Endosomal clathrin drives actin accumulation at the immunological synapse.

Antigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell-cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell-cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

Anti-CCR7 monoclonal antibodies as a novel tool for the treatment of chronic lymphocyte leukemia.

To date, chronic lymphocytic leukemia (CLL) remains incurable with current treatments, which include the monoclonal antibodies (mAbs) rituximab and alemtuzumab. The efficacy of rituximab is modest when used as single agent, and alemtuzumab induces severe immunosuppression. To develop more potent and specific therapies, we propose the CC chemokine receptor 7 (CCR7) as an attractive target molecule to treat CLL, as it not only fulfills the requirements of a high-surface expression and a good level of tissue specificity, but it also plays a crucial role in mediating the migration of the tumor cells to lymph nodes (LNs) and thus, in the development of clinical lymphadenopathy. In the current work, murine anti-human CCR7 mAb mediated a potent, complement-dependent cytotoxicity (CDC) against CLL cells while sparing normal T lymphocytes from the same patients. The sensitivity to CDC was related to the antigenic density of CCR7. Moreover, these mAb blocked the in vitro migration of CLL cells in response to CC chemokine ligand 19 (CCL19), one of the physiological ligands of CCR7. Conversely, CLL cells were poorly lysed through antibody-dependent, cell-mediated cytotoxicity (ADCC), probably as a result of the murine origin and the isotype of the anti-CCR7 mAb used. Molecular engineering techniques will allow us to obtain chimeric or humanized anti-CCR7 mAb to reach the best clinical response for this common and yet incurable leukemia.

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination.

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.

[Usefulness of brain natriuretic peptide to evaluate patients with heart failure treated with cardiac resynchronization]. / Utilidad del péptido natriurético BNP en la evaluación de pacientes con insuficiencia cardíca tratados con resincronización cardíaca.

INTRODUCTION AND OBJECTIVES: The aim of the present study was to document the evolution of the blood levels of brain natriuretic peptide (BNP) in patients with heart failure and their correlation with the clinical course after implantation of a biventricular pacemaker. PATIENTS AND METHOD: Twenty-eight patients with heart failure associated to left bundle branch block and left ventricular systolic dysfunction were included in the study. In each patient we performed laboratory tests, chest X-ray, electrocardiogram and echocardiogram, and measured blood levels of BNP. RESULTS: During follow-up (10 [6] months) functional capacity improved, decreasing from 3.3 (0.6) to 2.10 (0.4) (P=.03). The rate of hospitalizations for heart failure decreased from an average of 1.8 (0.7) (6 months before the procedure) to 0.8 (0.3) (6 months after the procedure; P=.04). The basal value of BNP decreased from 193 (98) pg/mL to 52 (14) at the end of the follow-up in the responder group (22 patients) and increased from 564 (380) to 650 (80) pg/mL in the nonresponder group (6 patients). Patients who responded showed significant clinical improvement and decreasing levels of BNP, which reached a plateau an average of 6 months after implantation. Multivariate logistic regression analysis identified lower levels of BNP, idiopathic dilated cardiomyopathy, and functional class as independent predictors of response to therapy. Age, QRS width and left ventricular ejection fraction were not predictors of response. CONCLUSIONS: Brain natriuretic peptide concentrations allowed us to monitor, in an objective manner, the clinical course of patients with biventricular resynchronization therapy.

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma.

BACKGROUND: The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. METHODS: NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 µg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. RESULTS: The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. CONCLUSIONS: The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

Increased circulating pro-inflammatory cytokines and Th17 lymphocytes in Hashimoto's thyroiditis.

CONTEXT: Th17 lymphocytes play an important role in different chronic inflammatory and autoimmune conditions. AIM: The aim of the study was to explore the status of Th17 cells in patients with autoimmune thyroid diseases (AITD). DESIGN: We assessed the serum levels and in vitro synthesis of IL-17 and IL-22 and of different cytokines (IL-6, IL-15, and IL-23) involved in the differentiation of Th17 cells in the peripheral blood and thyroid glands of 26 patients with AITD, eight with Graves' disease, and 18 with Hashimoto's thyroiditis (HT) as well as 10 healthy controls. RESULTS: We found enhanced levels of T cells synthesizing IL-17 and IL-22 in the peripheral blood from AITD patients, mainly in those with HT. In addition, a stronger expression of IL-17 and IL-22 and an enhanced number of IL-23R(+) cells was detected in thyroid glands from HT patients compared with Graves' disease or controls. Furthermore, increased concentrations of IL-6 and IL-15 were detected in sera from HT patients, whereas serum levels of IL-23 tended to be higher in these patients. Finally, an enhanced in vitro differentiation of T lymphocytes into Th17 cells induced by IL-23/IL-6 was observed in AITD patients. Accordingly, a strong induction of RORC2 gene was detected in lymphocytes from HT patients when stimulated with IL-23. CONCLUSION: Our results indicate that there is an increased differentiation of Th17 lymphocytes and an enhanced synthesis of Th17 cytokines in AITD, mainly in HT. These phenomena may have an important role in the pathogenesis of thyroid autoimmunity.

Analysis of migratory and prosurvival pathways induced by the homeostatic chemokines CCL19 and CCL21 in B-cell chronic lymphocytic leukemia.

OBJECTIVE: The CCR7 chemokine receptor has been reported to promote homing of B-cell chronic lymphocytic leukemia (CLL) cells into lymph nodes and support their survival, but the mechanisms mediating these effects are largely unknown. We investigated the role of different signaling pathways triggered by CCR7 engagement by its ligands, the chemokines CCL19 and CCL21, in the control of CLL migration and survival. MATERIALS AND METHODS: Chemotaxis and apoptosis assays were performed in the presence of pharmacologic inhibitors and genetic mutants of the phosphatidylinositol-3-OH kinase (PI3K), Rho guanosine triphosphatase, and mitogen-activated protein kinase (MAPK) signaling cascades to assess the role of these pathways on primary CLL migration and survival in response to CCR7 activation. Kinase activation was determined by immunoblotting and pull-down experiments. RESULTS: CLL chemotactic activity induced by CCL19 or CCL21 was markedly reduced by inhibitors of PI3K and the Rho effector molecule Rho-associated coiled-coil forming protein kinases (ROCK), and also by the expression of dominant negative forms of PI3K and RhoA, whereas constitutively activated PI3K and RhoA mutants strongly promoted CLL migration. In contrast, MAPKs were not significantly involved in CLL migration to CCL19/CCL21. Conversely, extracellular signal-regulated kinase and c-Jun-N-terminal kinase, along with PI3K, had a role in CCR7-mediated CLL cell survival. Biochemical experiments confirmed that CCL19/21 induced PI3K-dependent phosphorylation of Akt/protein kinase B, activation of the Rho/Rho-associated coiled-coil forming protein kinases/myosin light chain pathway and MAPKs phosphorylation. CONCLUSIONS: The role of PI3K, Rho guanosine triphosphatases, and MAPKs in CCR7-mediated CLL cells migration and survival suggests that these signal transduction pathways could represent promising targets for CLL therapy.

Tie-2 is overexpressed by monocytes in autoimmune thyroid disorders and participates in their recruitment to the thyroid gland.

CONTEXT: The angiopoietin/Tie system seems to have an important role in the pathogenesis of inflammatory diseases. Although Tie-2 is mainly expressed by endothelium, it is also detected in monocytes, which participate in the development of angiogenic and inflammatory phenomena. AIM: The aim was to study the expression and function of Tie-2 and their ligands, angiopoietin-1 (Ang-1) and Ang-2, in thyroid glands and monocytes from patients with autoimmune thyroid disease (AITD). DESIGN: We studied the expression of Tie-2, Ang-1, and Ang-2 by immunohistochemical techniques in surgical thyroid tissues from 17 patients with Graves' disease, 8 with Hashimoto's thyroiditis, and 3 healthy glands. In addition, we explored the expression and function of Tie-2 in peripheral blood monocytes from 17 patients with Graves' disease, 11 with Hashimoto's thyroiditis, and 14 healthy controls. RESULTS: We found that the expression of Ang-1, Ang-2, and Tie-2 was up-regulated in thyroid glands from AITD patients. Flow cytometry, immunofluorescence, ELISA, and RT-PCR analyses confirmed the synthesis and release of Ang-1, Ang-2, and Tie-2 by thyroid follicular cells (TFC) from AITD patients. In addition, these patients showed increased levels of Tie-2(+) monocytes in the peripheral blood, which exhibited an enhanced chemotactic response to Ang-2 or autologous TFC. CONCLUSIONS: Our data suggest that the Ang/Tie-2 system, through the participation of blood vessels, inflammatory cells, and TFC, may have an important role in the recruitment of monocytes to the thyroid gland and the pathogenesis of the tissue damage seen in AITD.