Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells.

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

Optimized "in vitro" culture conditions for human rheumatoid arthritis synovial fibroblasts.

The composition of synovial fluid in rheumatoid arthritis (RA) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, "in vitro" studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform "in vitro" studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in "in vitro" studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

The POF1B candidate gene for premature ovarian failure regulates epithelial polarity.

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton.

Mu opioid receptor activation modulates Toll like receptor 4 in murine macrophages.

Opioids have been shown to affect both innate and adaptive immunity. We previously showed that morphine affects the macrophage production of pro-inflammatory cytokines after LPS in a NFkB dependent manner. Toll like receptors (TLRs) play a crucial role in the signaling pathways which lead to NFkB activation. TLR4 is considered the Lipopolysaccaride (LPS) receptor. The data here presented show that, in murine macrophages, morphine impacts on the immune function acting on the early step of pathogen recognition. Morphine, when added to RAW 264.7 cells and when injected into mice (s.c. 20mg/kg) is in fact able to decrease TLR4 both at mRNA and protein level in RAW cells and peritoneal macrophages. In the same cells, the mu opioid receptor (MOR) antagonist Naltrexone increases TLR4 levels, thus suggesting a role of the endogenous opioid system in TLR4 regulation. The effect of the two drugs is moreover lost in case of co-administration. Experiments with MOR KO mice and with DAMGO (MOR specific agonist) confirm that the effect of morphine on TLR4 mRNA in peritoneal macrophages is due to the MOR activation. Moreover the effect on TLR4 is blocked by PTX thus indicating the involvement of a G(i) protein after MOR binding. This work unveils a clear link between MOR activation and TLR4, suggesting a new possible mechanism at the basis of the peripheral immunosuppressive effect of opioids.

Sex-dependent differences in the secretome of human endothelial cells.

BACKGROUND: Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and "omics" approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. METHODS: We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. RESULTS: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. CONCLUSIONS: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

Desacyl-ghrelin and synthetic GH-secretagogues modulate the production of inflammatory cytokines in mouse microglia cells stimulated by beta-amyloid fibrils.

Data from Alzheimer's disease (AD) patients and AD animal models demonstrate the accumulation of inflammatory microglia at sites of insoluble fibrillar beta-amyloid protein (fAbeta) deposition. It is known that fAbeta binds to CD36, a type B scavenger receptor also involved in internalization of oxidized low-density lipoprotein (LDL), and initiate a signaling cascade that regulates microglial recruitment, activation, and secretion of inflammatory mediators leading to neuronal dysfunction and death. The recent demonstration of a binding site for the growth hormone secretagogues (GHS) on CD36 prompted us to ascertain whether ghrelin and synthetic GHS could modulate the synthesis of inflammatory cytokines in fAbeta-activated microglia cells. We demonstrate that N9 microglia cells express the CD36 and are a suitable model to study the activation of inflammatory cytokines synthesis. In fact, in N9 cells exposed to fAbeta(25-35) for 24 hr, the expression of interleukin (IL)-1beta and IL-6 mRNA significantly increased. Interestingly, 10(-7) M desacyl-ghrelin, hexarelin, and EP80317 in the nanomolar range effectively counteracted fAbeta(25-35) stimulation of IL-6 mRNA levels, whereas ghrelin was ineffective. Similarly, the effects of fAbeta(25-35) on IL-1beta mRNA levels were attenuated by desacyl-ghrelin, hexarelin, and EP80317, but not ghrelin. Because we have observed that the specific GHS receptor GHS-R1a is not expressed in N9 cells, the actions of GHS should be mediated by different receptors. Reportedly, hexarelin and EP80317 are capable of binding the CD36 in mouse macrophages and reducing atherosclerotic plaque deposition in mice. We conclude that desacyl-ghrelin, hexarelin, and EP80317 might interfere with fAbeta activation of CD36 in microglia cells.

Potential therapeutic effect of curcumin loaded hyalurosomes against inflammatory and oxidative processes involved in the pathogenesis of rheumatoid arthritis: The use of fibroblast-like synovial cells cultured in synovial fluid.

In the present work curcumin loaded hyalurosomes were proposed as innovative systems for the treatment of rheumatoid arthritis. Vesicles were prepared using a one-step and environmentally friendly method. Aiming at finding the most suitable formulation in terms of size, surface charge and stability on storage, an extensive pre-formulation study was performed using different type and amount of phospholipids. Curcumin loaded vesicles prepared with 180 mg/ml of Phospholipon 90G (P90G) and immobilized with sodium hyaluronate (2 mg/ml) were selected because of their small size (189 nm), homogeneous dispersion (PI 0.24), negative charge (-35 mV), suitable ability to incorporate high amount of curcumin (E% ∼88%) and great stability on storage. The in vitro study using fibroblast-like synovial cells cultured in synovial fluid, demonstrated the ability of these vesicles to downregulate the production of anti-apoptotic proteins IAP1 and IAP2 and stimulate the production of IL-10, while the production of IL-6 and IL-15 and reactive oxygen species was reduced, confirming their suitability in counteracting pathogenesis of rheumatoid arthritis.

The prokineticin receptor agonist Bv8 decreases IL-10 and IL-4 production in mice splenocytes by activating prokineticin receptor-1.

BACKGROUND: Bv8, prokineticin-1, or endocrine gland-vascular endothelial growth factor, and prokineticin-2 are recently isolated peptide agonists of two G protein-coupled receptors, prokineticin receptor-1 (PROKR 1) and PROKR 2, and have been described as affecting a number of myeloid cell functions. We evaluated the impact of Bv8 on lymphoid cells by investigating its ability to modulate T cell cytokine balance in mouse. RESULTS: The production of T-helper1 cytokines (IL-2, IFN-gamma and IL-1beta), the T-helper 2 cytokine IL-4, and the anti-inflammatory cytokine IL-10 by mouse splenocytes was evaluated after polyclonal stimulation or immunisation with the keyhole limpet hemocyanin protein antigen by measuring cytokine levels. When added in vitro to Con-A-stimulated splenocytes, Bv8 significantly increased IL-1beta and decreased IL-4 and IL-10; IL-2 and IFN-gamma were not affected. Similar results were obtained when Bv8 was administered in vivo. In KLH-immunised mice, splenocytes restimulated in vitro with KLH and Bv8 produced significantly smaller amounts of IL-4 and IL-10. KLH-induced IL-10 and IL-4 production was also significantly blunted in animals administered Bv8 in vivo at the time of KLH immunisation or two weeks later. The Bv8-induced effects were lost in mice lacking the PROKR 1 gene, thus indicating that PROKR 1 is the receptor involved in the modulation of cytokines. CONCLUSION: These findings indicate that Bv8/prokineticin-1 is a novel modulator of lymphoid functions, and may be a suitable target for new immunopharmacological strategies.

Differential involvement of RelB in morphine-induced modulation of chemotaxis, NO, and cytokine production in murine macrophages and lymphocytes.

Acute morphine impairs innate and acquired immunity. The mechanisms involved in immunosuppression have not been well defined yet. The transcription factor NF-kappaB is a central regulator of immunity, and of the NF-kappaB family, RelB is particularly involved in the expression of genes important in immune responses. We investigated the involvement of RelB in morphine-induced immunosuppression in mice deficient for the RelB factor. RelB-/- mice and wild-type (WT) controls were injected s.c. with morphine 20 mg/Kg, and 1 h later, immune parameters were evaluated. Morphine significantly reduced macrophage production of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-12 in WT animals, and the drug failed to diminish the production of these cytokines in the RelB-/- mice. In contrast, the anti-inflammatory cytokine IL-10 was similarly affected in the two strains. Macrophage NO production was modulated by morphine in WT animals only, and morphine similarly decreased macrophage chemotaxis in the presence or in the absence of RelB. When Th1 and Th2 cytokines were evaluated, we observed a clear morphine-induced reduction of IL-2 and IFN-gamma production by WT splenocytes, whereas no effect of the drug was observed in RelB-/- mice. On the contrary, the production of the Th2 cytokines IL-4 and IL-10 was lessened to the same degree by morphine in WT and RelB-/- mice. In conclusion, our data suggest that RelB is an important target for morphine modulation of proinflammatory and Th1 cytokines. They also indicate that morphine uses multiple intracellular pathways to exert its generalized immunosuppression.

Anti-inflammatory Effect of Somatostatin Analogue Octreotide on Rheumatoid Arthritis Synoviocytes.

Somatostatin and its analogues are known to have modulatory effects on immune response and their anti-proliferative, anti-angiogenic, and analgesic properties make them attractive candidates for a therapeutic use in immune-mediated diseases, such as rheumatoid arthritis. Here, we demonstrate the ability of the somatostatin analogue octreotide to inhibit interleukin-15 and to increase interleukin-10 production by rheumatoid arthritis fibroblast-like synovial cells maintained in a chronic inflammatory state. We also prove that the inhibitory effect of octreotide on interleukin-15 and tumor necrosis factor-α production depended on the increase in interleukin-10, since neutralizing anti-interleukin-10 antibody was able to partially reverse this inhibition. In addition, our observations suggest an octreotide control on purinergic signaling, with an inhibitory effect on purinergic P2X and P2Y receptors activation. This would have great implications, considering the roles of P2 receptors in the onset of inflammation. Data here reported extend knowledge on the biological action of octreotide and underline its multiple effects on immune response, which could make octreotide an attractive and valid support for the therapy of diseases where several inflammatory mediators are involved, such as rheumatoid arthritis, and in which the simultaneous action on different aspects can be a successful strategy.

Potent Antiglioblastoma Agents by Hybridizing the Onium-Alkyloxy-Stilbene Based Structures of an α7-nAChR, α9-nAChR Antagonist and of a Pro-Oxidant Mitocan.

Adenocarcinoma and glioblastoma cell lines express α7- and α9α10-containing nicotinic acetylcholine receptors (nAChRs), whose activation promotes tumor cell growth. On these cells, the triethylammoniumethyl ether of 4-stilbenol MG624, a known selective antagonist of α7 and α9α10 nAChRs, has antiproliferative activity. The structural analogy of MG624 with the mitocan RDM-4'BTPI, triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridization among their three substructures (stilbenoxy residue, alkylene linker, and terminal onium) and elongation of the alkylene linker might result in novel antitumor agents with higher potency and selectivity. We found that lengthening the ethylene bridge in the triethylammonium derivatives results in more potent and selective toxicity toward adenocarcinoma and glioblastoma cells, which was paralleled by increased α7 and α9α10 nAChR antagonism and improved ability of reducing mitochondrial ATP production. Elongation of the alkylene linker was advantageous also for the triphenylphosphonium derivatives resulting in a generalized enhancement of antitumor activity, associated with increased mitotoxicity.

Characterization of a Monoclonal Antibody Specific for the Growth Hormone Secretagogue Receptor.

Ghrelin is an orexigenic peptide hormone that primarily regulates growth hormone secretion, food intake, and energy homeostasis. It has been shown to also play a role in numerous higher brain functions, such as the regulation of inflammation and cell proliferation. Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor highly expressed in brain and detectable in some peripheral tissues. The wide distribution of ghrelin receptor and the number of tissues and cell types known to respond to ghrelin suggest that a number of systems may be affected by treatment with this hormone or its analogues. In this study, we characterized a new GHSR specific monoclonal antibody recognizing specifically the ghrelin receptor. This could be a useful tool for immunoassays aimed at obtaining insights into the physiological and pathological significance of the GHSR/ghrelin system.

Specific Monoclonal Antibody Against Bcr/Abl Out-of-Frame Alternative Proteins as Diagnostic Tool in Chronic Myelogenous Leukemia Patients.

More recently, alternative splicing of specific genes are investigated for their therapeutic potential. In particular, we reported the existence of BCR-ABL alternative splicing isoforms, in about 80% of Philadelphia-positive patients, which lead to the expression of aberrant proteins. These fusion proteins are characterized by an orphan initial and correct Bcr portion attached to a 112 amino acid sequence, arising from the impairment in the reading frame (reading of ABL exon 4 and 5). We demonstrated that these Abl-out-of-frame (OOF) isoforms could have an immunological role with therapeutic implications. The aim of this study was to characterize a new monoclonal antibody (MAb) specific for Abl-OOF protein portion, for diagnostic use, to detect this biomarker in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) patients and to generate novel approaches in the immunotherapy. 5F11G11 MAb recognizes the OOF protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence. In addition, we demonstrate the MAb's ability to recognize the alternative hybrid Bcr/Abl fusion protein expressed in leukemic cells from CML patients, to support the possible use of 5F11G11 MAb as a diagnostic tool to select patients with Philadelphia chromosome-positive CML that could be eligible for an immunotherapeutic approach with this new antigen.

Regulation of human peripheral blood lymphocytes IL-10 BY SMS 201-995.

The somatostatin analog SMS 201-995 inhibits human peripheral blood lymphocytes (PBL) proliferation and here we demonstrate that it induces a significant increase in T cells IL-10 release as is evidenced in double fluorescence experiments. Seizing IL-10 by monoclonal antibody, SMS does not affect lymphocyte proliferation, suggesting that this cytokine is involved in the antiproliferative effect of these analog. We previously demonstrated that SMS inhibits T cells acting on the CD28 rather than the CD3-mediated signal in exactly the same way as does IL-10. Thus SMS inhibits human PBL activation by inducing IL-10 release and the consequent inhibition of the CD28 co-stimulatory pathway providing new perspectives on developing immunosuppressive strategies.

The expression of GHS-R in primary neurons is dependent upon maturation stage and regional localization.

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer's disease.

Immunologic evaluation of peptides derived from BCR/ABL-out-of-frame fusion protein in HLA A2.1 transgenic mice.

Philadelphia chromosome-positive chronic myelogenous leukemia and acute lymphocytic leukemia express, besides the main BCR/ABL transcripts, novel BCR/ABL transcripts derived from alternative splicing between BCR exons 1, 13, or 14 with ABL exons 4 and 5. Their translational products present at C-terminus an amino acid portion derived from out-of-frame (OOF) reading of the ABL gene. The presence of OOF-peptide-specific T cells in chronic myelogenous leukemia patients was demonstrated and a first study in in vivo model demonstrated that OOF ABL portion was immunogenic in human leukcocyte antigen (HLA)-A2.1 transgenic mice. Here we immunized HLA A2.1 mice with novel peptides designed on the ABL OOF sequence, containing epitopes with high affinity for HLA A2.1 molecule. The specific immune response, cellular and humoral, obtained ex vivo against HLA A2.1-positive human chronic myelogenous leukemia cells using peptide 22-53 and the cytotoxic activity induced by peptide 32mer confirm the possibility to use the ABL OOF portion as target to evoke a specific and multiple immune response in Philadelphia positive leukemic patients in cytogenetic remission.

Characterization of a monoclonal antibody specific for novel Bcr/Abl out-of-frame fusion proteins.

The new tumor-specific antigens Bcr/Abl-OOF, identified in Philadelphia chromosome (Ph)-positive leukemia cells, are derived from an alternative splicing event involving BCR exons 1, 13, or 14 and ABL exons 4 and 5. The COOH-terminus of these transcription products contain an amino acid portion derived from an out-of-frame (OOF) reading of the ABL gene; these variants are expressed in Ph-positive chronic myelogenous leukemia (CML) and acute lymphocytic leukemia patients. Previously, we confirmed the presence of out-of-frame peptide-specific T cells in the peripheral blood of CML patients with the ability to lyse primary autologous CML cells. We also demonstrated that the out-of-frame Abl portion was immunogenic in HLA-A2.1 transgenic mice. Here we describe the production and characterization of monoclonal antibody 1D8G8, a new tool for localization and functional studies of the tumor antigen Bcr/Abl-OOF. This antibody recognizes the out-of-frame protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence, and binds to a specific epitope of this antigen presented in association with HLA-A2.1 molecules at the surface of these cells, as demonstrated by flow cytometry. Thus this MAb could be useful to better understand how this new protein presents in Ph-positive cells beside the canonical Bcr/Abl fusion proteins.