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Table S1. Characteristics of the healthy subjects (HS) and patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC)

The bioimaging of cancer cells by the specific targeting of overexpressed biomarkers is an approach that holds great promise in the identification of selective diagnostic tools. Tumor-associated human carbonic anhydrase (hCA) isoforms IX and XII have been considered so far as well-defined biomarkers, with their expression correlating with cancer progression and aggressiveness. Therefore, the availability of highly performant fluorescent tools tailored for their targeting and able to efficiently visualize such key targets is in high demand. We report here on the design and synthesis of a kind of quantum dot (QD)-based fluorescent glyconanoprobe coated with a binary mixture of ligands, which, according to the structure of the terminal domains, impart specific property sets to the fluorescent probe. Specifically, monosaccharide residues ensured the dispersibility in the biological medium, CA inhibitor residues provided specific targeting of membrane-anchored hCA IX overexpressed on bladder cancer cells, and the quantum dots imparted the optical/fluorescence properties.

Purpose Low free testosterone (T) level in men is independently associated with presence and severity of Non-Alcoholic Steatohepatitis (NASH). The histological and molecular effects of oral testosterone prodrug LPCN 1144 treatment on hepatic fibrosis and NASH features are unknown. A metabolic syndrome-induced NASH model in rabbits consuming high fat diet (HFD) has been previously used to assess treatment effects of injectable T on hepatic fibrosis and NASH features. Here we present results on LPCN 1144 in this HFD-induced, NASH preclinical model. Methods Male rabbits were randomly assigned to five groups: regular diet (RD), HFD, HFD + 1144 vehicle (HFD + Veh), HFD + 1144 (1144), and HFD + 1144 + α-tocopherol (1144 + ALPHA). Rabbits were sacrificed after 12 weeks for liver histological, biochemical and genetic analyses. Histological scores were obtained through Giemsa (inflammation), Masson’s trichrome (steatosis and ballooning), and Picrosirius Red (fibrosis) staining. Results Compared to RD, HFD and HFD + Veh significantly worsened NASH features and hepatic fibrosis. Considering HFD and HFD + Veh arms, histological and biomarker features were not significantly different. Both 1144 and 1144 + ALPHA arms improved mean histological scores of NASH as compared to HFD arm. Importantly, percentage of fibrosis was improved in both 1144 (p p = 0.05) treatment arms vs. HFD. Both treatment arms also reduced HFD-induced inflammation and fibrosis mRNA markers. Furthermore, 1144 treatments significantly improved HFD-induced metabolic dysfunctions. Conclusions Histological and biomarker analyses demonstrate that LPCN 1144 improved HFD-induced hepatic fibrosis and NASH biochemical, biomolecular and histochemical features. These preclinical findings support a therapeutic potential of LPCN 1144 in the treatment of NASH and of hepatic fibrosis.

The expression of the nuclear receptor transcription factor (TF) COUP‑TFII is broadly associated with cell differentiation and cancer development, including of pancreatic ductal adenocarcinoma (PDAC), a devastating disease with one of the poorest prognoses among cancers worldwide. Recent studies have started to investigate the pathological and physiological roles of a novel COUP‑TFII isoform (COUP‑TFII_V2) that lacks the DNA‑binding domain. As the role of the canonical COUP‑TFII in PDAC was previously demonstrated, the present study evaluated whether COUP‑TFII_V2 may have a functional role in PDAC. It was demonstrated that COUP‑TFII_V2 naturally occurs in PDAC cells and in primary samples, where its expression is consistent with shorter overall survival and peripheral invasion. Of note, COUP‑TFII_V2, exhibiting nuclear and cytosolic expression, is linked to epithelial to mesenchymal transition (EMT) and cancer progression, as confirmed by nude mouse experiments. The present results demonstrated that COUP‑TFII_V2 distinctively regulates the EMT of PDAC and, similarly to its sibling, it is associated with tumor aggressiveness. The two isoforms have both overlapping and exclusive functions that cooperate with cancer growth and dissemination. By studying how PDAC cells switch from one isoform to the other, novel insight into cancer biology was gained, indicating that this receptor may serve as a novel possible target for PDAC management.

Germline mutations in more than 20 genes, including those encoding for the succinate dehydrogenase (SDH), predispose to rare tumours, such as pheochromocytoma/paraganglioma (PPGL). Despite encoding for the same enzymatic complex, SDHC and SDHD mutated PHEO/PGLs are generally benign, while up to 80% of SDHB mutated ones are malignant. In this study, we evaluated the different effects of tumour microenvironment on tumour cell migration/invasion, by co-culturing SDHB or SDHD silenced tumour spheroids with primary cancer-associated fibroblasts (CAFs). We observed that SDHD silenced spheroids had an intermediate migration pattern, compared to the highest migration capability of SDHB and the lowest one of the wild type (Wt) spheroids. Interestingly, we noticed that co-culturing Wt, SDHB and SDHD silenced spheroids with CAFs in low glucose (1 g/l) medium, caused a decreased migration of all the spheroids, but only for SDHB silenced ones this reduction was significant. Moreover, the collective migration, observed in high glucose (4.5 g/l) and characteristic of the SDHB silenced cells, was completely lost in low glucose. Importantly, migration could not be recovered even adding glucose (3.5 g/l) to low glucose conditioned medium. When we investigated cell metabolism, we found that low glucose concentration led to a reduction of oxygen consumption rate (OCR), basal and maximal oxidative metabolism, and ATP production only in CAFs, but not in tumour cells. These results suggest that CAFs metabolism impairment was responsible for the decreased invasion process of tumour cells, most likely preventing the release of the pro-migratory factors produced by CAFs. In conclusion, the interplay between CAFs and tumour cells is distinctive depending on the gene involved, and highlights the possibility to inhibit CAF-induced migration by impairing CAFs metabolism, indicating new potential therapeutic scenarios for medical therapy.

Chemotherapy-induced neuropathic pain is a clinically relevant adverse effect of several anticancer drugs leading to dose reduction or therapy discontinuation. The lack of knowledge about the mechanisms of neuropathy development and pain chronicization makes chemotherapy-induced neuropathic pain treatment an unmet medical need. In this context, the vascular endothelial growth factor A (VEGF-A) has emerged as a neurotoxicity biomarker in a model of chemotherapy-induced neuropathy, and its decrease has been related to pain relief. Aim of this study was to clarify the VEGF-A-dependent pain signaling in the CNS for individuating new targeted therapeutic approaches. In mice, the intrathecal infusion of VEGF-A induced a dose-dependent noxious hypersensitivity mediated by the VEGF receptor 1 (VEGFR-1) as demonstrated by pharmacological and genetic tools. In electrophysiological study, VEGF-A stimulated the spinal nociceptive neurons activity through VEGFR-1. In the dorsal horn of the spinal cord, VEGF-A increased in astrocytes of animals affected by neuropathy suggesting this cell population as a source of the potent pain mediator. Accordingly, the selective knockdown of astrocytic VEGF-A, by shRNAmir, blocked the development of oxaliplatin-induced neuropathic pain. Besides, the anti-VEGFR-1 mAb D16F7 (previously described as anticancer) effectively relieved neuropathic pain induced by chemotherapeutic agents. In conclusion, astrocyte-released VEGF-A is a new player in the complex neuron-glia network that oversees physiological and pathological pain and D16F7 mAb rises as a potent pain killer strategy.

Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.

Hybrid materials composed of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) and lipid self-assemblies possess a considerable applicative potential in the biomedical field, specifically, for drug/nutrients delivery. In recent works we have shown that SPION-doped lipid cubic liquid crystals undergo a cubic-to-hexagonal phase transition under the action of temperature or of an alternating magnetic fields (AMF). This transition triggers the release of drugs embedded in the lipid scaffold or in the water channels. In this contribution, we address this phenomenon in depth, to fully elucidate the structural details and optimize the design of hybrid multifunctional carriers for drug delivery. Combining Small-Angle X-Ray Scattering (SAXS) with a magnetic characteri-zation, we find that in bulk lipid cubic phases the cubic-to-hexagonal transition determines the magnetic response of SPIONs. We then extend the investigation from bulk liquid-crystalline phases to colloidal dispersions, i.e. to lipid/SPIONs nanoparticles with cubic internal structure (“magnetocubosomes”). Through Synchrotron SAXS, we monitored the structural response of magnetocubosomes while exposed to an AMF: the magnetic energy, converted into heat by SPIONs, activates the cubic-to-hexagonal transition, and can thus be used as a remote stimulus to spike drug release “on-demand”. In addition, we show that the AMF-induced phase transition in magnetocubosomes steers the re-alignment of SPIONs into linear string assemblies and connect this effect with the change in their magnetic properties, observed at the bulk level. Finally, we assess the internalization ability and cytotoxicity of magnetocubosomes in vitro on HT29 ade-nocarcinoma cancer cells, in order to test the applicability of these smart carriers in drug delivery applications

Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. Significance: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma.

Cancer-associated fibroblasts (CAFs) are one of the main components of the stromal compartment in the tumor microenvironment (TME) and the crosstalk between CAFs and cancer cells is essential for tumor progression and aggressiveness. Cancer cells mediate an activation process, converting normal fibroblasts into CAFs, that are characterized by modified expression of many proteins and increased production and release of microvesicles (MVs), extracellular vesicles generated by outwards budding from the cell membrane. Recent evidence underlined that the uptake of CAF-derived MVs changes the overall protein content of tumor cells. In this paper, we demonstrate that tumor activated fibroblasts overexpress Galectin-1 (Gal-1) and consequently release MVs containing increased levels of this protein. The uptake of Gal-1 enriched MVs by tumor cells leads to the upregulation of its intracellular concentration, that strongly affects cancer cell migration, while neither proliferation nor adhesion are altered. Accordingly, tumor cells co-cultured with fibroblasts silenced for Gal-1 have a reduced migratory ability. The present work reveals the key role of an exogenous protein, Gal-1, derived from activated fibroblasts, in cancer progression, and contributes to clarify the importance of MVs-mediated protein trafficking in regulating tumor-stroma crosstalk.

Aims Obesity represents a global health problem. Excessive caloric intake promotes the release of inflammatory mediators by hypertrophic adipocytes and obesity-induced inflammation is now recognized as a risk factor for the development of several diseases, such as cardiovascular diseases, insulin resistance, type-II diabetes, liver steatosis and cancer. Since obesity causes inflammation, we tested the ability of acetylsalicylic acid (ASA), a potent anti-inflammatory drug, in counteracting this inflammatory process and in mitigating obesity-associated health complications. Main Methods Mice were fed with standard (SD) or high fat diet (HFD) for 3 months and then treated with acetylsalicylic acid for the subsequent two months. We then analyzed the metabolic and inflammatory status of their adipose and liver tissue by histological, molecular and biochemical analysis. Key findings Although ASA did not exert any effect on body weight, quantification of adipocyte size revealed that the drug slightly reduced adipocyte hypertrophy, however not sufficient so as to induce weight loss. Most importantly, ASA was able to improve insulin resistance. Gene expression profiles of pro- and anti-inflammatory cytokines as well as the expression of macrophage and lymphocyte markers revealed that HFD led to a marked macrophage accumulation in the adipose tissue and an increase of several pro-inflammatory cytokines, a situation almost completely reverted after ASA administration. In addition, liver steatosis caused by HFD was completely abrogated by ASA treatment. Significance ASA can efficiently ameliorate pathological conditions usually associated with obesity by inhibiting the inflammatory process occurring in the adipose tissue.

Purpose In both preclinical and clinical settings, testosterone treatment (TTh) of hypogonadism has shown beneficial effects on insulin sensitivity and visceral and liver fat accumulation. This prospective, observational study was aimed at assessing the change in markers of fat and liver functioning in obese men scheduled for bariatric surgery. Methods Hypogonadal patients with consistent symptoms (n = 15) undergoing 27.63 ± 3.64 weeks of TTh were compared to untreated eugonadal (n = 17) or asymptomatic hypogonadal (n = 46) men. A cross-sectional analysis among the different groups was also performed, especially for data derived from liver and fat biopsies. Preadipocytes isolated from adipose tissue biopsies were used to evaluate insulin sensitivity, adipogenic potential and mitochondrial function. NAFLD was evaluated by triglyceride assay and by calculating NAFLD activity score in liver biopsies. Results In TTh-hypogonadal men, histopathological NAFLD activity and steatosis scores, as well as liver triglyceride content were lower than in untreated-hypogonadal men and comparable to eugonadal ones. TTh was also associated with a favorable hepatic expression of lipid handling-related genes. In visceral adipose tissue and preadipocytes, TTh was associated with an increased expression of lipid catabolism and mitochondrial bio-functionality markers. Preadipocytes from TTh men also exhibited a healthier morpho-functional phenotype of mitochondria and higher insulin-sensitivity compared to untreated-hypogonadal ones. Conclusions The present data suggest that TTh in severely obese, hypogonadal individuals induces metabolically healthier preadipocytes, improving insulin sensitivity, mitochondrial functioning and lipid handling. A potentially protective role for testosterone on the progression of NAFLD, improving hepatic steatosis and reducing intrahepatic triglyceride content, was also envisaged. Clinical Trial Registration ClinicalTrials.gov Identifier: NCT02248467, September 25th 2014

It has been well established, particularly in animal models, that oestrogens exert neuroprotective effects in brain areas linked to cognitive processes. A key protective role could reside in the capacity of oestrogen to modulate the inflammatory response. However, the direct neuroprotective actions of oestrogens on neurones are complex and remain to be fully clarified. In the present study, we took advantage of a previously characterised primary culture of human cholinergic neurones (hfNBM) from the foetal nucleus basalis of Meynert, which is known to regulate hippocampal and neocortical learning and memory circuits, aiming to investigate the direct effects of oestrogens under inflammatory conditions. Exposure of cells to tumour necrosis factor (TNF)α (10 ng mL-1 ) determined the activation of an inflammatory response, as demonstrated by nuclear factor-kappa B p65 nuclear translocation and cyclooxygenase-2 mRNA expression. These effects were inhibited by treatment with either 17β-oestradiol (E2 ) (10 nmol L-1 ) or G1 (100 nmol L-1 ), the selective agonist of the G protein-coupled oestrogen receptor (GPER1). Interestingly, the GPER1 antagonist G15 abolished the effects of E2 in TNFα-treated cells, whereas the ERα/ERβ inhibitor tamoxifen did not. Electrophysiological measurements in hfNBMs revealed a depolarising effect caused by E2 that was specifically blocked by tamoxifen and not by G15. Conversely, G1 specifically hyperpolarised the cell membrane and also increased both inward and outward currents elicited by a depolarising stimulus, suggesting a modulatory action on hfNBM excitability by GPER1 activation. Interestingly, pretreating cells with TNFα completely blocked the effects of G1 on membrane properties and also significantly reduced GPER1 mRNA expression. In addition, we found a peculiar subcellular localisation of GPER1 to focal adhesion sites that implicates new possible mechanisms of action of GPER1 in the neuronal perception of mechanical stimuli. The results obtained in the present study indicate a modulatory functional role of GPER1 with respect to mediating the oestrogen neuroprotective effect against inflammation in brain cholinergic neurones and, accordingly, may help to identify protective strategies for preventing cognitive impairments.

Azathioprine is a cornerstone of the therapy of Crohn’s disease. Unfortunately, infections and malignancies are relatively common adverse effects related to this drug; however, cirrhosis is exceptionally reported as a side effect. We report the case of a 49-year-old male patient with ileocolonic steno-penetrating Crohn’s disease who developed hepatic cirrhosis while treated with azathioprine. After taking azathioprine for 3 years with regular follow-up, he developed pancytopenia, and liver cirrhosis was diagnosed with ultrasound, abdomen computed tomography scan, transient elastography, and liver biopsy. As all other causes of liver damage were excluded, azathioprine was believed to be the cause of liver injury and therefore was interrupted.

Stevens–Johnson syndrome (SJS) is a severe mucocutaneous adverse drug reaction with a relatively high mortality rate. SJS is described during herpes simplex virus type 1 (HSV1) infection and, rarely, even during adalimumab therapy. We report the case of a patient with Crohn’s disease who developed SJS during an HSV1 infection and a contemporaneous anti-TNFα therapy with adalimumab. Remission was achieved with suspension of adalimumab and high doses of intravenous steroids and antivirals. Patients with HSV1 infection and on adalimumab therapy have a combined risk of SJS and should be monitored closely.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

Summary Background Capsule endoscopy has been considered the first-line approach for the investigation of obscure gastro-intestinal bleeding since its approval in 2001. Our study aims to evaluate the diagnostic yield of capsule endoscopy in the investigation of this condition. We also analyse the incidence of non-small-bowel lesions missed after conventional endoscopy and later detected by capsule endoscopy in patients with suspected obscure bleeding. Methods A total of 290 patients with negative conventional endoscopy referred to our centre to undergo a capsule endoscopy examination for the investigation of obscure gastro-intestinal bleeding. We considered as non-small-bowel lesions those outside the tract between the second duodenal portion and the ileocecal valve. We also looked for actively bleeding lesions at the time of the exam. Results Intestinal preparation was good, adequate or poor in 74.1%, 8.4%, and 17.5% of the tests, respectively. Caecum was reached in 92.4%. Capsule retention occurred in 0.7%. Mean small bowel transit time was 5 hours and 13 minutes. Diagnostic yield was 73.8%. An actively bleeding lesion was noticed in 39.3% of positive tests. Capsule endoscopy revealed clinically significant non-small-bowel lesions missed at gastroscopy or colonoscopy in 30.3% of patients, 43.2% of which were bleeding. Conclusions Capsule endoscopy has high diagnostic yield and safety in the investigation of obscure gastro-intestinal bleedings. Given the high percentage of non-small-bowel lesions detected, it may be appropriate to consider an endoscopic second look before performing a capsule endoscopy study.

Development of metabolically healthy adipocytes within dysfunctional adipose tissue may represent an attractive way to counteract metabolic syndrome (MetS). In an experimental animal model of high fat diet (HFD)-induced MetS, in vivo, long- and short-term tadalafil treatments were able to reduce visceral adipose tissue (VAT) accumulation and hypertriglyceridemia, and to induce the expression in VAT of the brown fat-specific marker, uncoupling protein 1 (UCP1). VAT preadipocytes (PAD), isolated from the tadalafil-treated HFD rabbits, showed: i) a multilocular morphology; ii) an increased expression of brown fat-specific genes (such as UCP1 and CIDEA); iii) improved mitochondrial structure and dynamic and reduced superoxide production; iv) improved insulin sensitivity. Similar effects were obtained after in vitro tadalafil treatment in HFD rPAD. In conclusion, tadalafil counteracted HFD-associated VAT alterations, by restoring insulin-sensitivity and prompting preadipocytes differentiation towards a metabolically healthy phenotype.

Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte’s organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte.

Humans are continually exposed to a large number of environmental carcinogens [...]

Cadmium (Cd) is a highly toxic environmental pollutant released from the smelting and refining of metals and cigarette smoking. Oral exposure to cadmium may result in adverse effects on a number of tissues, including the central nervous system (CNS). In fact, its toxicity has been related to neurological disorders, as well as neurodegenerative diseases such as Alzheimer&rsquo, s and Parkinson&rsquo, s diseases. Under normal conditions, Cd barely reaches the brain in adults because of the presence of the blood&ndash, brain barrier (BBB), however, it has been demonstrated that Cd-dependent BBB alteration contributes to pathogenesis of neurodegeneration. However, the mechanism underlying Cd-dependent BBB alteration remain obscure. Here, we investigated the signaling pathway of Cd-induced tight junction (TJ), F-actin, and vimentin protein disassembly in a rat brain endothelial cell line (RBE4). RBE4 cells treated with 10 &mu, M cadmium chloride (CdCl2) showed a dose- and time-dependent significant increase in reactive oxygen species (ROS) production. This phenomenon was coincident with the alteration of the TJ zonula occludens-1 (ZO-1), F-actin, and vimentin proteins. The Cd-dependent ROS increase elicited the upregulation of GRP78 expression levels, a chaperone involved in endoplasmic reticulum (ER) stress that induces caspase-3 activation. Further signal profiling by the pannexin-1 (PANX1) specific inhibitor 10Panx revealed a PANX1-independent increase in ATP spillage in Cd-treated endothelial cells. Our results point out that a ROS-dependent ER stress-mediated signaling pathway involving caspase-3 activation and ATP release is behind the BBB morphological alterations induced by Cd.

The bile acid receptors, farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5), regulate multiple pathways, including glucose and lipid metabolism. In a rabbit model of high-fat diet (HFD)-induced metabolic syndrome, long-term treatment with the dual FXR/TGR5 agonist INT-767 reduces visceral adipose tissue accumulation, hypercholesterolemia and nonalcoholic steatohepatitis. INT-767 significantly improves the hallmarks of insulin resistance in visceral adipose tissue (VAT) and induces mitochondrial and brown fat-specific markers. VAT preadipocytes isolated from INT-767-treated rabbits, compared to preadipocytes from HFD, show increased mRNA expression of brown adipogenesis markers. In addition, INT-767 induces improved mitochondrial ultrastructure and dynamic, reduced superoxide production and improved insulin signaling and lipid handling in preadipocytes. Bothin vivoandin vitrotreatments with INT-767 counteract, in preadipocytes, the HFD-induced alterations by upregulating genes related to mitochondrial biogenesis and function. In preadipocytes, INT-767 behaves mainly as a TGR5 agonist, directly activating dose dependently the cAMP/PKA pathway. However,in vitroexperiments also suggest that FXR activation by INT-767 contributes to the insulin signaling improvement. INT-767 treatment counteracts HFD-induced liver histological alterations and normalizes the increased pro-inflammatory genes. INT-767 also induces a significant reduction of fatty acid synthesis and fibrosis markers, while increasing lipid handling, insulin signaling and mitochondrial markers. In conclusion, INT-767 significantly counteracts HFD-induced liver and fat alterations, restoring insulin sensitivity and prompting preadipocytes differentiation toward a metabolically healthy phenotype.

Morphine-induced hyperalgesia is a pharmacological phenomenon often hindering its prolonged applications in the clinic. It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. Extracellular signal-regulated kinase (ERK) pathway contributes to pain sensitization, and its phosphorylation under pain conditions results in the induction and maintenance of pain hypersensitivity. The present study was designed to determine whether low dose morphine treatment in mice could influence the spinal activity of ERK. The data showed that morphine (1 µg/kg) induced a marked increase in ERK phosphorylation. Intrathecal pre-treatment with a selective mitogen-activated and extracellular signal-regulated kinase (MEK) inhibitor PD98059, attenuated morphine-associated thermal hyperalgesia. Morphine exposure increased phosphorylation of c-JUN, that was prevented by the inhibition of ERK pathway. In addition, double immunofluorescence studies revealed that, p-ERK and p-c-JUN are localized on neurons of the spinal dorsal horn expressing µ receptors. These data suggest that ERK contributes to the morphine-induced hyperalgesia by regulating the activation of c-JUN.

Nucleoside reverse transcriptase inhibitors (NRTIs) are known to produce painful neuropathies and to enhance states of pain hypersensitivity produced by HIV-1 infection in patients with AIDS leading to discontinuation of antiretroviral therapy, thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current study, we tested the hypothesis that HuD, an RNA binding protein known to be an essential promoter of neuronal differentiation and survival, might be involved in the response to NRTI-induced neuropathy. Antiretroviral neuropathy was induced by a single intraperitoneal administration of 2′,3′-dideoxycytidine (ddC) in mice. HuD was physiologically expressed in the cytoplasm of the soma and in axons of neurons within DRG and spinal cord and was considerably overexpressed following ddC treatment. ddC up-regulated spinal GAP43 protein, a marker of neuroregeneration, and this increase was counteracted by HuD silencing. GAP43 and HuD colocalize in DRG and spinal dorsal horn (SDH) axons and administration of an anti-GAP43 antibody aggravated the ddC-induced axonal damage. The administration of a protein kinase C (PKC) inhibitor or the PKCγ silencing prevented both HuD and GAP43 increased expression. Conversely, treatment with the PKC activator PDBu potentiated HuD and GAP43 overexpression, demonstrating the presence of a spinal PKC-dependent HuD–GAP43 pathway activated by ddC. These results indicated that HuD recruitment and GAP43 protein increase are mechanistically linked events involved in the response to antiretroviral-induced neurodegenerative processes.

Pheochromocytomas (Pheos) and paragangliomas (PGLs) are neuroendocrine tumors. Approximately 30–40% of Pheos/PGLs are due to germline mutations in one of the susceptibility genes, including those encoding the succinate dehydrogenase subunits A-D (SDHA-D). Up to 2/3 of patients affected bySDHBmutated Pheo/PGL develop metastatic disease with no successful cure at present. Here, for the first time, we evaluated the effects ofSDHBsilencing in a three dimension (3D) culture using spheroids of a mouse Pheo cell line silenced or not (wild type/wt/control) for the SDHB subunit. We investigated the role of the microenvironment on spheroid growth and migration/invasion by co-culturingSDHB-silenced or wt spheroids with primary cancer-activated fibroblasts (CAFs). When spheroids were co-cultured with fibroblasts,SDHB-silenced cells showed a significant increase in matrigel invasion as demonstrated by the computation of the migratory areas (P SDHB-silenced spheroids moved collectively, unlike the cells of wt spheroids that moved individually. Additionally,SDHB-silenced spheroids developed long filamentous formations along which clusters of cells migrated far away from the spheroid, whereas these structures were not present in wt spheroids. We found that lactate, largely secreted by CAFs, plays a specific role in promoting migration only ofSDHB-silenced cells. In this study, we demonstrated thatSDHBsilencingper seincreases tumor cell migration/invasion and that microenvironment, as represented by CAFs, plays a pivotal role in enhancing collective migration/invasion in PheoSDHB-silenced tumor cells, suggesting their role in increasing the tumor metastasizing potential.

G-protein coupled receptor H4 (H4R) is a histamine receptor subtype that is involved in a condition of pathological chronic pain, but its pathophysiological function is unknown. Here, we investigate the role of H4R in a model of traumatic nerve injury. H4R knockout (H4R-/-) mice exposed to spared nerve injury (SNI) developed a more prominent mechanical and thermal hypersensitivity than wild type mice. Western blotting and immunofluorescence were used to characterize the cellular mechanisms. Nerve injury increased phosphorylated pERK MAPK expression in the spinal cord that was further promoted in H4R-/- genotype. Additionally, the increase in the phosphorylated cAMP response element-binding protein (CREB) was significantly enhanced in neuropathic H4R-/- mice. In the same way, after SNI a remarkable increase of dopamine beta-hydroxylase (DβH) immunoreactive neurons was detected in spinal cord of H4R-/- mice. The number of injured DRG neurons after SNI of H4R-/- mice, identified by activating transcription factor 3 (ATF3) staining was comparable to that of wild type littermates. Similarly the density of intraepidermal nerve fibres in plantar skin after SNI was reduced with the same degree in H4R-/- mice and with wild type mice. We conclude that the phenotype of H4R-/- mice leads to increased neuropathic pain hypersensitivity promoting an overactivation of spinal ERK-CREB pathway in DβH expressing neurons without modifying the innervation of the hind paw skin and integrity of the primary sensory neurons. In summary, our results provide H4R as a potential new target for the clinical management of chronic neuropathic pain conditions.