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In situ synchrotron-based techniques are starting to be intensively exploited to investigate a range of electrochemical systems; in particular, X-ray photoelectron spectroscopy (XPS), owing to its utmost surface sensitivity and photon-in/electron-out nature, has been recognized as a powerful tool to achieve insight into phenomena occurring at the electrode surface and at the electrode/electrolyte interface of solid oxide–based electrochemical devices, in the presence of a reactive gas and under applied electrochemical polarization. Conventional XPS exhibits the key limitation of extensive space averaging that results in missing important details of the morphochemical structure of the materials forming the electrochemical device, that, instead, have a notable impact on electrocatalytic performance and durability. For this reason, combining the analytical power of XPS with space-resolution capability can provide unique and practically useful insight for the understanding and improvement of electrochemical devices. In this chapter, we focus on in situ XPS microspectroscopy, enabling elemental and chemical state mapping with high spatial resolution under electrochemical operating conditions. Specifically, we describe our recent work on a range of solid oxide fuel cell materials and configurations, performed with reactive gases and gas mixtures both in high-vacuum and near-ambient pressure conditions This chapter includes on the one hand details of instrumental solutions regarding cell engineering, gas feed modes, and pressure management, and on the other hand, a representative selection of the kind of electrochemical information that can be drawn from photoelectron microspectroscopy.

INTRODUCTION: The aim of this international collaborative effort was to report 36-month longitudinal changes using the 6MWT in ambulant patients affected by Duchenne muscular dystrophy amenable to skip exons 44, 45, 51 or 53. MATERIALS AND METHODS: Of the 92 patients included in the study, 24 had deletions amenable to skip exon 44, 27 exon 45, 18 exon 51, and 28 exon 53. Five patients with a single deletion of exon 52 were counted in both subgroups skipping exon 51 and 53. RESULTS: The difference between subgroups amenable to skip different exons was not significant at 12 months but became significant at both 24 (p≤0.05) and 36 months (p≤0.01). DISCUSSION: Mutations amenable to skip exon 53 had lower baseline values and more negative changes than the other subgroups while those amenable to skip exon 44 had better results both at baseline and at follow up. Deletions amenable to skip exon 45 were associated with a more variable pattern of progression. Single exon deletions were more often associated with less drastic changes but this was not always true in individual cases. CONCLUSION: Our results confirm that the progression of disease can differ between patients with different deletions, although the changes only become significant from 24 months onwards. This information is relevant because there are current clinical trials specifically targeting patients with these subgroups of mutations. ispartof: PLOS ONE vol:14 issue:6 ispartof: location:United States status: published

Plastic changes in the amygdala and limbic cortex networks have been widely shown in chronic pain. We have here investigated the role of group I metabotropic glutamate receptors (mGluRs) in the basolateral amygdala (BLA) pre-infra-limbic (PL-IL) divisions of the medial prefrontal cortex (mPFC) neuron connections after carrageenan-induced inflammatory pain in the rat. Intra-plantar injection of carrageenan decreased either spontaneous or mechanically/electrically evoked activity of PL cortex pyramidal neurons which responded with excitation in a way prevented by CPCOOEt, a selective mGluR1 antagonist, though not by MPEP, a selective mGluR5 antagonist. Accordingly, intra-BLA microinjection of DHPG, a group I mGluR agonist, caused PL cortex neuron activity depression, antagonized by CPCCOEt. CPCOOEt, but not MPEP, reduced also carrageenan-induced mechanical allodynia. The PL cortex cell deactivation in inflammatory pain condition was associated with increased GABA (conversely glutamate was decreased) in the PL/IL cortex. The local application of bicuculline, a GABAA receptor selective antagonist, reduced mechanical allodynia. An over-expression of mGluR1, but not mGluR5, have been observed in the PL-IL cortex after inflammatory pain suggesting an increased mGluR1-dependent cross-talk among BLA and IL-PL cortex neurons in inflammatory pain conditions. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. © 2012 Elsevier Ltd. All rights reserved.

Background: Osteosarcoma is an aggressive bone tumor. Itrepresents the principal cause of cancer-associated death in children.Considering the recent findings on the role of iron in cancer, iron chelation has been investigated for its antineoplastic properties in many tumors. Deferasirox is the most used iron chelator compound and in previous studies showed an anticancer effectinhematologic and solid malignancies. Eltrombopag is a Thrombopoietin receptor used in thrombocytopenia, that also binds and mobilize iron. It demonstrated an effect in iron overload conditions and also in contrasting cancer cells proliferation. Objective: We analyzed the effects of Deferasirox and Eltrombopag in Human Osteosarcoma cells, in the attempt to identify other therapeutic approaches for this tumor. Methods: We cultured and treated withDeferasirox and Eltrombopag, alone and in combination, two human osteosarcoma cell lines, MG63 and 143B. After 72h exposure, we performed RTqPCR, Western Blotting, Iron Assay and cytofluorimetric assays to evaluate the effect on viability, apoptosis, cell cycle progression and ROS production. Results: The iron chelating properties of the two compounds are confirmed also in Osteosarcoma, but we did not observe any direct effect on tumor progression. Discussion: We tested Deferasirox and Eltrombopag, alone and in combination, in Human Osteosarcoma cells for the first time and demonstrated that their iron chelating activity does not influence biochemical pathways related to cancer progression and maintenance. Conclusion: Although further investigations on possible effects mediated by cells of the tumor microenvironment could be of great interest, in vitro iron chelation in Osteosarcoma does not impair tumor progression.

Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents. Bortezomib (BTZ) is an approved anticancer drug, classified as a selective reversible inhibitor of the ubiquitin-dependent proteasome system, that leads to cancer cell cycle arrest and apoptosis reducing the invasion ability of Osteosarcoma cells in vitro. It also regulates the RANK/RANKL/OPG system, involved in the pathogenesis of bone tumors and in cell migration. A side effect of BTZ is to induce painful sensory peripheral neuropathy which lead to cessation of therapy or dose reduction. Recently BTZ has been evaluated in combination with Cannabinoids targeting CB1 receptor, demonstrating a promising synergic effect. The Endocannabinoid/Endovanilloid (EC/EV) system includes two G protein-coupled receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel and their endogenous ligands and enzymes. CB1 and CB2 are expressed mainly in Central Nervous System and Immune Peripheral cells respectively. TRPV1 is also expressed in primary sensory neurons and is involved in pain modulation. EC/EV system induces apoptosis, reduces invasion and cell proliferation in Osteosarcoma cell lines and is involved in bone metabolism. We analyzed the effects of BTZ, alone and in combination with selective agonists at CB2 (JWH-133) and TRPV1 (RTX) receptors, in the Osteosarcoma cell line (HOS) on Apoptosis, Cell Cycle progression, migration and bone balance. We observed that the stimulation of CB2 and TRPV1 receptors increase the efficacy of BTZ in inducing apoptosis and reducing invasion, cell cycle progression and by modulating bone balance. These data suggest the possibility to use BTZ, in combination with EC/EV agonists, in Osteosarcoma therapy reducing its dose and its side effects.

Tumor-associated macrophages and their alternative activation states together with cytokines and growth factors trapped in tumor microenvironment contribute to the progression of OS. In contrast to other tumor types, M2 polarized macrophages, reduce metastasis and improve survival in osteosarcoma patients. Mifamurtide is an immunomodulatory drug given together with standard adjuvant chemotherapy in high-grade osteosarcoma to improve outcome. Macrophages obtained from peripheral blood mononucleated cells of healthy donors and MG63 cells were cultured alone and together, and treated with Mifamurtide. We analyzed the effects of Mifamurtide on macrophage polarization and on MG63 proliferation, migration and differentiation, evaluating the expression of M1/M2 and osteoblast markers and molecules involved in metastasis and proliferation pathways. Our data suggest that Mifamurtide, switching macrophage polarization towards a TAM-like intermediate M1/M2 phenotype, may modulate the delicate balance between pro-inflammatory and immunomodulatory macrophage functions. Moreover, Mifamurtide may inhibit the cellular proliferation and induce the tumor cell differentiation, probably through the down regulation of pSTAT3, pAKT and IL-17R.

Tissino et al. demonstrate that in chronic lymphocytic leukemia, the VLA-4 (CD49d/CD29) integrin remains activable by B cell receptor stimulation also upon in vitro and in vivo ibrutinib exposure. Clinically, ibrutinib-treated CD49d-positive CLL patients experience reduced recirculation lymphocytosis and nodal response and inferior outcomes., The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

In addition to its role in neuronal survival, the brain neurotrophic factor (BDNF) has been shown to influence serotonin transmission and synaptic plasticity, events strongly implicated in the appearance of l-DOPA-induced dyskinesia (LID), a motor complication occurring in parkinsonian patients after long-term treatment with the dopamine precursor. In order to evaluate a possible influence of BDNF in the appearance of LID, 6-OHDA-lesioned rats received a striatal injection of different concentrations of an adeno-associated viral (AAV) vector over-expressing either BDNF or GFP, as control vector. Eight weeks later, animals started to receive a daily treatment with l-DOPA (4-6mg/kg plus benserazide 4-6mg/kg, s.c.) or saline, and dyskinesias, as well as l-DOPA-induced rotations, were evaluated at several time-points. Moreover, molecular changes in striatal D1 receptor-dependent cAMP/PKA and ERK/mTORC signaling pathways, as well as, sprouting of striatal serotonin axons, were measured. Results showed that the AAV-BDNF vector injection induced striatal over-expression of BDNF, as well as striatal and pallidal serotonin axon hyperinnervation. Moreover, rats that over-expressed BDNF were more prone to develop LID and l-DOPA-induced rotations, compared to the GFP-treated control group. Finally, rats that over-expressed BDNF showed increased levels of striatal D1R-dependent signaling phospho-proteins in response to l-DOPA administration. This study suggests that BDNF over-expression, by inducing changes in pre-synaptic serotonin axonal trophism, is able to exacerbate maladaptive responses to l-DOPA administration.

In this study, we investigated the role of the endovanilloid/endocannabinoid system in the glucocorticoid-induced osteoclast overactivity. Receptorial and enzymatic component of the endovanilloid/endocannabinoid system are expressed in bone cells, and dysregulated when bone mass is reduced. Moreover, blockade or desensitization of vanilloid receptor 1 (TRPV1) and/or stimulation of cannabinoid receptor 2 (CB2) are beneficial for reducing number and activity of the bone cells modulating resorption, the osteoclasts. We have treated in vitro healthy woman derived osteoclasts with methylprednisolone in presence or not of CB2 or TRPV1 agonists/antagonists, analysing the effect on osteoclast function and morphology through a multidisciplinary approach. Moreover, a treatment with a protein kinase C inhibitor to evaluate osteoclast activity and endovanilloid/endocannabinoid component expression levels was performed in osteoclasts derived from healthy subjects in presence of not of methylprednisolone. Our results show, for the first time, that the endovanilloid/endocannabinoid system is dysregulated by the treatment with methylprednisolone, that the osteoclast activity is increased and that pharmacological compounds stimulating CB2 or inhibiting TRPV1 might reduce, possible inhibiting protein kinase C beta II, the methylprednisolone-induced osteoclast over-activation, suggesting their therapeutic use for protecting from the glucocorticoid-induced bone mass loss.

The current leading causes of death in the world are cardiovascular diseases (in particular myocardial infarctions) and cancer. In Italy in 2016 almost 400,000 people died because of these two pathologies. Despite the great progress made by medicine in recent decades, the road to cure these diseases is still long, and cancer and cardiac diseases remain a serious socio-economic problem for our society. For this reason, this PhD research focuses on these two main topics; called in the thesis the oncology project and the cardiology project. Both projects were devoted to the synthesis of Silicon Carbide Nanowires (SiC NWs), aiming to devise their new possible biomedical applications in the field of oncology and cardiology. This PhD, funded by Cariparma Crédit Agricole, is part of a Bilateral Agreement for Joint Philosophy Doctor (PhD) Degrees between the University of Parma and CNR-IMEM with the University of Cape Town. The synthesis of compounds for potential applications in cancer treatment was the main research topic of the oncology project during the period spent abroad in South Africa. Modern anti-tumor therapies are very invasive for the patient and often based on the administration of drugs, generally cisplatin derivatives, very cytotoxic and harmful to the human body. Furthermore, such treatments are often not fully selective and kill the diseased but also the healthy cells of the organism. Therefore, the aim of many scientists in the world is to design and synthesize new molecules with excellent anticancer properties but with fewer side effects compared to the drugs already currently in use, for example, less toxicity and greater selectivity for the cancer cells. Thiosemicarbazones (TSCs) are Schiff bases which in recent years have attracted considerable attention thanks to their promising anticancer properties. These compounds are very active also in the fight against malaria, moreover they are used as antifungal and antibacterial agents. TSCs are widely documented in literature, and a correlation has often been found between the efficacy of the antitumor activity of the molecule and the presence of aromatic groups in its structure. Furthermore, exploiting the presence in the TSCs of a set of donor atoms (N and S) able to coordinate various types of metal ions to form the related complexes, many studies emphasize the importance of the presence of metal ions bound to the molecule to further increase the antitumor efficacy. Unfortunately, TSCs have poor solubility in aqueous matrices. This behavior can make their administration as drugs more complex and create possible problems of precipitation (due to the insolubility) or accumulation inside the human body. For this reason, in the oncology project of this PhD research a series of aromatic-TSCs were synthetized and then anchored on the chemically modified surface of SiC NWs, employed as carriers. According to previous results obtained by my research group at IMEM-CNR, these SiC-based NWs are biocompatible and can be conjugated with several organic molecules (e.g. porphyrins). The target of these new nanomaterial-drug systems are solid tumors, e.g. affecting breast, lungs, pancreas and liver, where they can be injected and be active to reduce the tumor mass in view of later surgical removal. To anchor these thiosemicarbazones to the nanowires, SiC NWs coated with a thin external layer of silica (nanomaterials called core-shell SiC/SiO2 NWs) were chosen. In fact, this external layer of silica can be chemically functionalized by appropriate silane linkers. The silane linkers used are (3-aminopropyl)triethoxysilane (APTES) and triethoxy(3-isothiocyanatopropyl) silane (Si-NCS). These alkoxysilanes react with the silica surface and covalently bind to the NWs. Thus, it was possible introduce on the NWs surface amino or isothiocyanate groups, able to react with proper TSCs in the anchoring step. To conjugate the NWs with TSCs, it was required the synthesis of thiosemicarbazones having suitable functional groups able to react with the groups present on NWs surface. In this way thiosemicarbazone molecule was anchored by covalent bonds to the surface of the nanowires. During the time spent at the University of Cape Town, working on the oncology project, various TSCs-metal complexes were prepared and successively anchored to NWs previously decorated with amino groups. Literature is rich in examples of platinum (and related metals) complexes with excellent anticancer activity. Therefore, reactions between the TSCs bound to the NWs surface and platinum, ruthenium and rhodium salts were carried out to obtain NWs functionalized with these complexes. Unfortunately, it was observed that this approach has a drawback due to the ability of the amino groups to give competitive complexes with transition metals. Therefore, the attention of the oncology project was focused on a specific thiosemicarbazone compound (Triapine), on a way to find a new procedure to anchor it to NWs and subsequently on the evaluation of the antitumor activity of this new nano-system by in vitro tests. Triapine is an α-(N)-heterocyclic thiosemicarbazone, which successfully passed Phase II of Clinical Trial. Currently, Triapine is tested as an anticancer drug in human patients. The precursor of Triapine was synthetized, with a suitable functional group able to react with the Si-NCS linker anchored to the NWs. In this way the NWs surface was covered by Triapine molecules connected to the SiC/SiO2 structure through a carbon chain as linker. Then, the anchored Triapine was used as a ligand to coordinate metal ions; specifically copper, a metal with low toxicity to the human body. Subsequently, these nano-systems (NWs-Triapine and NWs-Triapine-Cu complex) and the free molecules (Triapine and Triapine-Cu complex without NWs) were tested on A549 human lung adenocarcinoma cell line to evaluate their antitumor efficacy. The free molecules showed good IC50 values – in the micromolar range - with greater antitumor activity of the free complex compared to the free ligand in the first 24 h, but situation reversed in the subsequent 48 h and 72 h. Vice versa, about the compounds anchored to the NWs, only the NWs-complex has proved active, suggesting that the free molecules and the molecules anchored to the NWs have different mechanisms of action and different interactions with cancer cells. The mechanisms of action were better investigated studying the cell cycle. Regarding the other research topic of this PhD thesis, the problem of myocardial infarction has been addressed in the cardiology project. Infarction is the death (necrosis) of part of the heart muscle tissue (myocardium) due to the lack of adequate oxygenation by the arterial blood flow (ischemia). After the infarction, the affected area is no longer able to contract due to the death of cardiomyocytes, the cardiac cells responsible for the generation and transmission of the electro-cardiac pulse. The dead cardiac cells cannot be regenerated and are replaced by a fibrous connective tissue (collagen fibers) that forms a scar area in which the electrical pulse is no longer able to be diffused. Therefore, an insulating area is generated in the heart, with drastic consequences for the health of the organ and for the correct cardiac functioning. Previous in vitro experiments on cardiomyocytes had shown that SiC NWs (semiconductive nanowires, without the external shell of silica) were able to connect two distant cardiac cells, previously isolated from each other. Thanks to the presence of SiC NWs, the two cells, previously silent, were able to propagate the electric pulse to each other and to synchronize their action potential. Based on these results, in collaboration with a medical research team of the University of Parma, in this cardiology project SiC NWs were tested in vivo on infarcted rats’ hearts, to verify if the SiC NWs semiconductive properties were able to restore, even partially, the normal propagation of the cardiac electrical pulse, interrupted after the infarction in the ischemic tissues area. For this purpose, in ten rats the myocardial infarction was induced by creating an ischemic zone in the left ventricle through three different protocols: injection of formaldehyde solution at different concentrations (4% and 38%), deposition of a drop of formaldehyde 38% solution on the heart surface and cryoinjury (creation of a necrotic area by selective burning of cardiac tissues using a metal tip cooled by liquid nitrogen). Before and after the infarction the electro-cardiac activity was monitored with a special high-resolution electrode grid. This accurate epicardial mapping of the affected area showed important changes in the values related to the EGs parameters (waves and intervals) and in the isochrone maps (velocity and direction of propagation of the pulse wavefront) before and after the infarction. These data also allowed to understand that cryoinjury was the best technique in creating an effective ischemic zone, with well-defined edges, easily reproducible in rats. Subsequently, a saline solution containing SiC NWs was injected into the infarcted area and the cardiac signals were collected and analyzed. The aim of this cardiology project is to compare the cardiac functioning after the injection of semiconductive SiC NWs with the pre-infarction conditions to evaluate whether the use of SiC NWs helps restore normal cardiac functioning, lost as a result of a myocardial infarction. Based on preliminary data, in some rats after the injection of SiC NWs some important physioelectric parameters show promising changes compared to the values recorded after the infarction, returning similar to the normal pre-infarction conditions. However, the absence of significant statistical series makes it difficult to assert more specific statements. Le principali cause di morte nel mondo sono le malattie cardiovascolari (in particolare gli infarti del miocardio) e il cancro. In Italia nel 2016 sono morte quasi 400.000 persone a causa di queste due patologie. Nonostante i grandi progressi compiuti dalla medicina negli ultimi decenni, la strada per curare queste malattie è ancora lunga, e il cancro e le malattie cardiache restano un serio problema socio-economico per la nostra società. Per questo motivo, il tema della mia ricerca di dottorato è stata la sintesi di nanofili di carburo di silicio (SiC NWs), con l'obiettivo di ideare nuove possibili applicazioni biomediche nel campo della cardiologia e dell'oncologia. Il mio dottorato di ricerca, finanziato da Cariparma Crédit Agricole, fa parte del Bilateral Agreement for Joint Philosophy Doctor (PhD) Degrees tra l'Università di Parma e il CNR-IMEM con l'Università di Città del Capo, dove ho lavorato per sei mesi. Durante il mio periodo all'estero in Sud Africa, mi sono occupato principalmente della sintesi di composti con potenziali applicazioni nel trattamento del cancro. Le moderne terapie antitumorali sono molto invasive per il paziente e spesso basate sulla somministrazione di farmaci, generalmente derivati del cisplatino, molto citotossici e dannosi per il corpo umano. Inoltre, tali trattamenti spesso non sono completamente selettivi e uccidono sia le cellule malate che quelle sane dell'organismo. Pertanto, l'obiettivo di molti scienziati è progettare e sintetizzare nuove molecole con eccellenti proprietà antitumorali, ma con meno effetti collaterali rispetto ai farmaci già in uso, ad esempio, minore tossicità e maggiore selettività verso le cellule tumorali. I tiosemicarbazoni (TSCs) sono basi di Schiff che negli ultimi anni hanno attirato notevole attenzione grazie alle loro promettenti proprietà antitumorali. Questi composti sono molto attivi anche nella lotta contro la malaria, inoltre sono utilizzati come agenti antifungini e antibatterici. I TSCs sono ampiamente documentati in letteratura e spesso è stata trovata una correlazione tra l'efficacia dell'attività antitumorale della molecola e la presenza di gruppi aromatici nella sua struttura. Inoltre, sfruttando nei TSCs la presenza di un set di atomi donatori (N e S) in grado di coordinare vari tipi di ioni metallici per formare i relativi complessi, molti studi sottolineano l'importanza della presenza di tali ioni metallici legati alla molecola per ulteriormente aumentare l'efficacia antitumorale. Sfortunatamente, i TSCs hanno una scarsa solubilità in matrici acquose. Questo comportamento può rendere più complessa la loro somministrazione come farmaci e creare possibili problemi di precipitazione (a causa dell'insolubilità) o accumulo all'interno del corpo umano. Per questo motivo, ho deciso di sintetizzare una serie di TSCs aromatici e di ancorarli sulla superficie modificata chimicamente dei SiC NWs, impiegati come carriers. In base ai precedenti risultati ottenuti dal mio gruppo di ricerca presso IMEM-CNR, questi SiC NWs sono biocompatibili e possono essere coniugati con diverse molecole organiche (ad esempio le porfirine). L'obiettivo di questi nuovi sistemi (farmaco- nanomateriale) sono i tumori solidi, ad esempio quelli che colpiscono il seno, i polmoni, il pancreas e il fegato, dove possono essere iniettati ed essere attivi per ridurre la massa tumorale in vista della successiva rimozione chirurgica. Per ancorare questi tiosemicarbazoni ai nanofili, ho scelto di utilizzare SiC NWs rivestiti con un sottile strato esterno di silica (nanomateriali chiamati core-shell SiC/SiO2 NWs) in grado di essere funzionalizzati chimicamente da opportuni linker silanici. Come linker silanici ho usato (3-amminopropil) trietossisilano (APTES) e trietossi (3-isotiocianatopropil) silano (Si-NCS). Questi alcossisilani reagiscono con la superficie di silica e si legano in modo covalente ai NWs. Pertanto, è stato possibile introdurre sulla superficie dei NWs dei gruppi amminici o isotiocianato in grado di reagire con le molecole di TSCs nella fase di ancoraggio. Per coniugare i NWs con i TSCs è stata richiesta la sintesi di tiosemicarbazoni aventi opportuni gruppi funzionali in grado di reagire con i gruppi presenti sulla superficie dei NWs. In questo modo la molecola di tiosemicarbazone è stata ancorata da legami covalenti alla superficie dei nanofili. Durante il tempo trascorso presso l'Università di Città del Capo sono stati preparati vari complessi metallici di TSCs e successivamente sono stati ancorati a NWs precedentemente decorati con gruppi amminici. La letteratura è ricca di esempi di complessi di platino (e metalli correlati) con eccellente attività antitumorale. Pertanto, ho scelto di svolgere reazioni tra i TSCs legati alla superficie del NWs con dei sali di platino, rutenio e rodio per ottenere NWs funzionalizzati con questi complessi. Sfortunatamente, è stato osservato che questo approccio ha un inconveniente dovuto alla capacità dei gruppi amminici di dare complessi competitivi con i metalli di transizione. Pertanto, l'attenzione è stata concentrata su uno specifico tiosemicarbazone (Triapina), su un modo per trovare una nuova procedura per ancorarlo ai NWs e successivamente sulla valutazione dell'attività antitumorale del nuovo nano-sistema mediante test in vitro. La Triapina è un tiosemicarbazone α-(N)-eterociclico, che ha superato con successo la fase II della sperimentazione clinica (Clinical Trial). Attualmente, la Triapina è testata come farmaco antitumorale in pazienti umani. Ho sintetizzato il precursore della Triapine con un adatto gruppo funzionale in grado di reagire con il linker Si-NCS ancorato ai NWs. In questo modo ho ottenuto dei NWs la cui superficie era coperta da molecole di Triapina collegate alla struttura SiC/SiO2 attraverso la catena carboniosa del linker. Ho usato la Triapina ancorata come legante per coordinare ioni metallici; nello specifico, ho deciso di usare il rame, un metallo con bassa tossicità per il corpo umano. Successivamente, questi nano-sistemi (NWs-Triapina e NWs-Triapina-Cu complesso) e le molecole libere (Triapina e Triapina-Cu complesso senza i NWs) sono stati testati sulla linea cellulare di adenocarcinoma umano del polmone (A549) per valutare la loro efficacia antitumorale. Le molecole libere hanno mostrato buoni valori di IC50 - nell'intervallo micromolare - con una maggiore attività antitumorale del complesso libero rispetto al legante libero nelle prime 24 ore, ma la situazione si è invertita nelle successive 48 ore e 72 ore. Viceversa, sui composti ancorati ai NWs, solo il NWs-complesso si è dimostrato attivo, suggerendo che le molecole libere e le molecole ancorate ai NWs hanno meccanismi di azione differenti e diverse interazioni con le cellule tumorali. Successivamente i meccanismi di azione sono stati studiati meglio analizzando il ciclo cellulare. Per quanto riguarda il progetto di ricerca relativo alle malattie cardiache, ho voluto affrontare il problema dell'infarto al miocardio. L'infarto è la morte (necrosi) di una parte del tessuto muscolare del cuore (miocardio) a causa della mancanza di un'adeguata ossigenazione da parte del flusso sanguigno arterioso (ischemia). Dopo l'infarto, l'area interessata non è più in grado di contrarsi a causa della morte dei cardiomiociti, le cellule cardiache responsabili della generazione e della trasmissione dell'impulso elettrocardiaco. Le cellule cardiache morte non possono essere più rigenerate e vengono sostituite da un tessuto connettivo fibroso (fibre di collagene) che forma un'area cicatriziale in cui l'impulso elettrico non è più in grado di essere diffuso. Pertanto, nel cuore viene generata un'area di tessuto isolante, con conseguenze drastiche per la salute dell'organo e per il corretto funzionamento cardiaco. Precedenti esperimenti in vitro su cardiomiociti avevano dimostrato che i SiC NWs (nanofili semiconduttori, senza il guscio esterno di silica) erano in grado di collegare due cellule cardiache distanti, precedentemente isolate l'una dall'altra. Grazie alla presenza di SiC NWs, le due celle, inizialmente silenziose, erano in grado di propagare l'impulso elettrico tra loro e sincronizzare il loro potenziale d'azione. Sulla base di questi risultati, in collaborazione con un gruppo di ricerca medica dell'Università di Parma, abbiamo testato i SiC NWs in vivo su cuori di ratti infartuati, per verificare se le proprietà semiconduttive dei SiC NWs fossero in grado di ripristinare, anche parzialmente, la normale propagazione di l'impulso elettrocardiaco, interrotto dopo l'infarto nell'area dei tessuti ischemici. A questo scopo, in dieci ratti è stato indotto un infarto del miocardio creando una zona ischemica nel ventricolo sinistro attraverso tre diversi protocolli: iniezione di una soluzione di formaldeide a diverse concentrazioni (4% e 38%), deposizione di una goccia di una soluzione di formaldeide al 38% sulla superficie cardiaca esterna e cryoinjury (creazione di un'area necrotica mediante combustione selettiva dei tessuti cardiaci utilizzando una punta di metallo raffreddata con azoto liquido). Prima e dopo l'infarto, l'attività elettrocardiaca veniva monitorata con una speciale griglia ad elettrodi ad alta risoluzione. Questa accurata mappatura epicardica dell'area interessata ha mostrato importanti cambiamenti nei valori relativi ai parametri EGs (onde e intervalli) e nelle mappe di isocrone (velocità e direzione della propagazione del fronte d'onda dell’impulso) prima e dopo l'infarto. Questi dati hanno anche permesso di capire che la cryoinjury era la tecnica migliore per creare una zona ischemica efficace, con bordi ben definiti, facilmente riproducibili negli altri ratti. Successivamente, una soluzione salina contenente SiC NWs è stata iniettata nell'area infartuata e i segnali cardiaci sono stati raccolti e analizzati. Lo scopo del progetto è di confrontare il funzionamento cardiaco dopo l'iniezione dei SiC NWs con le condizioni pre-infarto per valutare se l'uso dei SiC NWs aiuta a ripristinare il normale funzionamento cardiaco perso. Questa ricerca non è ancora finita e altri esperimenti sui ratti sono programmati entro la fine dell'anno. Sulla base dei dati preliminari, posso affermare che in alcuni ratti dopo l'iniezione di SiC NWs alcuni importanti parametri fisioelettrici mostrano cambiamenti promettenti rispetto ai valori registrati dopo l'infarto, ritornando simili alle normali condizioni pre-infarto. Tuttavia, l'assenza di una serie statistica significativa rende difficile fare affermazioni più specifiche.

Recent new insights into the molecular basis of thyroid eye disease have led to the use of more specific therapies such as monoclonal antibodies This review explores the traditional immunosuppressant therapy for TED, highlighting the basis for emergent recent medications, possible treatment options and, eventually possible new general recommendation for management of TED. Data has been retrieved from the literature searching on Pubmed. Steroid therapy remains the first line therapy for moderate/severe and severe vision threatening TED The use of some traditional nonspecific immunosuppressant such as mycophenolate, cyclosporine and azathioprine seems useful in combination with steroid therapy to achieve stable results in the long term; methotrexate is useful as steroid-sparing medications and in steroid resistant or intolerant patients. In recent years, many scientific reports have showed the effectiveness of biological immunosuppressive agents in the management of TED. Etanercept, adalimumab, and tocilizumab have shown to be effective in reduction of the inflammatory signs with the possible advantage to prevent relapse of the disease. Particularly Tociliuzumab seems very effective as second line therapy, after steroid failure. Teprotumumab may control the disease activity and it seems to be very effective in preventing severity disease progression. Infliximab might be useful in severe TED with optic nerve compression resistant to steroid and decompression. Indeed, the actual incidence of adverse effects is not well assessed yet, therefore the use should be limited at those cases that really need an alternative therapy to steroid, handled by an expert multidisciplinary team.

Introduction Immunoassay (IA) tests should be able to detect low concentrations of illegal drugs when used for the screening of drugs in drivers. False negatives should be avoided, and false positives should be reduced as far as possible. In this study, semi-quantitative results for blood samples containing illicit drugs (cannabinoids, cocaine, amphetamines/methamphetamines, opiates and methadone) obtained with cloned enzyme donor immunoassay (CEDIA), were compared with results of confirmatory analysis performed through gas chromatography-mass spectrometry (GC–MS). Screening cut-off points for each class of drugs were retrospectively optimized. Methods Whole blood samples from drivers involved in road accidents in the period from January 2013-December 2017 were analyzed with CEDIA (4200 samples). Confirmatory analyses were performed through (GC–MS) on: (i) all samples with screening concentrations above 1 ng/ml for at least one drug (positive screening results); (ii) 800 samples with screening concentration lower than 1 ng/ml (negative screening results). Recommended per se limits in relation to driving under the influence of drugs were set as fixed values. Sensitivity, specificity, positive and negative predictive values were evaluated by contingency tables and compared to ROC-analysis in order to obtain ideal screening cut-offs. Results CEDIA results were available for 4200 blood samples and 1172 positive screening results were found. Among these, 1008 confirmation analysis were obtained through GC–MS. Optimized screening cut-offs obtained through ROC analysis were as follows: 8.0 ng/ml for THC; 5.5 ng/ml for THC-COOH; 21.1 ng/ml for cocaine; 6.9 ng/ml for benzoylecgonine; 33.1 ng/ml for opiates; 61.6 ng/ml for amphetamines; 5.0 ng/ml for methadone. Using these cut-offs, sensitivity was above 97% for THC-COOH, cocaine, benzoylecgonine, amphetamines, opiates and methadone, and 92% for THC; specificity was above 90% for cocaine, benzoylecgonine, amphetamines, opiates and methadone, 80% for THC and 89% for THC-COOH; negative predictive value was above 99% for all drugs and metabolites. Conclusion Previous studies have shown that CEDIA tests are useful for preliminary screening of serum and urine. Its implementation in whole blood is of primary importance for the assessment of impaired driving, since the per se limits of many European countries refer to whole blood, and preparation of the serum and/or the collection of urine is not always possible in the hospital emergency department, where blood samples are withdrawn. Our study shows that CEDIA tests on whole blood permit the definition of cut-off values with optimal sensitivity and negative predictive values for all analytes (near to 100%), including very good specificity.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis, alteration in the microvasculature and immunologic abnormalities. It has been hypothesized that an abnormal redox state could regulate the persistent fibrotic phenotype in SSc patients. N-Formyl peptide receptors (FPRs) are chemotactic receptors overexpressed in fibroblasts derived from SSc patients. In this study, we demonstrated that stimulation of FPRs promotes the generation of reactive oxygen species (ROS) in skin fibroblasts. In fibroblast cells, ROS production was due to FPRs interaction with the urokinase receptor (uPAR) and to β1 integrin engagement. FPRs cross-talk with uPAR and integrins led to Rac1 and ERKs activation. FPRs stimulation increased gp91phox and p67phox expression as well as the direct interaction between GTP-Rac1 and p67phox, thus promoting assembly and activation of the NADPH oxidase complex. FPRs functions occur through interaction with a specific domain of uPAR (residues 88SRSRY92) that can be exposed on the cell membrane by protease-mediated receptor cleavage. Immunohistochemistry analysis with a specific anti-SRSRY antibody showed increased expression of uPAR in a cleaved form, which exposes the SRSRY sequence at its N-terminus (DIIDIII-uPAR88–92) in skin biopsies from SSc patients. As expected by the increased expression of both FPRs and DII-DIII-uPAR88-92, fibroblasts derived from SSc patients showed a significantly increase in ROS generation both at a basal level than after FPRs stimulation, as compared to fibroblasts from normal subjects. C37, a small molecule blocking the interaction between FPRs and uPAR, and selumetinib, a clinically approved MAPKK/ERK inhibitor, significantly inhibited FPRs-mediated ROS production in fibroblasts derived from SSc patients. Thus, FPRs, through the interaction with the uPA/uPAR system, can induce ROS generation in fibroblasts by activating the NADPH oxidase, playing a role in the alteration of the redox state observed in SSc.

The early phase of systemic sclerosis (SSc) presents edema as one of the main features: this is clinically evident in the digital swelling (puffy fingers) as well as in the edematous skin infiltration of the early active diffuse subset. Other organs could be affected by this same disease process, such as the lung (with the appearance of ground glass opacities) and the heart (with edematous changes on cardiac magnetic resonance imaging). The genesis of tissue edema is tightly linked to pathological changes in the endothelium: various reports demonstrated the effect of transforming growth factor β, vascular endothelial growth factor and hypoxia-reperfusion damage with reactive oxygen species generation in altering vascular permeability and extravasation, in particular in SSc. This condition has an alteration in the glycocalyx thickness, reducing the protection of the vessel wall and causing non-fibrotic interstitial edema, a marker of vascular leak. Moreover, changes in the junctional adhesion molecule family and other adhesion molecules, such as ICAM and VCAM, are associated with an increased myeloid cells' extravasation in the skin and increased myofibroblasts transformation with further vascular leak and cellular migration. This mini-review examines current knowledge on determinants of vascular leak in SSc, shedding light on the role of vascular protection. This could enhance further studies in the light of drug development for early treatment, suggesting that the control of vascular leakage should be considered in the same way that vasodilation and inflammation reduction, as potential therapeutic targets.

Toluene, a liquid aromatic hydrocarbon, is one of the most widely used industrial solvents, and is present in numerous paints, paint thinners, glues and other industrial and household products. It has become the most abused solvent in the world due to its rapid effects following inhalation. However, the numerous cases of fatal and non-fatal toluene-related intoxication reported in the literature have not yet been collected and discussed in the forensic setting. In this paper we aim to provide a review of the cases of toluene abuse and intoxication and the state of the art of the forensic toxicological analysis of toluene intoxications in the living and in the dead subject, from the early identification to the medico-legal interpretation of the toxicological result. We have identified a total of 45 papers regarding different aspects of toluene abuse, and divided them into three sections, namely sampling, storage and techniques of analysis, assessment in living subjects and post-mortem assessment. This article reports toluene concentrations in blood from 202 living subjects, 23 fatal toluene intoxications and 85 toluene related deaths. Toxicological results are discussed in relation to the clinical presentation (living subjects, including impaired drivers), and the manner of death according to the medical examiner reports (post-mortem examinations). Finally, we discuss the strengths and limitations of the review.

Chronic blood transfusions are responsible to cause iron overload, which leads to several complications to end organs and osteoporosis. Iron chelation is needed to remove iron excess and to contain bone-mass loss. Deferasirox is the most recent oral iron chelator that prevents transfusion related iron overload complications. Recently Eltrombopag (ELT) iron chelating properties are emerging. ELT is an agonist at Thrombopoietin receptor, used in treatment of thrombocytopenia. We tested ELT and Deferasirox in iron overloaded osteoclasts from thalassemic patients and donors measuring intracellular iron, TRAP expression and osteoclast activity. We confirmed ELT iron chelation capacity also in bone tissue and a synergic effect when used with Deferasirox. Moreover, having demonstrated its effects on osteoclast activity, we suggest for the first time that ELT could ameliorate bone tissue's health reducing bone mass loss.

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Background Iron deficiency anemia in celiac disease is related to impaired duodenal mucosal uptake, due to villous atrophy. Iron enters the enterocytes through an apical divalent metal transporter, DMT1. Different DMT1 transcripts have been identified, depending on the presence of an iron-responsive element that allows DMT1 up-regulation during iron starvation. An intronic DMT1 polymorphism, IVS4+44C>A, has been associated with metal toxicity, and the CC-carriers show high iron levels. Aims This study investigates the association between DMT1 IVS4+44C>A and anemia in a cohort of 387 Italian celiac children, and the functional role of the polymorphism. Methods and results By association analysis, we found that DMT1 IVS4+44-AA genotype confers a four-fold risk of developing anemia, despite of atrophy degree. By analysis of mRNA from gastroesophageal biopsies, we found that total DMT1 is significantly upregulated in presence of mild, but not severe, atrophy, independently from IVS4+44C>A variant, and in normal but not in atrophic CC-biopsies. Moreover, we found that A-allele is associated to preferential expression of the DMT1 transcripts lacking the iron-responsive element, thus limiting the DMT1 overexpression that normally occurs to respond to iron starvation. Discussion Possibly, the IVS4+44-AA-related dysregulation of the iron-induced changes in DMT1 expression is not able to impair iron absorption in physiological condition. However, if exacerbated by the concomitant massive loss of functional absorbing tissue paralleling worsened stages of villus atrophy, it might be ineffective in counteracting iron deficiency, despite of DMT1 overexpression. Conclusion We suggest, for the first time, that celiac disease may unmask the contribution of the DMT1 IVS4+44C>A polymorphism to the risk of anemia.

BACKGROUND Osteosarcoma is the most frequent malignant bone tumor in childhood and young adulthood. Long-term survivors of osteosarcoma patients show high prevalence of osteoporosis and fractures. The immunomodulatory mifamurtide, which modulates macrophages activity, improves disease outcome. OBJECTIVE To evaluate the role of mifamurtide on macrophage component of bone, the osteoclasts, during chemotherapy in children with osteosarcoma. METHOD Osteoclasts, obtained from peripheral blood cells of healthy donors were harvested in the presence or not of mifamurtide. Moreover, osteoclast cultures were obtained from osteosarcoma patients, at onset and during chemotherapy, alone or with mifamurtide. Pro-osteoporotic tartrateresistant acid phosphatase (TRAP), phosphokinase-β-2 (PKCβ2), vanilloid receptor type 1 (TRPV1), and anti-osteoporotic cannabinoid receptor type 2 (CB2) biomarkers were analyzed by bio-molecular (qPCR), biochemical (Western Blotting), and morphological (TRAP assay) approaches. RESULTS Osteoclasts from osteosarcoma patients show significant increase of TRAP and decrease of CB2 with respect to osteoclasts from healthy donors. This osteoclast hyperactivity is more evident in osteoclasts from osteosarcoma patients during chemotherapy. Mifamurtide reduces pro-osteoporotic TRAP, PKCβ2, TRPV1 levels and increases CB2 in osteoclasts from healthy donors. Moreover, chemotherapy-induced effects on bone resorption markers are fully reverted in osteoclasts derived from osteosarcoma patients in chemotherapy plus mifamurtide. CONCLUSION Our data suggest a new therapeutic role for mifamurtide as possible anti-resorption agent in chemotherapy-induced osteoporosis in children with osteosarcoma.

Context: Obesity is associated with a low-grade inflammatory state and adipocyte (ADP) hyperplasia/hypertrophy. Obesity inhibits the “browning” of white adipose tissue. Cannabinoid receptor 2 (CB2) agonists reduce food intake and induce antiobesity effect in mice. A common missense CB2 variant, Q63R, causes CB2-reduced function. Objective: To evaluate the influence of CB2 receptor on the modulation of childhood obesity and of ADP activity and morphology. Design: CB2-Q63R variant was analyzed in obese Italian children. The effects of an inflammatory stimulus and those of drugs selectively acting on CB2 were investigated on in vitro ADPs obtained from mesenchymal stem cells of adult healthy donors or from sc adipose biopsies of adult nonobese and obese subjects. Setting: Department of Women, Child and General and Specialist Surgery of the Second University of Naples. Patients or Other Participants: A total of 501 obese Italian children (age 11 ± 2.75). Twelve healthy bone marrow donors (age 36.5 ± 15); and 17 subjects, 7 lean (age 42 ± 10) and 10 obese (age 37.8 ± 12) underwent sc adipose tissue biopsies. Main Outcome Measures: Effects of CB2 stimulation on adipokine, perilipin, and uncoupling protein-1 expression. Results: The less-functional CB2-R63 variant was significantly associated with a high z-score body mass index. CB2 blockade with AM630 reverse agonist increased inflammatory adipokine release and fat storage and reduced browning. CB2 stimulation with JWH-133 agonist reversed all of the obesity-related effects. Conclusion: CB2 receptor is a novel pharmacological target that should be considered for obesity.

BACKGROUND: The Nef protein can be detected in plasma of HIV-1-infected patients and plays a role in the pathogenesis of HIV-1. Nef produced during the early stages of infection is fundamental in creating the ideal environment for viral replication, e.g. by reducing the ability of infected cells to induce an immune response. AIM: Based on previous experience showing that both Tat and gp41 of HIV-1 are potent chemotactic factors for basophils and mast cells, and gp120 is a powerful stimulus for the release of histamine and cytokines (IL-4 and IL-13) from basophils, in this study we aimed to verify if the HIV Nef protein can exert some effects on basophils and mast cells purified from healthy volunteers through the interaction with the CXCL12 receptor, CXCR4. METHODS: Basophils purified from peripheral blood cells of 30 healthy volunteers and mast cells obtained from lung tissue of ten healthy volunteers were tested by flow cytometric analysis, chemotaxis and chemokine production by ELISA assays. RESULTS: Nef is a potent chemoattractant for basophils and lung mast cells obtained from healthy, HIV-1 and HIV-2 seronegative individuals. Incubation of basophils and mast cells with Nef induces the release of chemokines (CXCL8/IL-8 and CCL3/MIP-1α). The chemotactic activity of Nef on basophils and mast cells is mediated by the interaction with CXCR4 receptors, being blocked by preincubation of FcεRI+ cells with an anti-CXCR4 Ab. Stimulation with Nef or CXCL12/SDF-1α, a CXCR4 ligand, desensitizes basophils to a subsequent challenge with an autologous or heterologous stimulus. CONCLUSIONS: These results indicate that Nef, a HIV-1-encoded α-chemokine homolog protein, plays a direct role in basophils and mast cell recruitment and activation at sites of HIV-1 replication, by promoting directional migration of human FcεRI+ cells and the release of chemokines from these cells. Together with our previous results, these data suggest that FcεRI+ cells contribute to the dysregulation of the immune system in HIV-1 infection.

This study analyzed the effect of intra-ventrolateral periaqueductal grey (VL PAG) cannabinoid receptor (CB) stimulation on pain responses and rostral ventromedial medulla (RVM) neural activity in the chronic constriction injury (CCI) model of neuropathic pain in rats. Interaction between CB 1 and metabotropic glutamate 1 and 5 (mGlu 1 /mGlu 5 ) receptors was also investigated together with the expression of the CB 1 receptor associated Gai3 and cannabinoid receptor interacting 1a (CRIP 1a) proteins and the endocannabinoid synthesising and hydrolysing enzymes. In rats not subjected to CCI-induced pain, intra-VL PAG (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) (2-4-8 nmol), a CB receptor agonist, increased the tail flick latency and changed the ongoing activity of RVM OFF and the tail flick-related activity of the ON and OFF cells, accordingly. These effects were prevented by SR141716A and MPEP, selective CB 1 and mGlu 5 receptor antagonists, respectively, though not by CPCCOEt, a selective mGlu 1 receptor antagonist. A higher dose up to 16 nmol of WIN 55,212-2 was necessary to increase tail flick latency and change ON and OFF cell activity in CCI rats. Consistently, CCI rats showed a decrease in the expression of CB 1 receptors, NAPE-PLD, Gαi3 and CRIP 1a proteins;the expression of diacylglycerol lipase A (DAGLA) was increased while fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) did not change. As in control rats, MPEP and SR141716A also blocked WIN 55,212-2- induced effects in CCI rats. These data demonstrate a down regulation of the endocannabinoid system and a functional interaction between mGlu5 and CB 1 receptors for cannabinoid-mediated effect in the PAG-RVM pain circuitry in neuropathic pain inflicted rats.

The amygdala is a crucial area in controlling the threshold of pain and its emotional component. The present study has evaluated the effect of a metabotropic glutamate 8 receptor (mGluR8) stimulation in the central nucleus of the amygdala (CeA) on the thermoceptive threshold and on CeA serotonin (5-HT), glutamate (Glu), and GABA release in normal and carrageenan-induced inflammatory pain conditions in rats. Furthermore, the activity of rostral ventromedial medulla (RVM) putative “pronociceptive” ON and “antinociceptive” OFF cells has been evaluated. (S)-3,4-Dicarboxyphenylglycine [(S)-3,4-DCPG], a selective mGluR8 agonist, administered into the CeA, did not change 5-HT, Glu, and GABA release, or the thermoceptive threshold, nor did it modify the activity of ON and OFF cells of the RVM in normal animals. In rats treated with carrageenan, intra-CeA (S)-3,4-DCPG perfusion produced antinociception, and increased 5-HT and Glu, whereas it decreased GABA release. Intra-CeA (S)-3,4-DCPG inhibited ON and increased OFF cell activities. Furthermore, an increase inmGluR8gene, protein, and staining, the latter being associated with vesicular GABA transporter-positive profiles, has been found in the CeA after carrageenan-induced inflammatory pain. These results show that stimulation of mGluR8, which was overexpressed within the CeA in inflammatory pain conditions, inhibits nociceptive behavior. Such an effect is associated with an increase in 5-HT and Glu release, a decrease in GABA, and the inhibition of ON- and the stimulation of OFF-cell activities within RVM.

Background: Seminal vesicle protein number 4 (SV-IV) is a small, basic, multifunctional, intrinsically disordered secretory protein synthesized in large amounts by rat seminal vesicle epithelium under androgen transcriptional control. SV-IV-immunorelated proteins occur in other rat tissues and in humans. Methods: The in vitro effect of SV-IV on human FcΕRI+ cells was investigated by standard immunologic, biochemical and molecular biology procedures. Results: SV-IV-induced histamine release from human basophils and lung mast cells without any influence on leukotriene C4 release and cell migration. The histamine release rate was slower compared with that induced by anti-IgE, the temperature dependence of the event being similar. SV-IV-induced histamine release was Ca2+-dependent, suggesting a physiological interaction of the protein with FcΕRI+ cells. SV-IV and anti-IgE acted synergistically on the histamine release. SV-IV did not induce de novo synthesis of cytokines and growth factors (transforming growth factor-β1, interleukin-10, interleukin-13, tumor necrosis factor-α, vascular endothelial growth factor A) in FcΕRI+ cells. Conclusions: SV-IV protein induces in human FcΕRI+ cells the release of histamine, a proinflammatory, antiapoptotic and immunosuppressive biogenic amine. These data: (1) are consistent with the antiapoptotic and immunosuppressive properties of SV-IV; (2) confirm a regulatory feature of SV-IV on mammal inflammatory reactivity by either inhibiting the arachidonate cascade pathway or stimulating proinflammatory cytokine release from lymphocyte/monocytes and histamine from FcΕRI+ cells; (3) raise the possibility of a protective role of SV-IV on implanting hemiallogenic blastocysts against maternal reactive oxygen species and immunological attacks at the uterine implantation site.

Background Enhanced supraspinal glutamate levels following nerve injury are associated with pathophysiological mechanisms responsible for neuropathic pain. Chronic pain can interfere with specific brain areas involved in glutamate-dependent neuropsychological processes, such as cognition, memory, and decision-making. The medial prefrontal cortex (mPFC) is thought to play a critical role in pain-related depression and anxiety, which are frequent co-morbidities of chronic pain. Using an animal model of spared nerve injury (SNI) of the sciatic nerve, we assess bio-molecular modifications in glutamatergic synapses in the mPFC that underlie neuropathic pain-induced plastic changes at 30 days post-surgery. Moreover, we examine the effects of palmitoylethanolamide (PEA) administration on pain-related behaviours, as well as the cortical biochemical and morphological changes that occur in SNI animals. Results At 1 month, SNI was associated with mechanical and thermal hypersensitivity, as well as depression-like behaviour, cognitive impairments, and obsessive-compulsive activities. Moreover, we observed an overall glutamate synapse modification in the mPFC, characterized by changes in synaptic density proteins and amino acid levels. Finally, with regard to the resolution of pain and depressive-like syndrome in SNI mice, PEA restored the glutamatergic synapse proteins and changes in amino acid release. Conclusions Given the potential role of the mPFC in pain mechanisms, our findings may provide novel insights into neuropathic pain forebrain processes and indicate PEA as a new pharmacological tool to treat neuropathic pain and the related negative affective states. Graphical Abstract Palmitoylethanolamide: a new pharmacological tool to treat neuropathic pain and the related negative affective states. Electronic supplementary material The online version of this article (doi:10.1186/s13041-015-0139-5) contains supplementary material, which is available to authorized users.

In the current study, we have investigated the effect of CB 2 and TRPV 1 receptor ligands on in vitro osteoblasts from bone marrow of human healthy donors. A pivotal role for the endocannabinoid/endovanilloid system in bone metabolism has been highlighted. We have demonstrated a functional cross-talk between CB 2 and TRPV 1 in human osteoclasts, suggesting these receptors as new pharmacological target for the treatment of bone resorption disease as osteoporosis. Moreover, we have shown the presence of these receptors on human mesenchimal stem cells, hMSCs. Osteoblasts are mononucleated cells originated from hMSCs by the essential transcription factor runt-related transcription factor 2 and involved in bone formation via the synthesis and release of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. For the first time, we show that CB 2 and TRPV 1 receptors are both expressed on human osteoblasts together with enzymes synthesizing and degrading endocannabinoids/endovanilloids, and oppositely modulate human osteoblast activity in culture in a way that the CB 2 receptor stimulation improves the osteogenesis whereas TRPV 1 receptor stimulation inhibits it.

This study investigated the effects of a single administration of 6-(4-methoxyphenyl)-5-methyl-3-pyridinyl-4-isoxazolo[4,5-c]pyridin- 4(5H)-one (MMPIP), a negative allosteric modulator (NAM) of metabotropic glutamate receptor 7 (mGluR7), on pain and on affective and cognitive behavior in neuropathic mice. The activity of pyramidal neurons in the prelimbic cortex (PLC), which respond to stimulation of the basolateral amygdala (BLA) with either excitation or inhibition, was also investigated. The spared nerve injury (SNI) of the sciatic nerve induced, 14 days after surgery, thermal hyperalgesia and mechanical allodynia, reduced open-arm choice in the elevated plus-maze, increased time of immobility in the tail suspension, and increased digging and burying in the marble burying test. Cognitive performance was also significantly compromised in the SNI mice. Spared nerve injury induced phenotypic changes on pyramidal neurons of the PLC; excitatory responses increased, whereas inhibitory responses decreased after BLA stimulation. mGluR7 expression, mainly associated with vesicular glutamate transporter, increased in the hippocampus and decreased in the BLA, PLC, and dorsal raphe in SNI mice. MMPIP increased thermal and mechanical thresholds and open-arm choice. It reduced the immobility in the tail suspension test and the number of marbles buried and of digging events in the marble burying test. MMPIP also improved cognitive performance and restored the balance between excitatory and inhibitory responses of PLC neurons in SNI mice. 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one, XAP044, another selective mGluR7 NAM, reproduced the effects of MMPIP on thermal hyperalgesia, mechanical allodynia, tail suspension, and marble burying test. Altogether, these findings show that mGluR7 NAMs reduce pain responses and affective/cognitive impairments in neuropathic pain conditions. © 2015 International Association for the Study of Pain.

The present study investigated the role of metabotropic glutamate receptor subtype 8 (mGluR8) in the dorsal striatum (DS) in modulating thermonociception and rostral ventromedial medulla (RVM) ON and OFF cell activities in conditions of neuropathic pain induced by spared nerve injury (SNI) of the sciatic nerve in rats. The role of DS mGluR8 on mechanical allodynia was also investigated. Intra-DS ( S)-3,4-dicarboxyphenylglycine [( S)-3,4-DCPG], a selective mGluR8 agonist, did not modify the activity of the ON and OFF cells in sham-operated rats. In SNI rats, which showed a reduction of the mechanical withdrawal threshold, intra-DS microinjection of ( S)-3,4-DCPG inhibited the ongoing and tail flick-evoked activity of the ON cells while increasing the activity of the OFF cells. AZ12216052, a selective mGluR8 positive allosteric modulator (PAM), behaved like ( S)-3,4-DCPG in increasing tail flick latency and OFF cell activity and decreasing ON cell activity in SNI rats only but was less potent. VU0155041, a selective mGluR4 PAM, was ineffective in changing thermal nociception and ON and OFF cell activity in both sham-operated and SNI rats. ( S)-3,4-DCPG did not change mechanical withdrawal threshold in sham-operated rats but increased it in SNI rats. Furthermore, a decreased level of mGluR8 gene and immunoreactivity, expressed on GABAergic terminals, associated with a protein increase was found in the DS of SNI rats. These results suggest that stimulation of mGluR8 inhibits thermoceptive responses and mechanical allodynia. These effects were associated with inhibition of ON cells and stimulation of OFF cells within RVM.

Glycine is a candidate nociception inhibitory transmitter in specific brain regions, like for example the spinal cord, the thalamic nuclei and the periaqueductal gray matter. However, quantitative changes in glycine released in these brain regions during peripheral inflammation episodes have not been characterized in awake animals. To address this issue, an in vivo microdialysis study was carried out in freely moving rats in order to analyse periaqueductal gray matter extracellular glycine concentration following unilateral formalin injection into the dorsal skin of the right hind-paw. The extracellular concentration of glutamine was also evaluated in order to analyse whether or not a non-neurotransmitter amino acid was equally modified. Intra-periaqueductal gray matter tetrodotoxin perfusion reduced extracellular glycine concentration (−44±5%), but did not change the glutamine dialysate values. Peripheral injection of formalin reduced the glycine release during the early phase (−62±8%) and the late phase (−36±6%) of hyperalgesia, although not during the analgesic period. Perfusion with naloxone (300 μM) neither prevented the formalin-induced decreases in extacellular glycine concentration, nor modified the perfusate basal values of glycine and glutamine. These results show that, contrary to what has been recognized on the interactive role of opioids and GABA into the periaqueductal gray matter (i.e. opioid disinhibition), endogenous opioids seem not to modulate the activity of glycinergic neurons in the same midbrain area. In the light of these preliminary data, it is reasonable to suppose that GABA and glycine are probably not co-released at the level of periaqueductal gray matter of the rat.

Endothelins (ETs) are a family of peptide mediators that have a number of biological properties, including the ability to act as potent bronchoconstrictors of isolated human airways. Moreover, elevated concentrations of ET-1 in the bronchoalveolar lavage fluid from patients with symptomatic asthma have also been detected. We investigated the possible contribution of ET-1 in the development of bronchial hyperresponsiveness and the role of inflammatory cell accumulation in rabbit lungs. Our data show that ET-1 challenge to rabbits does not modify basal lung function but results in an increased airway responsiveness to inhaled histamine. Endothelin-treated rabbits were 3-fold (P

Eosinophils participate in the immune response against Helicobacter pylori, but little is known about their role in the gastritis associated to the infection. We recently demonstrated that the Hp(2-20) peptide derived from H. pylori accelerates wound healing of gastric mucosa by interacting with N-formyl peptide receptors (FPRs) expressed on gastric epithelial cells. The aim of the present study was to investigate whether eosinophils play a role in the repair of gastric mucosa tissue during H. pylori infection. Immunohistochemistry and transmission electron microscopy were used to detect eosinophils in gastric mucosal biopsies. Eosinophil re-distribution occurred in the gastric mucosa of H. pylori-infected patients: their density did not change in the deep mucosal layer, whereas it increased in the superficial lamina propria just below the foveolar epithelium; eosinophils entered the epithelium itself as well as the lumen of foveolae located close to the area harboring bacteria, which in turn were also engulfed by eosinophils. The H. pylori-derived peptide Hp(2-20) stimulated eosinophil migration through the engagement of FPR2 and FPR3, and also induced production of VEGF-A and TGF-β, two key mediators of tissue remodeling. We also demonstrate that Hp(2-20) in vivo induced eosinophil infiltration in rat gastric mucosa after injury brought about by indomethacin. This study suggests that eosinophil infiltrate could modulate the capacity of gastric mucosa to maintain or recover its integrity thereby shedding light on the role of eosinophils in H. pylori infection. Copyright © by BIOLIFE, s.a.s.

The metabotropic glutamate receptor 7 (mGluR7) negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), was locally microinjected into the ventrolateral periaqueductal gray (VL PAG) and the effect on pain responses in formalin and spare nerve injury (SNI) -induced neuropathic pain models was monitored in the rat. The activity of rostral ventromedial medulla (RVM) " pronociceptive" ON and " antinociceptive" OFF cells was also evaluated. Intra-VL PAG MMPIP blocked the first and second phase of nocifensive behaviour in the formalin pain model. MMPIP increased the tail flick latency and simultaneously increased the activity of the OFF cells while inhibiting that of ON cells in rats with SNI of the sciatic nerve. MMPIP failed to modify nociceptive responses and associated RVM ON and OFF cell activity in sham rats. An increase in mGluR7 gene, protein and staining, the latter being associated with vesicular glutamate transporter-positive profiles, has been found in the VL PAG in SNI rats. Blockade of mGluR7 within the VL PAG has an antinociceptive effect in formalin and neuropathic pain models. VL PAG mGluR7 blockade offers a target for dis-inhibiting the VL PAG-RVM pathway and silencing pain in inflammatory and neuropathic pain models. © 2013 Palazzo et al.; licensee BioMed Central Ltd.

Palmitoylethanolamide (PEA) is an endogenous cannabinoid-like compound in the central nervous system, which can modulate several functions in different pathological states, such as inflammation and pain response. We have here investigated the effect of PEA (5-10 mg/kg, intraperitoneally) on mechanical allodynia and thermal hyperalgesia 3 and 7 days following peripheral injection of formalin. Formalin induced a significant decrease of thermal and mechanical threshold in the injected and contralateral paw. PEA chronic treatment (once per day) significantly reduced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. Consistently, in vivo electrophysiological analysis revealed a significant increase of the duration and frequency, and a rapid decrease in the onset of evoked activity of the spinal nociceptive neurons 7 days after formalin. PEA normalized the electrophysiological parameters in a dose-dependent manner. Moreover, we investigated PEA effect on the glial/microglial phenotypical changes associated with spinal neuronal sensitization. We found that formalin induced a significant microglia and glia activation normalized by PEA, together with increased expression of glial interleukin 10. Finally, primary microglial cell cultures, conditioned with PEA or vehicle, where transplanted in naïve and formalin-treated mice, and nociceptive neurons were recorded. We observed that only PEA-conditioned cells normalized the activity of sensitized nociceptive neurons. In conclusion these data confirm the potent anti-inflammatory and anti-allodynic effect of PEA, and highlight a possible targeted microglial/glial effect of this drug in the spinal cord. © 2013 Bentham Science Publishers.

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1β release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1(-/-) mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1(-/-) mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid 2-arachidonoylglycerol. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB(1) agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-?? (PPAR-??) ligand, exerts antinociceptive and anti-inflammatory effects. PEA (3 and 6 nmol) was microinjected in the ventrolateral periaqueductal grey (VL PAG) of male rats and effects on nociceptive responses and ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) ON and OFF cells were recorded. Intra-PAG microinjection of PEA reduced the ongoing activity of ON and OFF cells and produced an increase in the latency of the nociceptive reaction. These effects were prevented by a selective PPAR-?? antagonist, GW6471 and by a large-conductance Ca(2+)-activated K(+) channel inhibitor, charybdotoxin. Cannabinoid 1 (CB(1)) receptor blockade by AM251 increased the PEA-induced effect both on the ongoing activity of the ON cell and on the latency to tail flick without affecting the effect of PEA on the OFF cell. Conversely, a transient receptor potential vanilloid type 1 (TRPV(1)) blocker, I-RTX, had no effect on the ON cell activity and tail flick latency, whereas it blocked the PEA-induced decrease in ongoing activity of the OFF cell. PEA decreased the burst and increased the latency of tail flick-evoked onset of ON cell activity in a manner antagonised by GW6471 and charybdotoxin. AM251 and I-RTX, instead, enhanced these latter effects. In conclusion, intra-VL PAG PEA induces antinociceptive effects associated with a decrease in RVM ON and OFF cell activities. PPAR-?? receptors mediate, and CB(1) and TRPV(1) receptors antagonise, PEA-induced effects within the PAG-RVM circuitry.

The receptor (CXCR4) for the stromal-derived factor-1 (SDF1) and the urokinase-receptor (uPAR) are up-regulated in various tumors. We show that CXCR4-transfected cells migrate toward SDF1 on collagen (CG) and do not on vitronectin (VN). Co-expression of cell-surface uPAR, which is a VN receptor, impairs SDF1-induced migration on CG and allows migration on VN. Blocking fMLP receptors (fMLP-R), alpha-v integrins or the uPAR region capable to interact with fMLP-Rs, impairs migration of uPAR/CXCR4-transfected cells on VN and restores their migration on CG. uPAR co-expression also reduces the adherence of CXCR4-expressing cells to various components of the extracellular matrix (ECM) and influences the partitioning of beta1 and alpha-v integrins to membrane lipid-rafts, affecting ECM-dependent signaling. uPAR interference in CXCR4 activity has been confirmed in cells from prostate carcinoma. Our results demonstrate that uPAR expression regulates the adhesive and migratory ability of CXCR4-expressing cells through a mechanism involving fMLP receptors and alpha-v integrins.

Human heart mast cells (HHMC), by elaborating vasoactive mediators, cytokines and chemokines, are the main primary effector cells of anaphylaxis. Mast cells have been identified perivascularly, close to myocytes and in the arterial intima in human heart tissue. Mast cells isolated from human heart tissue (HHMC) of patients undergoing cardiac transplantation express high-affinity receptors for IgE (FcepsilonRI) and C5a receptors. Activation of HHMC in vitro with anti-IgE or anti-FcepsilonRI induced the release of preformed mediators (histamine, tryptase, chymase, and renin) and the de novo synthesis of LTC(4) (approximately =18 ng/l0(6) cells) and PGD(2) (approximately =18 ng/l0(6) cells). Complement is activated and anaphylatoxin forms during anaphylaxis. C5a causes rapid release of histamine and tryptase from HHMC. These cells are activated in vitro by therapeutic (general anesthetics, protamine, etc.) and diagnostic agents (radiocontrast media, etc.) which can cause anaphylactoid reactions. Low concentrations of histamine and cysteinyl leukotrienes given to subjects undergoing diagnostic catheterization caused significant systemic and coronary hemodynamic effects. These results indicate that HHMC probably have a role in anaphylactic reactions.

The transient receptor potential vanilloid-1 (TRPV1) receptor is involved in peripheral and spinal nociceptive processing and is a therapeutic target for pain. We have shown previously that TRPV1 in the ventrolateral periaqueductal gray (VL-PAG) tonically contributes to brain stem descending antinociception by stimulating glutamate release into the rostral ventromedial medulla and off neuron activity. Because both opioid and vanilloid systems integrate and transduce pain sensation in these pathways, we studied the potential interaction between TRPV1 and μ-opioid receptors in the VL-PAG–rostral ventromedial medulla (RVM) system. We found that the TRPV1 agonist, capsaicin, and the μ-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin, when coadministered into the ventrolateral-PAG at doses nonanalgesic per se, produce 1) antinociception in tests of thermal nociception; 2) stimulation of glutamate release into the RVM; and 3) inhibition of on neuron activity in the RVM. These effects were all antagonized by the TRPV1 and opioid receptor antagonists 5′-iodo-resiniferatoxin and naloxone, respectively, thus suggesting the existence of a TRPV1–μ-opioid interaction in the VL-PAG–RVM system. By using double immunofluorescence techniques, we found that TRPV1 and μ-opioid receptors are coexpressed in several neurons of the VL-PAG. These findings suggest that μ-receptor activation not only acts on inhibitory neurons to disinhibit PAG output neurons but also interacts with TRPV1 activation at increasing glutamate release into the RVM, possibly by acting directly on PAG output neurons projecting to the RVM.

Helicobacter pylori-derived peptide RpL1 aa 2–20 (Hp(2–20)) in addition to its antimicrobial action exerts several immunomodulatory effects in eukaryotic cells by interacting with formyl peptide receptors (FPRs). It has recently been shown that activation of FPRs facilitates intestinal epithelial cell restitution. We investigated whether Hp(2–20) induces healing of injured gastric mucosa and assessed the mechanisms underlying any such effect. We investigated the expression of FPRs in two gastric epithelial cell lines (MKN-28 and AGS) at mRNA and protein level. To determine whether FPRs were functional we performed chemotaxis experiments and proliferation assays and studied the Hp(2–20)-activated downstream signaling pathway. The effect of Hp(2–20) on mucosal healing was evaluated in rats after indomethacin-induced injury. Here we show that: (1) FPRs were expressed in both cell lines; (2) Hp(2–20) stimulated migration and proliferation of gastric epithelial cells; (3) this effect was specifically mediated by formyl peptide receptor-like 1 (FPRL1) and FPRL2 and was associated with activation of FPR-related downstream signaling pathways; (4) Hp(2–20) up-regulated the expression and secretion of vascular endothelial growth factor; and (5) Hp(2–20) accelerated healing of rat gastric mucosa after injury brought about by indomethacin at both the macroscopic and microscopic levels. In conclusion, by interacting with FRPL1 and FPRL2, H. pylori-derived Hp(2–20) induces cell migration and proliferation, as well as the expression of vascular endothelial growth factor, thereby promoting gastric mucosal healing. This study provides further evidence of the complexity of the relationship between H. pylori and human gastric mucosa, and it suggests that a bacterial product may be used to heal gastric mucosal injury.

The effect of the non-selective, 1-aminoindan-1,5-dicarboxylic acid (AIDA), and selective (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl) methanone (JNJ16259685), metabotropic glutamate subtype 1 (mGlu1) receptor antagonists, on rat sciatic nerve chronic constrictive injury (CCI)-induced hyperalgesia, allodynia, spinal dorsal horn apoptosis, and gliosis was examined at 3 and 7 days post-injury. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), nestin, GFAP, and caspase-7 mRNA in the dorsal horn spinal cord by 3 days post-CCI. At 7 days post-CCI, only over-expression of bcl-2, nestin and GFAP mRNA was observed. Administration of AIDA reduced thermal hyperalgesia and mechanical allodynia at 3 and 7 days post-CCI; administration of JNJ16259685 reduced thermal hyperalgesia at 3 and 7 days post-CCI, but not mechanical allodynia. AIDA decreased the mRNA levels of bax, apaf-1, GFAP and caspase-7 genes. JNJ16259685 increased the mRNA levels of bcl-2 and GFAP gene, and decreased APAF-1 and caspases-7 genes. Inhibiting mGlu1 receptors also reduced TUNEL-positive profiles and immunohistochemical reactivity for caspase-7. We report here that despite inhibiting CCI-induced over-expression of pro-apoptotic genes in the spinal cord dorsal horn, the selective mGlu1 receptor antagonist JNJ16259685 exerted only a slight and transient allodynic effect. Moreover, JNJ16259685, but not the non-selective AIDA, increased astrogliosis which may account for its decreased analgesic efficacy. This study provides evidence that the contemporary and partial blockade of group I and likely ionotropic glutamate receptors may be a more suitable therapy than selective blockade of mGlu1 subtype receptors condition to decrease neuropathic pain symptoms. © 2008 Elsevier Ltd. All rights reserved.

Activation of transient receptor potential vanilloid-1 (TRPV1) channels in the periaqueductal gray (PAG) activates OFF antinociceptive neurons of the rostral ventromedial medulla (RVM). We examined in rats the effect of intra-ventrolateral (VL)-PAG injections of TRPV1 agonists and antagonists on the nocifensive response to heat in the plantar test, neurotransmitter (glutamate and GABA) release in the RVM, and spontaneous and tail flick-related activities of RVM neurons. The localization of TRPV1 in VL-PAG and RVM neurons was examined using various markers of glutamatergic and GABAergic neurons. Intra-VL-PAG injection of capsaicin increased the threshold of thermal pain sensitivity, whereas the selective TRPV1 antagonist 5′-iodo-resiniferatoxin (I-RTX) facilitated nociceptive responses, and blocked capsaicin analgesic effect at a dose inactive per se. Intra-VL PAG capsaicin evoked a robust release of glutamate in RVM microdialysates. I-RTX, at a dose inactive per se, blocked the effect of capsaicin, and inhibited glutamate release at a higher dose. Antinociception and hyperalgesia induced by capsaicin and I-RTX, respectively, correlated with enhanced or reduced activity of RVM OFF cells. Immunohistochemical analyses suggested that several TRPV1-immunoreactive (ir) neurons in both the VL-PAG and RVM are glutamatergic and surrounded by glutamatergic and GABAergic terminals. Our data suggest that VL-PAG neurons respond to TRPV1 stimulation by releasing glutamate into the RVM, thereby activating OFF cells and producing analgesia. The results obtained with the TRPV1 antagonist alone suggest that this pathway is tonically activated by endovanilloids.

N-arachidonoylserotonin (AA-5-HT, 1a) is an inhibitor of fatty acid amide hydrolase (FAAH) that acts also as an antagonist of transient receptor potential vanilloid-type 1 (TRPV1) channels and is analgesic in rodents. We modified the chemical structure of 1a with the aim of developing "hybrid" FAAH/TRPV1 blockers more potent than the parent compound or obtaining analogues with single activity at either of the two targets to study the mechanism of the analgesic action of 1a. Thirty-eight AA-5-HT analogues, containing a serotonin "head" bound to a variety of lipophilic moieties via amide, urea, or carbamate functionalities, were synthesized. Unlike 1a, most of the new compounds possessed activity at only one of the two considered targets. The amides 1b and 1c of alpha- and gamma-linolenic acid, however, showed "hybrid" activity similar to 1a. The carbamate 3f (OMDM106), although unable to antagonize TRPV1 receptors, was the most potent FAAH inhibitor in this study (IC50=0.5 microM). Compounds 3f and 1m (OMDM129), which exhibited activity at only FAAH or TRPV1, respectively, were 10-fold less potent than 1a at preventing formalin-induced hyperalgesia in mice.

The 911 amino acid band 3 (SLC4A1) is the major intrinsic membrane protein of red cells and is the principal Cl-/HCO3- exchanger. The N-terminal cytoplasmic domain of band 3 anchors the spectrin-based membrane skeleton to the lipid bilayer through its interaction with ankyrin and also binds glycolytic enzymes and hemoglobin. We identified a son of a consanguineous marriage with severe anemia in association with marked deficiency of band 3 (12% +/- 4% of normal). Direct nucleotide sequencing of SLC4A1 gene demonstrated a single base substitution (T --> C) at position + 2 in the donor splice site of intron 2, resulting in the generation of a novel mutant protein. Biochemical characterization of the mutant protein showed that it lacked the first 11 N-terminal amino acids (band 3 Neapolis). The expression of the mutant protein resulted in the complete absence of membrane-bound aldolase, and the mutant band 3 could not be tyrosine phosphorylated. The ability of the malarial parasite P falciparum to invade these red cells was significantly decreased. The identification of a novel band 3 mutant and its structural and functional characterization enabled us to identify pivotal roles for the 11 N-terminal amino acids in several protein functions and, in turn, in red-cell physiology.

Objective Studies of memory T cells transferred with the graft are relevant to better understand the early immune reconstitution of patients given autologous bone marrow transplantation (A-BMT). A critical question is whether memory T cells resident in bone marrow (BM) of patients with hematological malignancies are resistant to either pretransplant chemotherapy or ex vivo pharmacological purging. Patients and methods To address these issues, we evaluated the frequency of tetanus-toxoid (TT)-specific proliferating T-cell precursors (TT-PTCp) in BM and peripheral blood (PB) of eight patients with acute myeloid leukemia (AML) given A-BMT after in vitro purging of BM with mafosfamide. Patients were studied at the time of BM harvesting and five of them also after A-BMT. Results The range of TT-PTCp frequencies found after A-BMT were comparable with those observed in PB and in BM at the time of harvesting and did not differ significantly from those of eight age-matched healthy subjects who donated BM for a human leukocyte antigen-identical sibling. TT-PTCp frequencies in BM, studied before and after ex vivo purging, appeared not to be affected by incubation with mafosfamide. We also compared the T-cell receptor (TCR)-Vβ-repertoire usage of TT-specific T-cell lines (TT-TCL) in BM of patients at the time of harvesting and in their PB 2 months after transplantation. The same TCR-clonotypes were detected in TT-TCL at time of harvesting and after A-BMT. Conclusion These data indicate that BM-resident memory T cells of patients with AML are resistant to both pretransplant chemotherapy and ex vivo pharmacological purging and may contribute to immune reconstitution after A-BMT.

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10−12–10−9 M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84–95 (uPAR84–95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84–95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84–95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84–95, which is an endogenous ligand for FPRL2 and FPRL1.

In this study, we investigated the effects of group I metabotropic glutamate (mglu) receptor ligands on glutamate and gamma-aminobutyric acid (GABA) extracellular concentrations at the periaqueductal grey level by using in vivo microdialysis. An agonist of group I mglu receptors, (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG, 1 and 2 mM], as well as a selective agonist of mglu(5) receptors, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 2 and 4 mM), both increased dialysate glutamate and GABA concentrations. 7-(Hydroxyimino)cyclopropa-[b]-chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 1 mM), a selective mglu(1) receptor antagonist, and 2-methyl-6-(phenylethynyl)pyridine (MPEP, 0.5 mM), a selective mglu(5) receptor antagonist, perfused in combination with DHPG, antagonized the effect induced by DHPG on the extracellular glutamate and GABA concentrations. MPEP (0.5 mM), perfused in combination with CHPG, antagonized the increased glutamate and GABA extracellular levels induced by CHPG. MPEP (1 mM) decreased the extracellular concentrations of glutamate but did not modify the dialysate GABA concentrations. Moreover, as the intra-periaqueductal grey perfusion of (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [(RS)-CPP, 100 microM], a selective N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, did not change the extracellular concentrations of glutamate, this suggests that the MPEP-induced decrease in glutamate is not a consequence of NMDA receptor blockade. These data show that group I mglu receptors in the periaqueductal grey may modulate the release of glutamate and GABA in awake, freely moving rats. In particular, mglu(5), but not mglu(1), receptors seem to be functionally active on glutamate terminals.

This study investigates the effect of microinjections of capsaicin in the periaqueductal grey matter of rats on nociceptive behaviour and the possible interactions with NMDA and mGlu receptors. Intra-periaqueductal grey microinjection of capsaicin (1-3-6 nmol/rat) increased the latency of the nociceptive reaction in the plantar test. This effect was prevented by pretreatment with capsazepine (6 nmol/rat), which had no effect per se on the latency of the nociceptive reaction. 7-(Hydroxyimino)cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 50 nmol/rat) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 50 nmol/rat), antagonists of mGlu(1) and mGlu(5) receptors, respectively, completely blocked the effect of capsaicin. Similarly, pretreatment with DL-2-Amino-5-phosphonovaleric acid (DL-AP5, 5 nmol/rat) and riluzole (4 nmol/rat), an NMDA receptor antagonist and a voltage-dependent Na(+) channels blocker which inhibits glutamate release, respectively, completely antagonized the effect of capsaicin. However, pretreatment with (2S)-alpha-Ethylglutamic acid (30 nmol/rat) and (RS)-alpha-Methylserine-O-phosphate (MSOP, 30 nmol/rat), antagonists of group II and group III mGlu receptors, respectively, had no effects on capsaicin-induced analgesia. Similarly, pretreatment with N-(piperidin-1-yl)-5-(4-chlophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 5 pmol/rat), a selective cannabinoid CB(1) receptor antagonist, did not affect the capsaicin-induced antinociception. In conclusion, this study shows that capsaicin might produce antinociception at the periaqueductal grey level by increasing glutamate release, which activates postsynaptic group I mGlu and NMDA receptors.