Valentina Vivo

Elucidating the gut microbiome of ulcerative colitis: bifidobacteria as novel microbial biomarkers

Ulcerative colitis (UC) is associated with a substantial alteration of specific gut commensals, some of which may be involved in microbiota-mediated protection. In this study, microbiota cataloging of UC patients by 16S rRNA microbial profiling revealed a marked reduction of bifidobacteria, in particular the Bifidobacterium bifidum species, thus suggesting that this taxon plays a biological role in the aetiology of UC. We investigated this further through an in vivo trial by testing the effects of oral treatment with B. bifidum PRL2010 in a wild-type murine colitis model. TNBS-treated mice receiving 10(9) cells of B. bifidum PRL2010 showed a marked reduction of all colitis-associated histological indices as well as maintenance of mucosal integrity as it was shown by the increase in the expression of many tight junction-encoding genes. The protective role of B. bifidum PRL2010, as well as its sortase-dependent pili, appears to be established through the induction of an innate immune response of the host. These results highlight the importance of B. bifidum as a microbial biomarker for UC, revealing its role in protection against experimentally induced colitis.

Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: Synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity

In this study, we synthesized and tested in vitro and in vivo two groups of bis(ammonio)alkane-type compounds, 6a-9a and 6b-9b, which incorporate the orthosteric muscarinic agonist iperoxo into a molecular fragment of the M2-selective allosteric modulators W84 and naphmethonium. The agonist potency and efficacy of these hybrid derivatives at M1, M2 and M3 muscarinic receptor subtypes and their anticholinesterase activity were evaluated on isolated tissue preparations. Their analgesic action was then assayed in vivo in the acetic acid writhing test and the occurrence of peripheral and central cholinergic side effects was also determined. The investigated hybrids behaved as potent muscarinic agonists and weak cholinesterase inhibitors. These effects were more pronounced for bisquaternary salts bearing the naphmethonium moiety than for the W84-containing analogs, and resulted in a significant analgesic activity in vivo. A promising profile was displayed by the naphmethonium-related compound 8b, which combined the most potent antinociception among the test compounds with the absence of relevant cholinergic side effects.

Combination of Gefitinib and Pemetrexed Prevents the Acquisition of TKI Resistance in NSCLC Cell Lines Carrying EGFR-Activating Mutation

Introduction: Development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors is a clinical issue in patients with epidermal growth factor receptor gene (EGFR)‐mutated non–small cell lung cancer (NSCLC). The aim of this study was to investigate the potential of combining gefitinib and pemetrexed in preventing the acquisition of resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines harboring EGFR exon 19 deletion. Methods: The effect of different combinatorial schedules of gefitinib and pemetrexed on cell proliferation, cell cycle, apoptosis, and acquisition of gefitinib resistance in PC9 and HCC827 NSCLC cell lines and in PC9 xenograft models was investigated. Results: Simultaneous treatment with gefitinib and pemetrexed enhanced cell growth inhibition and cell death and prevented the appearance of gefitinib resistance mediated by T790M mutation or epithelial‐to‐mesenchymal transition (EMT) in PC9 and HCC827 cells, respectively. In PC9 cells and in PC9 xenografts the combination of gefitinib and pemetrexed, with different schedules, prevented gefitinib resistance only when pemetrexed was the first treatment, given alone or together with gefitinib. Conversely, when gefitinib alone was administered first and pemetrexed sequentially alternated, a negative interaction was observed and no prevention of gefitinib resistance was documented. The mechanisms of resistance that developed in vivo included T790M mutation and EMT. The induction of EMT was a feature of tumors treated with gefitinib when given before pemetrexed, whereas T790M was recorded only in tumors treated with gefitinib alone. Conclusions: The combination of gefitinib and pemetrexed is effective in preventing gefitinib resistance; the application of intermittent treatments requires that gefitinib not be administered before pemetrexed.

Suppression of inflammatory events associated to intestinal ischemia–reperfusion by 5-HT1A blockade in mice

Intestinal ischemia and reperfusion (I/R) is a potentially life-threatening disease, ensuing from various clinical conditions. Experimentally, either protective or detrimental roles have been attributed to 5-HT in the functional and morphological injury caused by mesenteric I/R. Recently, we proved the involvement of 5-HT2A receptors in the intestinal dysmotility and leukocyte recruitment induced by 45min occlusion of the superior mesenteric artery (SMA) followed by 24h reperfusion in mice. Starting from these premises, the aim of our present work was to investigate the role played by endogenous 5-HT in the same experimental model where 45min SMA clamping was followed by 5h reflow. To this end, we first observed that ischemic preconditioning before I/R injury (IPC+I/R) reverted the increase in 5-HT tissue content and in inflammatory parameters induced by I/R in mice. Second, the effects produced by intravenous administration of 5-HT1A ligands (partial agonist buspirone 10mgkg(-1), antagonist WAY100135 0.5-5mgkg(-1)), 5-HT2A antagonist sarpogrelate (10mgkg(-1)), 5-HT3 antagonist alosetron (0.1mgkg(-1)), 5-HT4 antagonist GR125487 (5mgkg(-1)) and 5-HT re-uptake inhibitor fluoxetine (10mgkg(-1)) on I/R-induced inflammatory response were investigated in I/R mice and compared to those obtained in sham-operated animals (S). Our results confirmed the significant role played by 5-HT2A receptors not only in the late but also in the early I/R-induced microcirculatory dysfunction and showed that blockade of 5-HT1A receptors protected against the intestinal leukocyte recruitment, plasma extravasation and reactive oxygen species formation triggered by SMA occlusion and reflow. The ability of α7 nicotinic receptor (α7nAchR) antagonist methyllycaconitine (5mgkg(-1)) to counteract the beneficial action provided by buspirone on I/R-induced neutrophil infiltration suggests that the anti-inflammatory effect produced by 5-HT1A receptor antagonism could be partly ascribed to the indirect activation of α7nAch receptors.

Amino Acid Derivatives as Palmitoylethanolamide Prodrugs: Synthesis, In Vitro Metabolism and In Vivo Plasma Profile in Rats

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization.

Enhancement of the anti-tumor activity of FGFR1 inhibition in squamous cell lung cancer by targeting downstream signaling involved in glucose metabolism

Fibroblast Growth Factor Receptor (FGFR) signaling is a complex pathway which controls several processes, including cell proliferation, survival, migration, and metabolism. FGFR1 signaling is frequently deregulated via amplification/over-expression in NSCLC of squamous histotype (SQCLC), however its inhibition has not been successfully translated in clinical setting. We determined whether targeting downstream signaling implicated in FGFR1 effects on glucose metabolism potentiates the anti-tumor activity of FGFR1 inhibition in SQCLC. In FGFR1 amplified/over-expressing SQCLC cell lines, FGF2-mediated stimulation of FGFR1 under serum-deprivation activated both MAPK and AKT/mTOR pathways and increased glucose uptake, glycolysis, and lactate production, through AKT/mTOR-dependent HIF-1α accumulation and up-regulation of GLUT-1 glucose transporter. These effects were hindered by PD173074 and NVP-BGJ398, selective FGFR inhibitors, as well as by dovitinib, a multi-kinase inhibitor. Glucose metabolism was hampered by the FGFR inhibitors also under hypoxic conditions, with consequent inhibition of cell proliferation and viability. In presence of serum, glucose metabolism was impaired only in cell models in which FGFR1 inhibition was associated with AKT/mTOR down-regulation. When the activation of the AKT/mTOR pathway persisted despite FGFR1 down-regulation, the efficacy of NVP-BGJ398 could be significantly improved by the combination with NVP-BEZ235 or other inhibitors of this signaling cascade, both in vitro and in xenotransplanted nude mice. Collectively our results indicate that inhibition of FGFR1 signaling impacts on cancer cell growth also by affecting glucose energy metabolism. In addition, this study strongly suggests that the therapeutic efficacy of FGFR1 targeting molecules in SQCLC may be implemented by combined treatments tackling on glucose metabolism.Fibroblast Growth Factor Receptor (FGFR) signaling is a complex pathway which controls several processes, including cell proliferation, survival, migration, and metabolism. FGFR1 signaling is frequently deregulated via amplification/over-expression in NSCLC of squamous histotype (SQCLC), however its inhibition has not been successfully translated in clinical setting. We determined whether targeting downstream signaling implicated in FGFR1 effects on glucose metabolism potentiates the anti-tumor activity of FGFR1 inhibition in SQCLC. In FGFR1 amplified/over-expressing SQCLC cell lines, FGF2-mediated stimulation of FGFR1 under serum-deprivation activated both MAPK and AKT/mTOR pathways and increased glucose uptake, glycolysis, and lactate production, through AKT/mTOR-dependent HIF-1α accumulation and up-regulation of GLUT-1 glucose transporter. These effects were hindered by PD173074 and NVP-BGJ398, selective FGFR inhibitors, as well as by dovitinib, a multi-kinase inhibitor. Glucose metabolism was hampered by the FGFR inhibitors also under hypoxic conditions, with consequent inhibition of cell proliferation and viability. In presence of serum, glucose metabolism was impaired only in cell models in which FGFR1 inhibition was associated with AKT/mTOR down-regulation. When the activation of the AKT/mTOR pathway persisted despite FGFR1 down-regulation, the efficacy of NVP-BGJ398 could be significantly improved by the combination with NVP-BEZ235 or other inhibitors of this signaling cascade, both in vitro and in xenotransplanted nude mice. Collectively our results indicate that inhibition of FGFR1 signaling impacts on cancer cell growth also by affecting glucose energy metabolism. In addition, this study strongly suggests that the therapeutic efficacy of FGFR1 targeting molecules in SQCLC may be implemented by combined treatments tackling on glucose metabolism.

Esomeprazole immediate release tablets: Gastric mucosa ex vivo permeation, absorption and antisecretory activity in conscious rats.

The aim of this work was to study the esomeprazole activity on the control of gastric secretion after administration of a novel immediate release tablet. The ex vivo permeation of esomeprazole across porcine gastric mucosa from immediate release tablets, containing sodium carbonate or magnesium oxide as alkalinizing agents, was firstly assessed. Pharmacokinetics and pharmacodynamics studies in conscious rats following the administration of immediate release tablets with sodium carbonate, in comparison with delayed-release tablets having the same formula, were also conducted. The results showed an important effect of sodium carbonate and magnesium oxide on the drug release, on the ex vivo trans-mucosal transport and the stability in acid environment. In particular, the presence of sodium carbonate in esomeprazole tablet formulation provided the maximum increase of the drug in vitro transport across the mucosa. Then, the absorption and the antisecretory activity of this proton pump inhibitor orally administered in rats as immediate release tablets containing Na2CO3, was superior but not significantly different compared to delayed-release tablets having the same formula. In the adopted animal model, an activity of esomeprazole from immediate release alkaline formulation was seen also in presence of partial gastric absorption allowing inhibition of proton pumps reached via systemic circulation. This esomeprazole immediate release formulation could be used for the on-demand treatment of acid-related disorders such as gastro-esophageal reflux disease.

Dual Role of Endogenous Serotonin in 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis

Background and Aims: Changes in gut serotonin (5-HT) content have been described in Inflammatory Bowel Disease (IBD) and in different experimental models of colitis: the critical role of this monoamine in the pathogenesis of chronic gastrointestinal inflammation is gradually emerging. Aim of the present study was to evaluate the contribution of endogenous 5-HT through the activation of its specific receptor subtypes to the local and systemic inflammatory responses in an experimental model of IBD. Materials and Methods: Colitis was induced by intrarectal 2,4,6-TriNitroBenzene Sulfonic acid in mice subacutely treated with selective antagonists of 5-HT1A (WAY100135), 5-HT2A (Ketanserin), 5-HT3 (Ondansetron), 5-HT4 (GR125487), 5-HT7 (SB269970) receptors and with 5-HT1A agonist 8-Hydroxy-2-(di-n-propylamino)tetralin. Results: Blockade of 5-HT1A receptors worsened TNBS-induced local and systemic neutrophil recruitment while 5-HT1A agonist delayed and mitigated the severity of colitis, counteracting the increase in colonic 5-HT content. On the contrary, blockade of 5-HT2A receptors improved global health conditions, reduced colonic morphological alterations, down-regulated neutrophil recruitment, inflammatory cytokines levels and colonic apoptosis. Antagonism of 5-HT3, 5-HT4, and 5-HT7 receptor sites did not remarkably affect the progression and outcome of the pathology or only slightly improved it. Conclusion: The prevailing deleterious contribution given by endogenous 5-HT to inflammation in TNBS-induced colitis is seemingly mediated by 5-HT2A and, to a lesser extent, by 5-HT4 receptors and coexists with the weak beneficial effect elicited by 5-HT1A stimulation. These findings suggest how only a selective interference with 5-HT pro-inflammatory actions may represent an additional potential therapeutic option for intestinal inflammatory disorders.

Hake fish bone as a calcium source for efficient bone mineralization.

Abstract Calcium is recognized as an essential nutritional factor for bone health. An adequate intake is important to achieve or maintain optimal bone mass in particular during growth and old age. The aim of the present study was to evaluate the efficiency of hake fish bone (HBF) as a calcium source for bone mineralization: in vitro on osteosarcoma SaOS-2 cells, cultured in Ca-free osteogenic medium (OM) and in vivo on young growing rats fed a low-calcium diet. Lithotame (L), a Ca supplement derived from Lithothamnium calcareum, was used as control. In vitro experiments showed that HBF supplementation provided bone mineralization similar to standard OM, whereas L supplementation showed lower activity. In vivo low-Ca HBF-added and L-added diet similarly affected bone deposition. Physico-chemical parameters concerning bone mineralization, such as femur breaking force, tibia density and calcium/phosphorus mineral content, had beneficial effects from both Ca supplementations, in the absence of any evident adverse effect. We conclude HBF derived from by-product from the fish industry is a good calcium supplier with comparable efficacy to L.

Bifidobacterium bifidum PRL2010 alleviates intestinal ischemia/reperfusion injury

Mesenteric ischemia/reperfusion is a clinical emergency with high morbidity and mortality due to the transient reduction of blood supply to the bowel. In recent years, the critical contribution of gut microbiome to human health and proper gastrointestinal functions has gradually emerged. In the current study, we investigated the protective effects of five days supplementation with Bifidobacterium bifidum PRL2010 in a murine model of gut ischemia/reperfusion. Our findings indicate that animals pretreated with B. bifidum PRL2010 showed lower neutrophil recruitment in the lungs, remarkably reduced bacterial translocation and decreased transcription levels of TNFalpha and IL-10 both in liver and kidneys, at the same time increasing those of IL-12 in kidneys. Inhibiting the adhesion of pathogenic bacteria and boosting host innate immunity responses are among the possible protective mechanisms enacted by the probiotic. These results demonstrate that short-period treatment with B. bifidum PRL2010 is a potential strategy to dampen remote organ injury due to mesenteric ischemia/reperfusion.