Gluten induces rapid reprogramming of natural memory αß and γδ intraepithelial T cells to induce cytotoxicity in celiac disease.

Celiac disease (CD) is an autoimmune disease in which intestinal inflammation is induced by dietary gluten. The means through which gluten-specific CD4+ T cell activation culminates in intraepithelial T cell (T-IEL)-mediated intestinal damage remain unclear. Here, we performed multiplexed single-cell analysis of intestinal and gluten-induced peripheral blood T cells from patients in different CD states and healthy controls. Untreated, active, and potential CD were associated with an enrichment of activated intestinal T cell populations, including CD4+ follicular T helper (TFH) cells, regulatory T cells (Tregs), and natural CD8+ αß and γδ T-IELs. Natural CD8+ αß and γδ T-IELs expressing activating natural killer cell receptors (NKRs) exhibited a distinct TCR repertoire in CD and persisted in patients on a gluten-free diet without intestinal inflammation. Our data further show that NKR-expressing cytotoxic cells, which appear to mediate intestinal damage in CD, arise from a distinct NKR-expressing memory population of T-IELs. After gluten ingestion, both αß and γδ T cell clones from this memory population of T-IELs circulated systemically along with gluten-specific CD4+ T cells and assumed a cytotoxic and activating NKR-expressing phenotype. Collectively, these findings suggest that cytotoxic T cells in CD are rapidly mobilized in parallel with gluten-specific CD4+ T cells after gluten ingestion.

Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a.

Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a-PG complex showed that the acyl chains were buried within the A'- and F'-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F'-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a-(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.

Matrix metalloproteinase-2 mediates ribosomal RNA transcription by cleaving nucleolar histones.

Cell proliferation and survival require continuous ribosome biogenesis and protein synthesis. Genes encoding ribosomal RNA are physically located in a specialized substructure within the nucleus known as the nucleolus, which has a central role in the biogenesis of ribosomes. Matrix metalloproteinase-2 was previously detected in the nucleus, however, its role there is elusive. Herein we report that matrix metalloproteinase-2 resides within the nucleolus to regulate ribosomal RNA transcription. Matrix metalloproteinase-2 is enriched at the promoter region of ribosomal RNA gene repeats, and its inhibition downregulates preribosomal RNA transcription. The N-terminal tail of histone H3 is clipped by matrix metalloproteinase-2 in the nucleolus, which is associated with increased ribosomal RNA transcription. Knocking down/out matrix metalloproteinase-2, or inhibiting its activity, prevents histone H3 cleavage and reduces both ribosomal RNA transcription and cell proliferation. In addition to the known extracellular roles of matrix metalloproteinase-2 in tumor growth, our data reveal an epigenetic mechanism whereby intranucleolar matrix metalloproteinase-2 regulates cell proliferation through histone clipping and facilitation of ribosomal RNA transcription.

Updates on the hepatocyte growth factor/c-Met axis in hepatocellular carcinoma and its therapeutic implications.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and is the second leading cause of cancer death. Since the diagnosis of HCC is difficult, in many cases patients with HCC are diagnosed advanced stage of development. Hepatocyte growth factor (HGF)/c-mesenchymal-epithelial transition receptor (c-Met) axis is a key signaling pathway in HCC, either via canonical or non-canonical pathways. Available treatments against HCC based upon HGF/c-Met inhibition can increase patient lifespan, but do not reach the expected therapeutic benefits. In HCC, c-Met monomers can bind other receptor monomers, activating several noncanonical signaling pathways, leading to increased cell proliferation, invasion, motility, and drug resistance. All of these processes are enhanced by the tumor microenvironment, with stromal cells contributing to boost tumor progression through oxidative stress, angiogenesis, lymphangiogenesis, inflammation, and fibrosis. Novel treatments against HCC are being explored to modulate other targets such as microRNAs, methyltransferases, and acetyltransferases, which are all involved in the regulation of gene expression in cancer. This review compiles basic knowledge regarding signaling pathways in HCC, and compounds already used or showing potential to be used in clinical trials.

(+)-Aeroplysinin-1 Modulates the Redox Balance of Endothelial Cells.

The bioactive natural compound from marine origin, (+)-aeroplysinin-1, has been shown to exhibit potent anti-inflammatory and anti-angiogenic effects. The aim of the present study was to identify new targets for (+)-aeroplysinin-1 in endothelial cells. The sequential use of 2D-electrophoresis and MALDI-TOF-TOF/MS allowed us to identify several differentially expressed proteins. Four of these proteins were involved in redox processes and were validated by Western blot. The effects of (+)-aeroplysinin-1 were further studied by testing the effects of the treatment with this compound on the activity of several anti- and pro-oxidant enzymes, as well as on transcription factors involved in redox homeostasis. Finally, changes in the levels of total reactive oxygen species and mitochondrial membrane potential induced by endothelial cell treatments with (+)-aeroplysinin-1 were also determined. Taken altogether, these findings show that (+)-aeroplysinin-1 has multiple targets involved in endothelial cell redox regulation.

In silico prediction of targets for anti-angiogenesis and their in vitro evaluation confirm the involvement of SOD3 in angiogenesis.

Biocomputational network approaches are being successfully applied to predict and extract previously unknown information of novel molecular components of biological systems. In the present work, we have used this approach to predict new potential targets of anti-angiogenic therapies. For experimental validation of predictions, we made use of two in vitro assays related to two key steps of the angiogenic process, namely, endothelial cell migration and formation of "tubular-like" structures on Matrigel. From 7 predicted candidates, experimental tests clearly show that superoxide dismutase 3 silencing or blocking with specific antibodies inhibit both key steps of angiogenesis. This experimental validation was further confirmed with additional in vitro assays showing that superoxide dismutase 3 blocking produces inhibitory effects on the capacity of endothelial cells to form "tubular-like" structure within type I collagen matrix, to adhere to elastin-coated plates and to invade a Matrigel layer. Furthermore, angiogenesis was also inhibited in the en vivo aortic ring assay and in the in vivo mouse Matrigel plug assay. Therefore, superoxide dismutase 3 is confirmed as a putative target for anti-angiogenic therapy.

Hydroxytyrosol targets extracellular matrix remodeling by endothelial cells and inhibits both ex vivo and in vivo angiogenesis.

The health benefits of olive oil are attributed to their bioactive compounds, such as hydroxytyrosol. Previously, we demonstrated that hydroxytyrosol inhibits angiogenesis in vitro. The present study aimed to: i) get further insight into the effects of hydroxytyrosol on extracellular matrix remodeling; and ii) test whether hydroxytyrosol is able to inhibit angiogenesis ex vivo and in vivo. Hydroxytyrosol induced a shift toward inhibition of proteolysis in endothelial cells, with decreased expression of extracellular matrix remodeling-enzyme coding genes and increased levels of some of their inhibitors. Furthermore, this work demonstrated that hydroxytyrosol, at concentrations within the range of its content in virgin olive oil that can be absorbed from moderate and sustained virgin olive oil consumption, is a strong inhibitor of angiogenesis ex vivo and in vivo. These results suggest the need for translational studies to evaluate the potential use of hydroxytyrosol for angio-prevention and angiogenesis inhibition in clinical setting.

The noni anthraquinone damnacanthal is a multi-kinase inhibitor with potent anti-angiogenic effects.

The natural bioactive compound damnacanthal inhibits several tyrosine kinases. Herein, we show that -in fact- damancanthal is a multi kinase inhibitor. A docking and molecular dynamics simulation approach allows getting further insight on the inhibitory effect of damnacanthal on three different kinases: vascular endothelial growth factor receptor-2, c-Met and focal adhesion kinase. Several of the kinases targeted and inhibited by damnacanthal are involved in angiogenesis. Ex vivo and in vivo experiments clearly demonstrate that, indeed, damnacanthal is a very potent inhibitor of angiogenesis. A number of in vitro assays contribute to determine the specific effects of damnacanthal on each of the steps of the angiogenic process, including inhibition of tubulogenesis, endothelial cell proliferation, survival, migration and production of extracellular matrix remodeling enzyme. Taken altogether, these results suggest that damancanthal could have potential interest for the treatment of cancer and other angiogenesis-dependent diseases.

Aeroplysinin-1, a Sponge-Derived Multi-Targeted Bioactive Marine Drug.

Organisms lacking external defense mechanisms have developed chemical defense strategies, particularly through the production of secondary metabolites with antibiotic or repellent effects. Secondary metabolites from marine organisms have proven to be an exceptionally rich source of small molecules with pharmacological activities potentially beneficial to human health. (+)-Aeroplysinin-1 is a secondary metabolite isolated from marine sponges with a wide spectrum of bio-activities. (+)-Aeroplysinin-1 has potent antibiotic effects on Gram-positive bacteria and several dinoflagellate microalgae causing toxic blooms. In preclinical studies, (+)-aeroplysinin-1 has been shown to have promising anti-inflammatory, anti-angiogenic and anti-tumor effects. Due to its versatility, (+)-aeroplysinin-1 might have a pharmaceutical interest for the treatment of different pathologies.

Damnacanthal inhibits IgE receptor-mediated activation of mast cells.

Damnacanthal, an anthraquinone obtained from the noni fruit (Morinda citrifolia L.), has been described to possess anti-cancer and anti-inflammatory properties. Since mast cells are key players in various inflammatory conditions as well as in cancer, we considered the possibility that the biological actions of damnacanthal, at least partly, could be due to effects on mast cells. Many of the biological activities of mast cells are mediated by IgE receptor cross-linking, which results in degranulation with release of preformed granule mediators, as well as de novo synthesis and release of additional compounds. Here we show that damnacanthal has profound inhibitory activity on mast cell activation through this pathway. The release of the granule compounds beta-hexosaminidase and tryptase release was completely abrogated by damnacanthal at doses that were non-toxic to mast cells. In addition, damnacanthal inhibited activation-dependent pro-inflammatory gene induction, as well as cytokine/chemokine release in response to mast cell stimulation. The mechanism underlying damnacanthal inhibition was linked to impaired phosphorylation of Syk and Akt. Furthermore, damnacanthal inhibited mast cell activation in response to calcium ionophore A23187. Altogether, the data presented here demonstrate that damnacanthal inhibits mast cell activation induced by different stimuli and open a new window for the use of this compound as a mast cell stabilizer.

Damnacanthal, a noni anthraquinone, inhibits c-Met and is a potent antitumor compound against Hep G2 human hepatocellular carcinoma cells.

Damnacanthal, an anthraquinone present in noni plants, targets several tyrosine kinases and has antitumoral effects. This study aims at getting additional insight on the potential of damnacanthal as a natural antitumor compound. The direct effect of damnacanthal on c-Met was tested by in vitro activity assays. Additionally, Western blots of c-Met phosphorylation in human hepatocellular carcinoma Hep G2 cells were performed. The antitumor effects of damnacanthal were tested by using cell growth, soft agar clonogenic, migration and invasion assays. Their mechanisms were studied by Western blot, and cell cycle, apoptosis and zymographic assays. Results show that damnacanthal targets c-Met both in vitro and in cell culture. On the other hand, damnacanthal also decreases the phosphorylation levels of Akt and targets matrix metalloproteinase-2 secretion in Hep G2 cells. These molecular effects are accompanied by inhibition of the growth and clonogenic potential of Hep G2 hepatocellular carcinoma cells, as well as induction of Hep G2 apoptosis. Since c-Met has been identified as a new potential therapeutical target for personalized treatment of hepatocellular carcinoma, damnacanthal and noni extract supplements containing it could be potentially interesting for the treatment and/or chemoprevention of hepatocellular carcinoma through its inhibitory effects on the HGF/c-Met axis.

4-methylumbelliferone inhibits angiogenesis in vitro and in vivo.

4-Methylumbelliferone (4-MU) is a hyaluronic acid biosynthesis inhibitor with antitumoral and antimetastatic effects. The objective of the present study was to determine the potential of 4-MU as an antiangiogenic compound. To fulfill this aim, cultured endothelial cells were used to perform an array of in vitro assays, as well as two different in vivo angiogenesis assays. This study demonstrates that, in fact, 4-MU behaves as a new inhibitor of both in vitro and in vivo angiogenesis. In vitro, 4-MU affects several key steps of angiogenesis, including endothelial cell proliferation, adhesion, tube formation, and extracellular matrix remodeling. Half-maximal inhibitory concentrations (IC50) values in the proliferation assay were 0.65 ± 0.04 and 0.37 ± 0.03 mM for HMEC and RF-24 endothelial cells, respectively. 4-MU (2 mM) treatment for 24 h induced apoptosis in 13% of HMEC and 5% of RF-24 cells. The number of adherent endothelial cells decreased by >20% after 24 h of treatment with 1 mM 4-MU. Minimal inhibitory concentrations in the tube formation assay were 2 and 0.5 mM 4-MU for HMEC and RF-24, respectively. Matrix metalloproteinase-2 expression was differentially altered upon 4-MU treatment in both tested endothelial cell lines. Taken together, the results suggest that 4-MU may have potential as a new candidate multitargeted bioactive compound for antiangiogenic therapy.

The brominated compound aeroplysinin-1 inhibits proliferation and the expression of key pro- inflammatory molecules in human endothelial and monocyte cells.

Aeroplysinin-1 is a brominated antibiotic used by some sponges for defense against bacterial pathogen invasion. Aeroplysinin-1 has a wide spectrum of anti-tumoral action and behaves as a potent anti-angiogenic compound for bovine aortic endothelial cells. In this study, we demonstrate anti-angiogenic effects of aeroplysinin-1 on human endothelial cells. Furthermore, the response of angiogenesis related genes to aeroplysinin-1 treatment was studied in human endothelial cells by using gene arrays. The major changes were observed in thrombospondin 1 (TSP-1) and monocyte chemoattractant protein-1 (MCP-1), both of which were down-regulated. These inhibitory effects of aeroplysinin-1 were confirmed by using independent experimental approaches. To have a deeper insight on the anti-inflammatory effects of aeroplysinin-1 in endothelial cells, cytokine arrays were also used. This experimental approach confirmed effects on MCP-1 and TSP-1 and showed down-regulation of several other cytokines. Western blotting experiments confirmed down-regulation of ELTD1 (EGF, latrophilin and seven transmembrane domain-containing protein 1), interleukin 1α and matrix metalloproteinase 1 (MMP-1). These results along with our observation of a dramatic inhibitory effect of aeroplysinin-1 on cyclooxygenase-2 protein expression levels in endothelial cells and a human monocyte cell line suggest that aeroplysinin-1 could be a novel anti-inflammatory compound with potential pharmacological interest.