenting cells that link the innate and acquired immune system. In our previous study, we identified that the upregulation of miR-369-3p suppresses the LPS-induced inflammatory response, reducing C/EBP-β, TNFα and IL-6 production. With the aim of gaining further insight into the biological function of miR-369-3p during acute inflammatory response, in the present study we identified novel gene targets of miR-369-3p and demonstrated the suppressive ability of these genes on the inflammatory dendritic cells. Bioinformatic analyses revealed that iNOS is a potential target of miR-369-3p. We demonstrated that the ectopic induction of miR-369-3p markedly reduced iNOS mRNA and protein as well as NO production. Moreover, we found that the upregulation of miR-369-3p decreased the release of TNFα, IL-6, IL-12, IL-1α, IL-1β in response to LPS, and increased the production of anti-inflammatory cytokines such as IL-10 and IL-1RA. In addition, LPS-induced nuclear translocation of NF-kB was inhibited by miR-369-3p. Levels of miR-369-3p were decreased in human inflamed regions of human intestine obtained from IBD patients. Our results provide novel additional information on miR-369-3p as a potential core of the signaling regulating the inflammatory response. These findings suggest that miR-369-3p should be considered as a potential target for the future development of new molecular therapeutic approaches.

Colorectal cancer (CRC) is the third most common cancer worldwide. There is a need for the early diagnosis of CRC for a better prognostic outcome. It is, therefore, crucial to understand the CRC pathogenesis in all its aspects. In many cases, one of the main causes of cancer-related deaths is the presence of metastases. In this context, an often overlooked aspect is the metastatic tropism, since CRC, like other cancers, is more prone to metastasize some organs rather than others. Beyond the liver and lung, and differently from other types of cancers, a not usual site of CRC metastases is the bone. However, it may assume a crucial role in the development and the outcome of the disease. Therefore, this review aims to discuss the complex relations between bone markers and CRC pathogenesis, suggesting the use of these molecules as potential targets for therapeutic purposes. Different osteogenic molecules, some of whom are growth factors and are implicated in the different osteogenic pathways, have been proved to also be involved in CRC progression. Some of them are oncogenes, while others oncosuppressors, and in a future perspective, some of them may represent new potential CRC biomarkers.

Grapes contain many flavonoid and non-flavonoid compounds with anticancer effects. In this work we fully characterized the polyphenolic profile of two grape skin extracts (GSEs), Autumn Royal and Egnatia, and assessed their effects on Polyunsaturated Fatty Acid (PUFA) membrane levels of Caco2 and SW480 human colon cancer cell lines. Gene expression of 15-lipoxygenase-1 (15-LOX-1), and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as cell morphology, were evaluated. The polyphenolic composition was analyzed by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. PUFA levels were evaluated by gas chromatography, and gene expression levels of 15-LOX-1 and PPAR-γ were analyzed by real-time Polymerase Chain Reaction (PCR). Morphological cell changes caused by GSEs were identified by field emission scanning electron microscope (FE-SEM) and photomicrograph examination. We detected a different profile of flavonoid and non-flavonoid compounds in Autumn Royal and Egnatia GSEs. Cultured cells showed an increase of total PUFA levels mainly after treatment with Autumn Royal grape, and were richer in flavonoids when compared with the Egnatia variety. Both GSEs were able to affect 15-LOX-1 and PPAR-γ gene expression and cell morphology. Our results highlighted a new antitumor mechanism of GSEs that involves membrane PUFAs and their downstream pathways.

The polyphenolic compounds present in grape extracts have chemopreventive and anticancer properties. Here, we studied the ability of two grape skin extracts (GSEs), Autumn Royal and Egnatia, to influence the cell motility and membrane fluidity regulated by the enzyme Stearoyl-CoA desaturase-1 (SCD1) which increases with the cancer aggressiveness. Caco2 and SW480 human colon cancer cell lines were treated with increasing concentrations of GSEs to evaluate cell proliferation and motility. SCD1 levels were evaluated in both treated cell lines, by membrane lipidomic analysis conducted by gas chromatography. The expression levels of SCD1 and other factors involved in the reorganization of the cytoskeleton and focal adhesions were assessed by Real-time PCR, Western Blotting, and Immunofluorescence staining. High-performance liquid chromatography (HPLC) analyses were performed to determine the phenolic composition in the GSEs, finding them more expressed in Autumn Royal than in Egnatia. Both treatments reduced the levels of SCD1, phospho-Rac1/Cdc42/Rac1/Cdc42 ratio, Cofilin, Vimentin, and phospho-Paxillin especially in Caco2 compared to SW480, showing a different behavior of the two cell lines to these natural compounds. Our findings show that GSEs block the cell migration and membrane fluidity through a new mechanism of action involving structural cellular components., Competing Interests: The authors declare no conflict of interest.

Background: Inflammatory bowel disease (IBD), including Crohn disease (CD) and ulcerative colitis (UC), is a multifactorial disorder characterized by chronic inflammation and altered gut barrier function. Dysbiosis, a condition defined by dysregulation of the gut microbiome, has been reported in patients with IBD and in experimental models of colitis. Although several factors have been implicated in directly affecting gut microbial composition, the genetic determinants impacting intestinal dysbiosis in IBD remain relatively unknown., Methods: We compared the microbiome of normal, uninflamed wild-type (WT) mice with that of a murine model of UC (ie, Winnie strain). Winnie mice possess a missense mutation in Muc2 that manifests in altered mucus production as early as 4 weeks of age, with ensuing colonic inflammation. To better address the potential role of mutant Muc2 in promoting dysbiosis in Winnie mice, we evaluated homozygous mutant mice (Winnie-/-) with their WT littermates that, after weaning from common mothers, were caged separately according to genotype. Histologic and inflammatory status were assessed over time, along with changes in their respective microbiome compositions., Results: Dysbiosis in Winnie mice was already established at 4 weeks of age, before histologic evidence of gut inflammatory changes, in which microbial communities diverged from that derived from their mothers. Furthermore, dysbiosis persisted until 12 weeks of age, with peak differences in microbiome composition observed between Winnie and WT mice at 8 weeks of age. The relative abundance of Bacteroidetes was greater in Winnie compared with WT mice. Verrucomicrobia was detected at the highest relative levels in 4-week-old Winnie mice; in particular, Akkermansia muciniphila was among the most abundant species found at 4 weeks of age., Conclusions: Our results demonstrate that mutant genetic determinants involved in the complex regulation of intestinal homeostasis, such as that observed in Winnie mice, are able to promote early gut dysbiosis that is independent from maternal microbial transfer, including breastfeeding. Our data provide evidence for intestinal dysbiosis attributed to a Muc2-driven mucus defect that leads to colonic inflammation and may represent an important target for the design of future interventional studies., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.)

Celiac disease (CD) presents as chronic low-grade inflammation of the small intestine often characterized by psychiatric comorbidities. The brain-derived neurotrophic factor (BDNF), which we have shown to be reduced in the serum of CD patients, acts as the bridge between immune activation and the nervous system adaptive response. Since Lactobacillus has been shown to upregulate BDNF, this study aimed to evaluate whether the administration of Lactobacillus rhamnosus GG (L.GG) could positively affect the brain BDNF system in rats mimicking the CD lesions. Data have shown that the administration of pepsin-trypsin digested gliadin (PTG) and L.GG alter the levels of mature BDNF (mBDNF), as evaluated by Western blotting. PTG provoked a reduction of mBDNF compared to controls, and a compensatory increase of its receptor TrkB. L.GG induced a slight positive effect on mBDNF levels under normal conditions, while it was able to rescue the PTG-induced reduced expression of mBDNF. The curative effect of L.GG was finely tuned, accompanied by the reduction of TrkB, probably to avoid the effect of excessive BDNF.

Objective: Polyphenols extracted by table grape have been demonstrated to decrease cell proliferation in vitro and to exert anti-atherosclerotic and antithrombotic activities, regulating cell functions. A grape polyphenolic profile is affected by climate as well as a grape cultivar. This study was aimed to characterize the berry skin polyphenolic composition, antioxidant activity and antiproliferative properties of two black grape cultivars, Autumn Royal and Egnatia., Methods: The phenolic composition of Grape Skin Extracts (GSEs) was determined by HPLC analyses. The antioxidant activity was determined using DPPH, ABTS and ORAC tests. Caco2, HT29 and SW480 human colon cancer cell lines were used to test the effects of GSEs in vitro. Cell proliferation and cell cycle were assessed with the MTT method and a Muse cell analyzer, respectively. qPCR and Western Blotting analysis were used to evaluate gene and protein expression, respectively., Results: The total polyphenolic content and the total antioxidant capacity were significantly higher in Autumn Royal than in Egnatia. However, table grape Egnatia showed greater ability to affect cell proliferation and apoptosis, as well as to exert a growth arrest in the S phase of the cell cycle, particularly in the Caco2 cell line., Conclusion: These data suggest that the new grape variety Egnatia is an interesting source of phenolic compounds that could be of interest in the food and pharmaceutical industries., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Changes in the regulation of endocannabinoid production, together with an altered expression of their receptors are hallmarks of cancer, including colorectal cancer (CRC). Although several studies have been conducted to understand the biological role of the CB1 receptor in cancer, little is known about its involvement in the metastatic process of CRC. The aim of this study was to investigate the possible link between CB1 receptor expression and the presence of metastasis in patients with CRC, investigating the main signaling pathways elicited downstream of CB1 receptor in colon cancer. Fifty-nine consecutive patients, with histologically proven colorectal cancer, were enrolled in the study, of which 30 patients with synchronous metastasis, at first diagnosis and 29 without metastasis. A low expression of CB1 receptor were detected in primary tumor tissue of CRC patients with metastasis and consequently, we observed an alteration of CB1 receptor downstream signaling. These signaling routes were also altered in intestinal normal mucosa, suggesting that, normal mucosa surrounding the tumor provides a realistic picture of the molecules involved in tissue malignant transformation. These observations contribute to the idea that drugs able to induce CB1 receptor expression can be helpful in order to set new anticancer therapeutic strategies., Competing Interests: The authors declare no conflict of interest.

Background: Adherence to a healthy diet has been reported to be essential for the primary prevention of colorectal cancer, through a reduction of tissue inflammation, a low concentration of circulating lipoproteins and lower levels of serum cholesterol. Since an altered expression of the fatty acids pattern has been demonstrated to be a crucial event in colorectal carcinogenesis, lipidomic analysis is considered able to identify early diagnostic and prognostic biomarkers of complex diseases such as colorectal cancer., Methods: cell membrane fatty acid profile and serum lipoproteins pattern were evaluated by gas chromatography and electrophoresis method respectively., Results: There is a close association between diet and lipidomic profile in colorectal cancer, both in pre-clinical and clinical studies. A modified serum lipoproteins pattern has been demonstrated to be predominant in intestinal tumors., Conclusions: The study of fatty acids profile in cell membrane and the evaluation of serum lipoproteins subfractions could be useful to have an integrate vision on the interactions between lipids and the pathogenesis of colorectal cancer and to understand the mechanisms of action and the consequences of these interactions on human health status.

Background/aim: The expression of cannabinoid receptor-1 (CB1-R) seems to be modulated by bioactive natural components such as the flavonoid quercetin. The aim of this study was to determine in an animal model of induced-colon cancer, whether quercetin inhibits colon carcinogenesis through changes in the expression of CB1-R., Materials and Methods: C57BL/6J male mice were randomly assigned to standard diet or experimental diet supplemented with 0.5% quercetin. Azoxymethane (AOM) (10 mg/kg body weight) or saline solution (PBS) was intraperitoneally injected, once weekly for 6 weeks., Results: The diet supplemented with quercetin induced CB1-R gene expression and protein, inhibiting the protein levels of STAT3 and p-STAT3 (both mediators of cell proliferation). Dietary quercetin also caused a significant increase in Bax/Bcl2 ratio protein expression., Conclusion: The anti-proliferative and pro-apoptotic effects of quercetin in AOM-treated mice are mediated by induction of the protein and gene expression levels of CB1-R., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14 -/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8 + T cells in Tnfsf14 -/- mice. LIGHT stimulation increases OPG levels in B, CD8 + T, and osteoblastic cells, as well as Wnt10b expression in CD8 + T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag - /Tnfsf14 - ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in western countries. The major risk factors for HCC are hepatitis C or B viruses, alcohol and metabolic disorders. The increasing risk of HCC in patients with metabolic disorders (i.e. obesity, diabetes and non-alcoholic steatohepatitis/NASH) regardless of the presence of liver cirrhosis is becoming relevant. Nevertheless, molecular mechanisms linking these risk factors to liver oncogenesis are unclear. This review focuses on the pathogenic role of the lysophosphatidic acid (LPA) pathway in HCC, highlighting the implications of this bioactive phospholipid in liver cancer biology and metabolism and as potential therapeutic target., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Food industries are increasingly oriented toward new foods to improve nutritional status and/or to combat nutritional deficiency diseases. In this context, silicon biofortification could be an innovative tool for obtaining new foods with possible positive effects on bone mineralization. In this paper, an alternative and quick in vitro approach was applied in order to evaluate the potential health-promoting effects of five silicon-biofortified leafy vegetables (tatsoi, mizuna, purslane, Swiss chard and chicory) on bone mineralization compared with a commercial silicon supplement. The silicon bioaccessibility and bioavailability of the five leafy vegetables (biofortified or not) and of the supplement were assessed by applying a protocol consisting of in vitro gastrointestinal digestion coupled with a Caco-2 cell model. Silicon bioaccessibility ranged from 0.89 to 8.18 mg/L and bioavailability ranged from 111 to 206 μg/L of Si for both vegetables and supplement. Furthermore, the bioavailable fractions were tested on a human osteoblast cell model following the expression of type 1 collagen and alkaline phosphatase. The results obtained highlighted that the bioavailable fraction of biofortified purslane and Swiss chard improved the expression of both osteoblast markers compared with the supplement and other vegetables. These results underline the potentially beneficial effect of biofortified leafy vegetables and also indicate the usefulness of in vitro approaches for selecting the best vegetable with positive bone effects for further in vivo research.

CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.

LIGHT, a TNF superfamily member, is involved in T-cell homeostasis and erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT has a role in Multiple Myeloma (MM)-bone disease. We found that LIGHT was overproduced by CD14+ monocytes, CD8+ T-cells and neutrophils of peripheral blood and bone marrow (BM) from MM-bone disease patients. We also found that LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis. In cultures from healthy-donors, LIGHT induced osteoclastogenesis in RANKL-dependent and -independent manners. In the presence of a sub-optimal RANKL concentration, LIGHT and RANKL synergically stimulated osteoclast formation, through the phosphorylation of Akt, NFκB and JNK pathways. In cultures of BM samples from patients with bone disease, LIGHT inhibited the formation of CFU-F and CFU-OB as well as the expression of osteoblastic markers including collagen-I, osteocalcin and bone sialoprotein-II. LIGHT indirectly inhibited osteoblastogenesis in part through sclerostin expressed by monocytes. In conclusion, our findings for the first time provide evidence for a role of LIGHT in MM-bone disease development.

Periodontal disease (PD), or periodontitis, is defined as a bacterially induced disease of the tooth-supporting (periodontal) tissues. It is characterized by inflammation and bone loss; therefore understanding how they are linked would help to address the most efficacious therapeutic approach. Bacterial infection is the primary etiology but is not sufficient to induce the disease initiation or progression. Indeed, bacteria-derived factors stimulate a local inflammatory reaction and activation of the innate immune system. The innate response involves the recognition of microbial components by host cells, and this event is mediated by toll-like receptors (TLRs) expressed by resident cells and leukocytes. Activation of these cells leads to the release of proinflammatory cytokines and recruitment of phagocytes and lymphocytes. Activation of T and B cells initiates the adaptive immunity with Th1 Th2 Th17 Treg response and antibodies production respectively. In this inflammatory scenario, cytokines involved in bone regulation and maintenance have considerable relevance because tissue destruction is believed to be the consequence of host inflammatory response to the bacterial challenge. In the present review, we summarize host factors including cell populations, cytokines, and mechanisms involved in the destruction of the supporting tissues of the tooth and discuss treatment perspectives based on this knowledge.