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Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN-α

2011-12-20T16:40:39Z

Title: Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN-α Author(s) : Rouzaut, A. (Ana); Garasa, S. (Saray); Teijeira, A. (Álvaro); Gonzalez, I. (Iranzu); Martinez-Forero, I. (Iván); Suarez, N. (Natalia); Larrea, E. (Esther); Alfaro, C. (Carlos); Palazon, A. (Asís); Dubrot, J. (Juan); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio) Abstract: Migration of DC into lymphatic vessels ferries antigenic cargo and pro-inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF-α were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-α2b or IFN-α5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro-adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA-1. Furthermore, incubation of DC with IFN-α led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN-α also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.

Repetitive nicotine exposure leads to a more malignant and metastasis-prone phenotype of SCLC: a molecular insight into the importance of quitting smoking during treatment

2011-12-20T15:07:21Z

Title: Repetitive nicotine exposure leads to a more malignant and metastasis-prone phenotype of SCLC: a molecular insight into the importance of quitting smoking during treatment Author(s) : Martinez-Garcia, E. (Eva); Irigoyen, M. (Marta); Gonzalez-Moreno, O. (Óscar); Corrales, L. (Leticia); Teijeira, A. (Álvaro); Salvo, E. (Elizabeth); Rouzaut, A. (Ana) Abstract: Cigarette smoking is strongly correlated with the onset of lung cancer. Nicotine, a major component in cigarette smoke, has been found to promote tumor growth and angiogenesis, as well as protect cancer cells from apoptosis. Among all lung cancer cases, small cell lung cancer (SCLC) is found almost exclusively in smokers; metastasis and chemoresistance are the main reasons for the high mortality rates associated with SCLC. Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnosis display lower response rates and a shorter median survival compared with those who stop smoking. In the current work, we examined the effects of acute and repetitive exposure to nicotine, in the concentrations found in the lungs of active smokers, on the malignant properties of N417 SCLC cells in vitro. We observed that repetitive nicotine exposure induced a neuronal-like appearance in N417 cells along with increased adhesion to the extracellular matrix and chemoresistance. These changes were accompanied by enhanced migration through collagen matrices and adhesion to and transmigration across lymphatic endothelial cell monolayers. SCLC differentiation reverted after cessation of nicotine exposure. Here, we provide evidence for the leading role of the CXCR4/CXCL12 axis in these phenomena. Finally, we show how nicotine-differentiated N417 cells produced bigger and more vascularized tumors in mice, with lower apoptotic rates, than their nondifferentiated counterparts. In short, these findings identify the mechanisms through which nicotine increases SCLC malignancy and provide further evidence that CXCR4 is a potential anticancer target for nicotine-associated SCLC.

Identity between the PCPH proto-oncogene and the CD39L4 (ENTPD5) ectonucleoside triphosphate diphosphohydrolase gene

2012-04-27T10:18:33Z

Title: Identity between the PCPH proto-oncogene and the CD39L4 (ENTPD5) ectonucleoside triphosphate diphosphohydrolase gene Author(s) : Paez, J.G. (J. Guillermo); Recio, J.A. (Juan A.); Rouzaut, A. (Ana); Notario, V. (Vicente) Abstract: PCPH was initially defined as a proto-oncogene on the basis of its frequent detection as an activated oncogene in tumorigenic Syrian hamster embryo fibroblast cell lines converted to the neoplastic state by a single treatment with the carcinogen 3-methylcholanthrene (MC). Further studies identified the translation product of the PCPH gene as a ribonucleotide-binding protein with special affinity for ribonucleoside diphosphates. Later, we showed that the PCPH protein was homologous to the product of the yeast GDA1 gene and demonstrated that it had intrinsic guanosine diphosphatase activity, although it did not complement the disrupted phenotype when expressed in gda1 null Saccharomyces cerevisiae strains. These results indicated that the primary function of PCPH was unlikely to be related to the ribonucleotide recycling function that its yeast counterpart performs in the Golgi during the process of protein glycosylation. However, taken together, our data strongly suggested that the normal cellular function of PCPH was related to ribonucleotide metabolism. We now report that PCPH is structurally and functionally identical to the mammalian ectonucleoside triphosphate diphosphohydrolase CD39L4 (ENTPD5), recently described as a member of the lymphoid activation antigen () CD39 protein family. These results may help to establish the normal cellular function of the PCPH proto-oncogene product and its role in neoplastic development during carcinogenesis.

Co-expression of inducible nitric oxide synthase and arginases in different human monocyte subsets. Apoptosis regulated by endogenous NO

2012-04-04T12:37:27Z

Title: Co-expression of inducible nitric oxide synthase and arginases in different human monocyte subsets. Apoptosis regulated by endogenous NO Author(s) : Rouzaut, A. (Ana); Subira, M.L. (María L.); Miguel, C. (Carlos) de; Domingo-de-Miguel, E. (Eduardo); Gonzalez, A. (Alvaro); Santiago, E. (Esteban); Lopez-Moratalla, N. (Natalia) Abstract: Human monocyte subsets, isolated from cultures of mononuclear cells, or freshly obtained from patients with multiple sclerosis, Graves' disease or pemphigus vulgaris, differed in phenotype, apoptotic features, mRNA levels of arginase II (A-II) and the inducible form of nitric oxide synthase (iNOS). Liver-type arginase I mRNA was present in all subsets. Apoptosis was followed by the expression of T cell intracellular antigen (TIA) and the simultaneous detection of DNA stainability by propidium iodine and annexin V binding. Apoptosis was practically absent both in activated CD14(++)CD33(++)DR(++)CD25(++)CD69(++)CD71(++/+) CD16(-) cells, expressing A-II mRNA and having arginase activity, but not iNOS mRNA, and in not fully mature large CD14(++)CD16(+)CD23(+)DR(++) monocytes, expressing simultaneously both mRNAs and having both enzyme activities. However, differentiated small CD14(+/++)CD16(+)CD69(+)CD25(+/-)CD71(++)CD23(+) DR(++) monocytes, expressing high levels of iNOS mRNA, exhibited apoptotic signs. Amounts of NO synthesised by monocytes co-expressing iNOS and arginase changed with the addition of arginine or an iNOS inhibitor; in that case a correlation of NO production and apoptotic features was observed. Data suggest a regulatory role for endogenous NO in apoptosis of stimulated and differentiated monocytes, and also that iNOS and A-II, when simultaneously present, could control the production of NO as a consequence of their competition for arginine.

Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes

2011-12-23T13:35:48Z

Title: Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes Author(s) : Palazon, A. (Asís); Teijeira, A. (Álvaro); Martinez-Forero, I. (Iván); Hervas-Stubbs, S. (Sandra); Roncal, C. (Carmen); Peñuelas, I. (Iván); Dubrot, J. (Juan); Morales-Kastresana, A. (Aizea); Perez-Gracia, J.L. (José Luis); Ochoa, M.C. (María Carmen); Ochoa, L. (Laura); Martinez, A. (Alfredo); Luque, A. (Alfonso); Dinchuk, J. (Joseph); Rouzaut, A. (Ana); Jure-Kunkel, M. (María); Melero, I. (Ignacio) Abstract: Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies.

Lymphangiogenesis and lung cancer

2012-08-07T06:32:33Z

Title: Lymphangiogenesis and lung cancer Author(s) : Rouzaut, A. (Ana); Irigoyen, M. (Marta); Montuenga, L.M. (Luis M.)

Differential gene expression in the activation and maturation of human monocytes

2012-04-04T12:37:26Z

Title: Differential gene expression in the activation and maturation of human monocytes Author(s) : Rouzaut, A. (Ana); Lopez-Moratalla, N. (Natalia); Miguel, C. (Carlos) de Abstract: Differential-display or RNA fingerprint was applied to identify genes differentially expressed in monocyte maturation induced by an immunomodulating peptide on human peripheral blood mononuclear cells. Two unknown sequences (06c22 and 06c71) and p21 protein (cyclin dependent kinase inhibitor) were repressed, and three genes activated: Cathepsin D, DRP2 (dihydropirimidinase related protein 2), and gp91phox (91-kDa subunit of citochrome b(558)). Phenotype of evolving monocytes was analyzed by flow cytometry and mRNA level of identified genes determined by reverse transcription-PCR. The expression pattern of identified genes seemed to correlate with different monocyte subsets, monocyte-derived cells, and expected functional changes. After peptide addition, immature monocytes were initially activated, increasing the expression of CD25, CD69, and HLA-DR markers. This was accompanied by repression of p21 and the two unknown sequences, along with the simultaneous activation of Cathepsin D and DRP2. Later, the differentiation marker CD16 rose, and gp91phox gene expression activated. Further maturation led certain monocytes to express marker CD23 and gp91phox expression to reach a maximum, while Cathepsin D and DRP2 dropped to preactivation levels. Results reflect part of the evolution of immature monocytes toward macrophages and monocyte-derived dendritic cell precursors.

Nitric oxide activates granule-associated DNase in human monocytes

2012-04-28T00:07:24Z

Title: Nitric oxide activates granule-associated DNase in human monocytes Author(s) : Pio, R. (Rubén); Lopez-Zabalza, M. (María J.); Rouzaut, A. (Ana); Santiago, E. (Esteban); Lopez-Moratalla, N. (Natalia) Abstract: Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activity

Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation

2011-12-20T12:13:47Z

Title: Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation Author(s) : Martinez-Forero, I. (Iván); Rouzaut, A. (Ana); Palazon, A. (Asís); Dubrot, J. (Juan); Melero, I. (Ignacio) Abstract: Covalent and reversible post-translational modifications of proteins are a common theme in signaling. Ubiquitin conjugation was originally described to target proteins to proteasomal degradation by ubiquitin polymerization involving lysine (K) 48 residues. Differently linked polymers of polyubiquitin have been found that modify proteins without targeting to proteasomal degradation. Instead this pathway creates docking sites for signaling scaffolds that are key to control the nuclear factor-kappaB (NF-kappaB) pathway. Indeed TRAF-2, TRAF-6, and TRAF-3 are E3 ubiquitin ligases that form K63-linked ubiquitin polymers. Therefore signaling via TNF family receptors, IL1R, IL-18R, T-cell receptor (TCR), and Toll-like receptors (TLR) use this type of post-translational modification. Specific enzymes exist (DUBs) that deactivate this system, degrading K63 polyubiquitin chains. Interestingly, mice deficient in these deubiquitinases develop autoimmunity and inflammation. In carcinogenesis, the K63 polyubiquitin pathway is possibly critical for inflammation-driven tumor promotion. The pathway is also critically involved in costimulation of tumor immunity/immunotherapy as well as in the biology of malignant cells themselves. The elements of this new signaling paradigm offer the opportunity for therapeutic exploitation and drug discovery.

TGFbeta-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions

2011-12-20T15:06:33Z

Title: TGFbeta-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions Author(s) : Irigoyen, M. (Marta); Anso, E. (Elena); Salvo, E. (Elizabeth); Dotor, J. (Javier); Martinez-Irujo, J.J. (Juan José); Rouzaut, A. (Ana) Abstract: TGFbeta-induced protein (TGFBI) is an extracellular protein that mediates cell adhesion to collagen, laminin and fibronectin through its interaction with different beta integrins. We had previously reported that hypoxia-induced TGFBI mRNA expression in lymphatic endothelial cells (LEC). Here, we demonstrate that TGFBI can contribute to hypoxia-induced increases in LEC adhesion to the ECM. We show that while there are no changes in alpha1, alpha4, alphav, beta1, beta2, beta3, alpha5beta1, alphavbeta3, alphavbeta5 integrin expression on the LEC surface after hypoxia exposure, there exists an accumulation of TGFBI adaptor protein in LEC supernatants. We also demonstrate that hypoxia driven TGBFI expression is dependent on TGFbeta production by LEC. Furthermore, we show that TGFBI mediated LEC adhesion and migration through the ECM by its binding to the beta3 integrin. The identification of the specific mechanisms regulating LEC-ECM interactions may help us design new therapeutic applications for diseases in which lymphatic vessel function is compromised.

Hypoxia alters the adhesive properties of lymphatic endothelial cells. A transcriptional and functional study

2011-12-20T11:59:29Z

Title: Hypoxia alters the adhesive properties of lymphatic endothelial cells. A transcriptional and functional study Author(s) : Irigoyen, M. (Marta); Anso, E. (Elena); Martínez, E. (Enrique); Garayoa, M. (Mercedes); Martinez-Irujo, J.J. (Juan José); Rouzaut, A. (Ana) Abstract: Recent advances in our understanding of the molecular biology of lymphatic endothelial cells have revealed that these vessels, besides their known function in tissue homeostasis and immunity, constitute conduits for the tumor cells to metastasize. One of the factors that contribute to tumor spread is the acquisition of an angiogenic phenotype as a response to the onset of tumor hypoxia. To our knowledge, little is known about the effects of low oxygen levels on the lymphatic vasculature. Therefore, we used cDNA microarrays to study the transcriptional changes occurring in hypoxia exposed lymphatic endothelial cells. Our analysis was then complemented by functional assays showing that these cells responded with increased attachment to the extracellular matrix, delayed proliferation and production of reactive oxygen species. Differential expression of genes involved in these processes such as NADPH oxidase 4, the tissue inhibitor of metalloproteinase 3, and TGFbeta induced protein I, was found. Hypoxia was also found to increase mRNA levels of the cytokine CXCL-12 and its receptor CXCR4. Moreover, adhesion experiments revealed that hypoxia increased the binding of non-small cell lung carcinoma cells to this endothelium in a CXCR4 dependent way. We thus illustrate the response of lymphatic endothelial cells to hypoxia and suggest targets to study tumor metastasis through these vessels.

Direct effects of type I interferons on cells of the immune system

2012-01-10T12:23:26Z

Title: Direct effects of type I interferons on cells of the immune system Author(s) : Hervas-Stubbs, S. (Sandra); Perez-Gracia, J.L. (José Luis); Rouzaut, A. (Ana); Sanmamed, M.F. (Miguel F.); Bon, A. (Agnes) Le; Melero, I. (Ignacio) Abstract: Type I interferons (IFN-I) are well-known inducers of tumor cell apoptosis and antiangiogenesis via signaling through a common receptor interferon alpha receptor (IFNAR). IFNAR induces the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway in most cells, along with other biochemical pathways that may differentially operate, depending on the responding cell subset, and jointly control a large collection of genes. IFNs-I were found to systemically activate natural killer (NK) cell activity. Recently, mouse experiments have shown that IFNs-I directly activate other cells of the immune system, such as antigen-presenting dendritic cells (DC) and CD4 and CD8 T cells. Signaling through the IFNAR in T cells is critical for the acquisition of effector functions. Cross-talk between IFNAR and the pathways turned on by other surface lymphocyte receptors has been described. Importantly, IFNs-I also increase antigen presentation of the tumor cells to be recognized by T lymphocytes. These IFN-driven immunostimulatory pathways offer opportunities to devise combinatorial immunotherapy strategies.

Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells

2012-04-04T12:37:25Z

Title: Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells Author(s) : Herblot, S. (S.); Vekris, A. (A.); Rouzaut, A. (Ana); Najeme, F. (F.); Miguel, C. (Carlos) de; Bezian, J.H. (J.H.); Bonnet, J. (J.) Abstract: In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles.

All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation

2011-12-20T11:50:02Z

Title: All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation Author(s) : Fontan, L. (Lorena); Oliemuller, E. (Erik); Martinez-Irujo, J.J. (Juan José); Miguel, C. (Carlos) de; Rouzaut, A. (Ana) Abstract: Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.

Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells

2011-12-20T11:42:45Z

Title: Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells Author(s) : Dubrot, J. (Juan); Portero, A. (Aitziber); Orive, G. (Gorka); Hernandez, R.M. (Rosa María); Palazon, A. (Asís); Rouzaut, A. (Ana); Perez-Gracia, J.L. (José Luis); Hervas-Stubbs, S. (Sandra); Pedraz, J.L. (José Luis); Melero, I. (Ignacio) Abstract: Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat-mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb.