Endoscopic Submucosal Dissection for Subepithelial Tumor Treatment in the Upper Digestive Tract: A Western, Multicenter Study.

Background/Aims: Endoscopic submucosal dissection (ESD) has been proposed for removal of gastrointestinal subepithelial tumors (GI-SETs), but data are still scanty. This study aimed to report a case series from a western country. Patients and Methods: Data of patients with upper GI-SETs suitable for ESD removal observed in 4 centers were retrospectively reviewed. Before endoscopic procedure, the lesion was characterized by endosonographic evaluation, histology, and CT scan. The en bloc resection and the R0 resection rates were calculated, as well as incidence of complications, and the 1-year follow-up was reported. Results: Data of 84 patients with esophageal (N = 13), gastric (N = 61), and duodenal (N = 10) GI-SETs were collected. The mean diameter of lesions was 26 mm (range: 12-110 mm). There were 17 gastrointestinal stromal tumors, 12 neuroendocrine tumors, 35 leiomyomas, 18 lipomas, and 2 hamartomas. En bloc and R0 resection were achieved in 83 (98.8%) and in 80 (95.2%) patients, respectively. Overall, a complication occurred in 11 (13.1%) patients, including bleeding (N = 7) and perforation (N = 4). Endoscopic approach was successful in all bleedings, but 1 patient who required radiological embolization, and in 2 perforations, while surgery was performed in the other patients. Overall, a surgical approach was eventually needed in 5 (5.9%), including 3 in whom R0 resection failed and 2 with perforation. Conclusions: Our study found that ESD may be an effective and safe alternative to surgical intervention for both benign and localized malignant GI-SETs.

Introdução/objetivos: A dissecção endoscópica da submucosa (ESD) tem sido proposta para a exérese de tumores subepiteliais gastrointestinais (GI-SETs), embora a literatura seja escassa. Este estudo teve como objetivo reportar uma série de casos de um país ocidental. Métodos: Coorte retrospectiva incluindo doentes com SETs do tubo digestivo superior submetidos a ESD em 4 centros (1 ano de follow-up). Antes do procedimento, a lesão foi caracterizada por ecoendoscopia, histologia e tomografia computadorizada. Foram avaliadas as taxas de ressecção em bloco e R0, bem como a incidência de complicações. Resultados: Incluídos 84 doentes com GI-SETs esofágicos (N = 13), gástricos (N = 61) e duodenais (N = 10). O diâmetro médio das lesões foi de 26 mm (intervalo 12­110 mm) ­ 17 tumores do estroma gastrointestinal, 12 tumores neuroendócrinos, 35 leiomiomas, 18 lipomas e 2 hamartomas. A resseção foi em bloco e R0 em 83 (98.8%) e em 80 (95.2%) doentes, respectivamente. Globalmente, ocorreram complicações em 11 (13.1%) doentes, incluindo hemorragia (N = 7) e perfuração (N = 4). A terapêutica endoscópica foi eficaz em todas as hemorragias exceto em 1 doente que necessitou de embolização radiológica e em 2 perfurações (submetidas a cirurgia). No geral, a abordagem cirúrgica foi necessária em 5 (5.9%) ­ 3 doentes com resseção R1 e 2 com perfuração. Conclusões: A ESD pode ser uma alternativa eficaz e segura à intervenção cirúrgica para GI-SETs benignos e malignos localizados.

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Endoscopy management of sleeve gastrectomy stenosis: what we learned from 202 consecutive patients.

BACKGROUND: Gastric sleeve stenosis (GSS) is described in 1%-4% of patients. OBJECTIVE: To evaluate the role of endoscopy in the management of stenosis after laparoscopic sleeve gastrectomy using a standardized approach according to the characteristic of stenosis. SETTING: Retrospective, observational, single-center study on patients referred from several bariatric surgery departments to an endoscopic referral center. METHODS: We enrolled 202 patients. All patients underwent endoscopy in a fluoroscopy setting, and a systematic classification of the type, site, and length of the GSS was performed. According to the characteristics of the stenosis, patients underwent pneumatic dilatation or placement of a self-expandable metal stent or a lumen-apposed metal stent. Failure of endoscopic treatment was considered an indication for redo surgery, whereas patients with partial or complete response were followed up for 2 years. In the event of a recurrence, a different endoscopic approach was used. RESULTS: We found inflammatory strictures in 4.5% of patients, pure narrowing in 11%, and functional stenosis in 84.5%. Stenosis was in the upper tract of the stomach in 53 patients, whereas medium and distal stenosis was detected in 138 and 11 patients, respectively, and short stenosis in 194 patients. A total of 126 patients underwent pneumatic dilatation, 8 self-expandable metal stent placement, 64 lumen-apposed metal stent positioning, and 36 combined therapy. The overall rate of endoscopy success was 69%. CONCLUSION: GSS should be considered to be a chronic disease, and the endoscopic approach seems to be the most successful treatment, with a prolonged positive outcome of 69%. Characteristics of the stenosis should guide the most suitable endoscopic approach.

Role of Endoscopic Internal Drainage in Treating Gastro-Bronchial and Gastro-Colic Fistula After Sleeve Gastrectomy.

BACKGROUND: Gastro-bronchial and gastro-colic fistulas (GB-GC) represent a rare, but serious complication after laparoscopic sleeve gastrectomy (LSG). The aim of this study is to evaluate the efficacy of endoscopic first-line approach with endoscopic internal drainage (EID) by inserting double pigtail stents (DPS) METHODS: We retrospectively analyzed data from 40 consecutive patients referred at two tertiary centers for gastro-bronchial (N=30) and gastrocolic (N=10) fistulas following LSG. Nineteen patients previously experienced emergency surgical drainage. The mean interval between the index surgery and endoscopic fistula treatment was 265.6±521 days. RESULTS: Healing of the fistulous tract was achieved in 19 patients (47.5%), with complete resolution at an average follow-up of 16 months. Mean time of treatment duration was 157.8±141 days with 5.0±2.9 endoscopic sessions. No major adverse events were registered. CONCLUSIONS: Despite complete fistula healing was achieved in less than 50% of our population, EID for GB/GC fistula after LSG still represents the most conservative approach with low complications rate. Previous surgical drainage seems to be a positive prognostic factor for endoscopic healing. While the longer the interval between the index surgery and endoscopic treatment, the lower was the rate of treatment success.

Impaired Duodenal Palmitoylethanolamide Release Underlies Acid-Induced Mast Cell Activation in Functional Dyspepsia.

BACKGROUND & AIMS: Acid hypersensitivity is claimed to be a symptomatic trigger in functional dyspepsia (FD); however, the neuroimmune pathway(s) and the mediators involved in this process have not been investigated systematically. Palmitoylethanolamide (PEA) is an endogenous compound, able to modulate nociception and inflammation, but its role in FD has not been assessed. METHODS: Duodenal biopsy specimens from FD and control subjects, and peroxisome proliferator-activated receptor-α (PPARα) null mice were cultured at a pH of 3.0 and 7.4. Mast cell (MC) number, the release of their mediators, and the expression of transient receptor potential vanilloid receptor (TRPV)1 and TRPV4, were evaluated. All measurements also were performed in the presence of a selective blocker of neuronal action potential (tetradotoxin). FD and control biopsy specimens in acidified medium also were incubated in the presence of different PEA concentrations, alone or combined with a selective PPARα or PPAR-γ antagonist. RESULTS: An acid-induced increase in MC density and the release of their mediators were observed in both dyspeptic patients and controls; however, this response was amplified significantly in FD. This effect was mediated by submucosal nerve fibers and up-regulation of TRPV1 and TRPV4 receptors because pretreatment with tetradotoxin significantly reduced MC infiltration. The acid-induced endogenous release of PEA was impaired in FD and its exogenous administration counteracts MC activation and TRPV up-regulation. CONCLUSIONS: Duodenal acid exposure initiates a cascade of neuronal-mediated events culminating in MC activation and TRPV overexpression. These phenomena are consequences of an impaired release of endogenous PEA. PEA might be regarded as an attractive therapeutic strategy for the treatment of FD.

Endoscopic submucosal dissection versus endoscopic mucosal resection for type 0-II superficial gastric lesions larger than 20 mm.

BACKGROUND: Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are increasingly used for the treatment of superficial gastrointestinal neoplasia. However, the limits and the indications for each technique are still debated. Our retrospective study aimed to compare these techniques in patients with gastric flat lesions larger than 20 mm without the non-lifting sign. METHODS: Between January 2013 and July 2016, a total of 36 patients with early gastric flat lesions larger than 20 mm and without the non-lifting sign were resected by ESD and were followed up by endoscopy. As a control group, 40 EMR cases from our database were matched. En bloc and curative resection were compared between the two groups according to histological assessment, tumor size, recurrence, complication rate, and procedure time. A Kaplan-Meier comparison was performed for both groups with a log-rank test to compare the survival curves; the chi-square test was employed for other parameters. RESULTS: En bloc resection rate and curative resection rate were significantly higher in the ESD group than in the EMR group. Procedure time was significantly longer in the ESD group. No significant differences were found in the recurrence and complication rates, although the former were higher in the EMR group and the latter in the ESD group. Survival curves were similar for both groups. CONCLUSIONS: Our retrospective analysis seems to confirm a clear advantage for ESD over EMR in removing early superficial gastric neoplasm. Although ESD has expanded the endoscopic resectability of endoscopic gastric lesions, EMR may still be considered one of the therapeutic options for flat gastric lesions without the non-lifting sign.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia.

BACKGROUND: Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity. METHODS: Diarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα-/- mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα-/- mice. RESULTS: HIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα-/- mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation. CONCLUSIONS: EGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

A randomized, open-label, comparative, crossover trial on preference, efficacy, and safety profiles of lispro insulin u-100 versus concentrated lispro insulin u-200 in patients with type 2 diabetes mellitus: a possible contribution to greater treatment adherence.

BACKGROUND: Several outstanding pharmacological advances making innovative drugs sophisticateddevices available during the last few years. Nevertheless too many patients still disappointingly fail to meetthe metabolic targets suggested by current guidelines. Incorrect insulin administration techniques may greatly affect metabolic control in T2DM people. The aim of our study was to compare glycemic control associated with a concentrated insulin analog preparation (U-200 lispro) in people with T2DM to the one observed with standard U-100 lispro. The secondary endpoint of our study was patients' preference and performance ratings of U-200 lispro. METHODS: 126 patients with T2DM were enrolled. They were also assessed for limited joint mobility syndrome (LJMS),defined as limitation in at least two anatomical areas of the dominant upper extremity. After a 4-weekstructured insulin injection education period. Half of them were randomized to U-100 lispro, half to U-200 and after 12 weeks they were switched to the other preparation for 12 weeks. At the end a questionnaire was also administered to investigate patient preference. RESULTS: No significant variation in fasting blood glucose, HbA1c, severe or mild hypoglycemic rate and daily fast-acting insulin analog dose was observed with U-100 lispro while U-200 lispro treatment was associated with a significant improvement of all the above mentioned parameters and with around 20% decrease in insulin requirement. Moreover patients' answers to the questionnaire pointed out a higher preference for U-200 lispro for continuing treatment due to fewer difficulties completing injection. DISCUSSION: The explanation of better metabolic results with the U-200 device might be the lower inner piston inertia and volume and shorter duration of a complete injection. CONCLUSIONS: Checking for LJIMS before insulin prescription could be adopted as a standard practiceaimed at choosing the most suitable device for patient's specific characteristics and abilities. The use of U-200 lispro might improve treatment adherence and metabolic control. This would also result intocost reduction by saving about half the amount of pens per year and of time spent to both fill prescriptionand dump the pharmacy.

Endocannabinoid-related compounds in gastrointestinal diseases.

The endocannabinoid system (ECS) is an endogenous signalling pathway involved in the control of several gastrointestinal (GI) functions at both peripheral and central levels. In recent years, it has become apparent that the ECS is pivotal in the regulation of GI motility, secretion and sensitivity, but endocannabinoids (ECs) are also involved in the regulation of intestinal inflammation and mucosal barrier permeability, suggesting their role in the pathophysiology of both functional and organic GI disorders. Genetic studies in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease have indeed shown significant associations with polymorphisms or mutation in genes encoding for cannabinoid receptor or enzyme responsible for their catabolism, respectively. Furthermore, ongoing clinical trials are testing EC agonists/antagonists in the achievement of symptomatic relief from a number of GI symptoms. Despite this evidence, there is a lack of supportive RCTs and relevant data in human beings, and hence, the possible therapeutic application of these compounds is raising ethical, political and economic concerns. More recently, the identification of several EC-like compounds able to modulate ECS function without the typical central side effects of cannabino-mimetics has paved the way for emerging peripherally acting drugs. This review summarizes the possible mechanisms linking the ECS to GI disorders and describes the most recent advances in the manipulation of the ECS in the treatment of GI diseases.

Cannabidiol restores intestinal barrier dysfunction and inhibits the apoptotic process induced by Clostridium difficile toxin A in Caco-2 cells.

BACKGROUND: Clostridium difficile toxin A is responsible for colonic damage observed in infected patients. Drugs able to restore Clostridium difficile toxin A-induced toxicity have the potential to improve the recovery of infected patients. Cannabidiol is a non-psychotropic component of Cannabis sativa, which has been demonstrated to protect enterocytes against chemical and/or inflammatory damage and to restore intestinal mucosa integrity. OBJECTIVE: The purpose of this study was to evaluate (a) the anti-apoptotic effect and (b) the mechanisms by which cannabidiol protects mucosal integrity in Caco-2 cells exposed to Clostridium difficile toxin A. METHODS: Caco-2 cells were exposed to Clostridium difficile toxin A (30 ng/ml), with or without cannabidiol (10-7-10-9 M), in the presence of the specific antagonist AM251 (10-7 M). Cytotoxicity assay, transepithelial electrical resistence measurements, immunofluorescence analysis and immunoblot analysis were performed in the different experimental conditions. RESULTS: Clostridium difficile toxin A significantly decreased Caco-2 cells' viability and reduced transepithelial electrical resistence values and RhoA guanosine triphosphate (GTP), bax, zonula occludens-1 and occludin protein expression, respectively. All these effects were significantly and concentration-dependently inhibited by cannabidiol, whose effects were completely abolished in the presence of the cannabinoid receptor type 1 (CB1) antagonist, AM251. CONCLUSIONS: Cannabidiol improved Clostridium difficile toxin A-induced damage in Caco-2 cells, by inhibiting the apoptotic process and restoring the intestinal barrier integrity, through the involvement of the CB1 receptor.

HIV-1 Tat-induced diarrhea evokes an enteric glia-dependent neuroinflammatory response in the central nervous system.

Despite the effectiveness of combined anti-retroviral therapy, human immunodeficiency virus (HIV) infected-patients frequently report diarrhea and neuropsychological deficits. It is claimed that the viral HIV-1 Trans activating factor (HIV-1 Tat) protein is responsible for both diarrhea and neurotoxic effects, but the underlying mechanisms are not known. We hypothesize that colonic application of HIV-1 Tat activates glial cells of the enteric nervous system (EGCs), leading to a neuroinflammatory response able to propagate to the central nervous system. We demonstrated that HIV-1 Tat-induced diarrhea was associated with a significant activation of glial cells within the colonic wall, the spinal cord and the frontal cortex, and caused a consistent impairment of the cognitive performances. The inhibition of glial cells activity by lidocaine, completely abolished the above-described effects. These observations point out the role of glial cells as putative effectors in HIV-1 Tat-associated gastrointestinal and neurological manifestations and key regulators of gut-brain signaling.

Allele-specific transcriptional activity of the variable number of tandem repeats of the inducible nitric oxide synthase gene is associated with idiopathic achalasia.

BACKGROUND: Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory neurotransmission. OBJECTIVE: The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric oxide synthase (NOS2) gene promoter polymorphism in achalasia. METHODS: Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n. RESULTS: The alleles' distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3-0.5 and OR 1.6, 95% CI 1-2.4, all p < 0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13-2.53, p = 0.01). Transfection experiments revealed a similar allele-specific iNOS transcriptional activity. CONCLUSION: The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production.

Rifaximin Improves Clostridium difficile Toxin A-Induced Toxicity in Caco-2 Cells by the PXR-Dependent TLR4/MyD88/NF-κB Pathway.

BACKGROUND: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. AIM: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. METHODS: Caco-2 cells were incubated with TcdA and treated with rifaximin (0.1-10 µM) with or without ketoconazole (10 µM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens-1 (ZO-1), toll-like receptor 4 (TLR4), Bcl-2-associated X protein (Bax), transforming growth factor-ß-activated kinase-1 (TAK1), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-κB) was determined. RESULTS: Rifaximin treatment (0.1, 1.0, and 10 µM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360, 480, and 680% vs. TcdA treatment) 24 h after the treatment and improved their viability (61, 79, and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (-29, -65, and -77%) and increased the expression of ZO-1 (25, 54, and 87%) and occludin (71, 114, and 262%) versus TcdA treatment. The expression of TLR4 (-33, -50, and -75%), MyD88 (-29, -60, and -81%) and TAK1 (-37, -63, and -79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. CONCLUSION: Rifaximin improved TcdA-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway mechanism, and may be useful in the treatment of CDIs.

Rifaximin, a non-absorbable antibiotic, inhibits the release of pro-angiogenic mediators in colon cancer cells through a pregnane X receptor-dependent pathway.

Activation of intestinal human pregnane X receptor (PXR) has recently been proposed as a promising strategy for the chemoprevention of inflammation-induced colon cancer. The present study was aimed at evaluating the effect of rifaximin, a non-absorbable antibiotic, in inhibiting angiogenesis in a model of human colorectal epithelium and investigating the role of PXR in its mechanism of action. Caco-2 cells were treated with rifaximin (0.1, 1.0 and 10.0 µM) in the presence or absence of ketoconazole (10 µM) and assessed for cell proliferation, migration and expression of proliferating cell nuclear antigen (PCNA). The release of vascular endothelial growth factor (VEGF) and nitric oxide (NO), expression of Akt, mechanistic target of rapamycin (mTOR), p38 mitogen activated protein kinases (MAPK), nuclear factor κB (NF-κB) and metalloproteinase-2 and -9 (MMP-2 and -9) were also evaluated. Treatment with rifaximin 0.1, 1.0 and 10.0 µM caused significant and concentration-dependent reduction of cell proliferation, cell migration and PCNA expression in the Caco-2 cells vs. untreated cells. Treatment downregulated VEGF secretion, NO release, VEGFR-2 expression, MMP-2 and MMP-9 expression vs. untreated cells. Rifaximin treatment also resulted in a concentration-dependent decrease in the phosphorylation of Akt, mTOR, p38MAPK and inhibition of hypoxia-inducible factor 1-α (HIF-1α), p70S6K and NF-κB. Ketoconazole (PXR antagonist) treatment inhibited these effects. These findings demonstrated that rifaximin causes PXR-mediated inhibition of angiogenic factors in Caco-2 cell line and may be a promising anticancer tool.

Palmitoylethanolamide Modulates Inflammation-Associated Vascular Endothelial Growth Factor (VEGF) Signaling via the Akt/mTOR Pathway in a Selective Peroxisome Proliferator-Activated Receptor Alpha (PPAR-α)-Dependent Manner.

BACKGROUND AND AIM: Angiogenesis is emerging as a pivotal process in chronic inflammatory pathologies, promoting immune infiltration and prompting carcinogenesis. Ulcerative Colitis (UC) and Crohn's Disease (CD) represent paradigmatic examples of intestinal chronic inflammatory conditions in which the process of neovascularization correlates with the severity and progression of the diseases. Molecules able to target the angiogenesis have thus the potential to synergistically affect the disease course. Beyond its anti-inflammatory effect, palmitoylethanolamide (PEA) is able to reduce angiogenesis in several chronic inflammatory conditions, but no data about its anti-angiogenic activity in colitis have been produced, yet. METHODS: The effects of PEA on inflammation-associated angiogenesis in mice with dextran sulphate sodium (DSS)-induced colitis and in patients with UC were assessed. The release of Vascular Endothelial Growth Factor (VEGF), the hemoglobin tissue content, the expression of CD31 and of phosphatidylinositol 3-kinase/Akt/mammalian-target-of-rapamycin (mTOR) signaling axis were all evaluated in the presence of different concentrations of PEA and concomitant administration of PPAR-α and -γ antagonists. RESULTS: Our results demonstrated that PEA, in a selective peroxisome proliferator activated receptor (PPAR)-α dependent mechanism, inhibits colitis-associated angiogenesis, decreasing VEGF release and new vessels formation. Furthermore, we demonstrated that the mTOR/Akt axis regulates, at least partly, the angiogenic process in IBD and that PEA directly affects this pathway. CONCLUSIONS: Our results suggest that PEA may improve inflammation-driven angiogenesis in colonic mucosa, thus reducing the mucosal damage and potentially affecting disease progression and the shift towards the carcinogenesis.

Palmitoylethanolamide Exerts Antiproliferative Effect and Downregulates VEGF Signaling in Caco-2 Human Colon Carcinoma Cell Line Through a Selective PPAR-α-Dependent Inhibition of Akt/mTOR Pathway.

Palmitoylethanolamide (PEA) is a nutraceutical compound that has been demonstrated to improve intestinal inflammation. We aimed at evaluating its antiproliferative and antiangiogenic effects in human colon adenocarcinoma Caco-2 cell line. Caco-2 cells were treated with increasing concentrations of PEA (0.001, 0.01 and 0.1 µM) in the presence of peroxisome proliferator-activated receptor-a (PPAR-α) or PPAR-γ antagonists. Cell proliferation was evaluated by performing a MTT assay. Vascular endothelial growth factor (VEGF) release was estimated by ELISA, while the expression of VEGF receptor and the activation of the Akt/mammalian target of rapamycin (mTOR) pathway were evaluated by western blot analysis. PEA caused a significant and concentration-dependent decrease of Caco-2 cell proliferation at 48 h. PEA administration significantly reduced in a concentration-dependent manner VEGF secretion and VEGF receptor expression. Inhibition of Akt phosphorylation and a downstream decrease of phospho-mTOR and of p-p70S6K were observed as compared with untreated cells. PPAR-α, but not PPAR-γ antagonist, reverted all effects of PEA. PEA is able to decrease cell proliferation and angiogenesis. The antiangiogenic effect of PEA depends on the specific inhibition of the AkT/mTOR axis, through the activation of PPAR-α pathway. If supported by in vivo models, our data pave the way to PEA co-administration to the current chemotherapeutic regimens for colon carcinoma. Copyright © 2016 John Wiley & Sons, Ltd.

Endoscopic treatment of esophageal achalasia.

Achalasia is a motility disorder of the esophagus characterized by dysphagia, regurgitation of undigested food, chest pain, weight loss and respiratory symptoms. The most common form of achalasia is the idiopathic one. Diagnosis largely relies upon endoscopy, barium swallow study, and high resolution esophageal manometry (HRM). Barium swallow and manometry after treatment are also good predictors of success of treatment as it is the residue symptomatology. Short term improvement in the symptomatology of achalasia can be achieved with medical therapy with calcium channel blockers or endoscopic botulin toxin injection. Even though few patients can be cured with only one treatment and repeat procedure might be needed, long term relief from dysphagia can be obtained in about 90% of cases with either surgical interventions such as laparoscopic Heller myotomy or with endoscopic techniques such pneumatic dilatation or, more recently, with per-oral endoscopic myotomy. Age, sex, and manometric type by HRM are also predictors of responsiveness to treatment. Older patients, females and type II achalasia are better after treatment compared to younger patients, males and type III achalasia. Self-expandable metallic stents are an alternative in patients non responding to conventional therapies.

Eosinophilic esophagitis: From pathophysiology to treatment.

Eosinophilic esophagitis (EoE) is a chronic immune disease, characterized by a dense eosinophilic infiltrate in the esophagus, leading to bolus impaction and reflux-like symptoms. Traditionally considered a pediatric disease, the number of adult patients with EoE is continuously increasing, with a relatively higher incidence in western countries. Dysphagia and food impaction represent the main symptoms complained by patients, but gastroesophageal reflux-like symptoms may also be present. Esophageal biopsies are mandatory for the diagnosis of EoE, though clinical manifestations and proton pump inhibitors responsiveness must be taken into consideration. The higher prevalence of EoE in patients suffering from atopic diseases suggests a common background with allergy, however both the etiology and pathophysiology are not completely understood. Elimination diets are considered the first-line therapy in children, but this approach appears less effective in adults patients, who often require steroids; despite medical treatments, EoE is complicated in some cases by esophageal stricture and stenosis, that require additional endoscopic treatments. This review summarizes the evidence on EoE pathophysiology and illustrates the safety and efficacy of the most recent medical and endoscopic treatments.

S100B-p53 disengagement by pentamidine promotes apoptosis and inhibits cellular migration via aquaporin-4 and metalloproteinase-2 inhibition in C6 glioma cells.

S100 calcium-binding protein B (S100B) is highly expressed in glioma cells and promotes cancer cell survival via inhibition of the p53 protein. In melanoma cells, this S100B-p53 interaction is known to be inhibited by pentamidine isethionate, an antiprotozoal agent. Thus, the aim of the present study was to evaluate the effect of pentamidine on rat C6 glioma cell proliferation, migration and apoptosis in vitro. The change in C6 cell proliferation following treatment with pentamidine was determined by performing a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide-formazan assay. Significant dose-dependent decreases in proliferation were observed at pentamidine concentrations of 0.05 µM (58.5±5%; P<0.05), 0.5 µM (40.6±7%; P<0.01) and 5 µM (13±4%; P<0.001) compared with the control (100% viability). Furthermore, treatment with 0.05, 0.5 and 5 µM pentamidine was associated with a significant increase in apoptosis versus the untreated cells, as determined by DNA fragmentation assays, immunofluorescence analysis of C6 chromatin using Hoechst staining, and immunoblot analysis of B-cell lymphoma-2 (Bcl-2)-associated X protein (100%, P<0.05; 453%, P<0.01; and 1000%, P<0.001, respectively) and Bcl-2 (-60%, P<0.001; -80.13%, P<0.001; -95%, P<0.001, respectively). In addition, the administration of 0.05, 0.5 and 5 µM pentamidine significantly upregulated the protein expression levels of p53 (681±87.5%, P<0.05; 1244±94.3%, P<0.01; and 2244±111%, P<0.001, respectively), and significantly downregulated the expression levels of matrix metalloproteinase-2 (42±2.3%, P<0.05; 71±2.5%, P<0.01; and 95.8±3.3%, P<0.001, respectively) and aquaporin 4 (38±2.5%, P<0.05; 69±2.6%, P<0.01; and 88±3.0%, P<0.001, respectively), compared with the untreated cells. The wound healing assay demonstrated that cell migration was significantly impaired by treatment with 0.05, 0.5 and 5 µM pentamidine compared with untreated cells (88±4.2%, P<0.05; 64±2%, P<0.01; and 42±3.1%, P<0.001, respectively). Although additional in vivo studies are required to clarify the current in vitro data, the present study indicates that pentamidine and S100B-p53 inhibitors may represent a novel approach for the treatment of glioma.