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Zurita M.,Hospital Virgen Of Las Nieves | Lara P.C.,Hospital Dr Negrin | del Moral R.,Hospital Virgen Of Las Nieves | Torres B.,CIBER ISCIII | And 6 more authors.
BMC Cancer | Year: 2010

Background: Numerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of Estrogen Receptor1 (ESR1) and Stratifin (14-3-3-σ) gene promoters in disease-free and metastatic breast cancer patients.Methods: We studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR.Results: Serum levels of methylated gene promoter 14-3-3-σ significantly differed between Control and Metastatic Breast Cancer groups (P < 0.001), and between Disease-Free and Metastatic Breast Cancer groups (P < 0.001). The ratio of the 14-3-3-σ level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 ± 0.074) indicates that this test is a good approach to post-treatment prognosis.Conclusions: The relationship of 14-3-3-σ with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-σ as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response. © 2010 Zurita et al; licensee BioMed Central Ltd.


Bello-Morales R.,Autonomous University of Madrid | Perez-Hernandez M.,Autonomous University of Madrid | Rejas M.T.,Autonomous University of Madrid | Matesanz F.,Institute Parasitologia y Biomedicina | And 2 more authors.
PLoS ONE | Year: 2011

The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP), the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP)-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested. © 2011 Bello-Morales et al.


Broen J.C.A.,Radboud University Nijmegen | Bossini-Castillo L.,Institute Parasitologia y Biomedicina | Van Bon L.,Radboud University Nijmegen | Vonk M.C.,Radboud University Nijmegen | And 21 more authors.
Arthritis and Rheumatism | Year: 2012

Objective To investigate whether polymorphisms in Toll-like receptor (TLR) genes, previously reported to be associated with immune-mediated diseases, are involved in systemic sclerosis (SSc). Methods We genotyped 14 polymorphisms in the genes for TLRs 2, 4, 7, 8, and 9 in a discovery cohort comprising 452 SSc patients and 537 controls and a replication cohort consisting of 1,170 SSc patients and 925 controls. In addition, we analyzed 15-year followup data on 964 patients to assess the potential association of TLR variants with the development of disease complications. We analyzed the functional impact of the associated polymorphism on monocyte-derived dendritic cells. Results In the discovery cohort, we observed that a rare functional polymorphism in TLR2 (Pro631His) was associated with antitopoisomerase (antitopo) positivity (odds ratio 2.24 [95% confidence interval 1.24-4.04], P = 0.003). This observation was validated in the replication cohort (odds ratio 2.73 [95% confidence interval 1.85-4.04], P = 0.0001). In addition, in the replication cohort the TLR2 variant was associated with the diffuse subtype of the disease (P = 0.02) and with the development of pulmonary arterial hypertension (PAH) (Cox proportional hazards ratio 5.61 [95% confidence interval 1.53-20.58], P = 0.003 by log rank test). Functional analysis revealed that monocyte-derived dendritic cells carrying the Pro63His variant produced increased levels of inflammatory mediators (tumor necrosis factor α and interleukin-6) upon TLR-2-mediated stimulation (both P < 0.0001). Conclusion Among patients with SSc, the rare TLR2 Pro631His variant is robustly associated with antitopoisomerase positivity, the diffuse form of the disease, and the development of PAH. In addition, this variant influences TLR-2-mediated cell responses. Further research is needed to elucidate the precise role of TLR-2 in the pathogenesis of SSc. © 2012 by the American College of Rheumatology.


PubMed | Institute Parasitologia y Biomedicina, Autonomous University of Madrid, University of Rome La Sapienza and University of Cantabria
Type: Journal Article | Journal: International immunology | Year: 2016

Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3 that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3 chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.


Ienne S.,University of Sao Paulo | Pappas G.,Catholic University of Brasília | Benabdellah K.,Institute Parasitologia y Biomedicina | Benabdellah K.,University of Granada | And 2 more authors.
Infection, Genetics and Evolution | Year: 2012

Among trypanosomatids, the genus . Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. . Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of . P. serpens, that displays high amino acid sequence similarity with phytobacteria and . Leishmania enzymes. No orthologous . PDC/IPDC genes were found in . Trypanosoma cruzi or . T. brucei. Conservation of the . PDC/IPDC gene sequence was verified in 14 . Phytomonas isolates. A phylogenetic analysis shows that . Phytomonas protein is robustly monophyletic with . Leishmania spp. and . C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of γ-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between . Phytomonas, . Leishmania and . Crithidia. We have measured the PDC activity in . P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4. mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. © 2012 Elsevier B.V..


Munoz-Fernandez R.,Institute Parasitologia y Biomedicina | Prados A.,University of Granada | Leno-Duran E.,University of Granada | Blazquez A.,Hospital Universitario San Cecilio | And 4 more authors.
Human Reproduction | Year: 2012

BACKGROUND Decidual stromal cells (DSCs) have classically been considered fibroblastic cells, although their function, cell lineage and origin are not fully understood. We previously demonstrated that human DSCs showed similarities with follicular dendritic cells (FDCs): DSCs expressed FDC-associated antigens, both types of cells are contractile and both are related to mesenchymal stem cells (MSCs). To further characterize DSCs, we investigated whether DSCs and FDCs share any distinctive phenotypical and functional characteristics. METHODS Human FDC lines were obtained from tonsillectomy samples, human DSC lines from elective termination of pregnancy samples and human MSC lines from bone marrow aspirates. We isolated DSC, FDC and MSC lines and compared their characteristics with flow cytometry and enzyme-linked immunosorbent assay. Cell lines were cultured with tumour necrosis factor (TNF) and lymphotoxin (LT)α1β2, cytokines involved in FDC differentiation. Cell lines were also differentiated in culture after exposure to progesterone and cAMP, factors involved in the differentiation (decidualization) of DSC. RESULTS Like MSCs, DSCs and FDCs expressed MSC-associated antigens (CD10, CD29, CD54, CD73, CD106, α-smooth muscle actin and STRO-1) and lacked CD45 expression, and all three types of cell line showed increased expression of CD54 (ICAM-1) and CD106 (VCAM-1) when cultured TNF and LTα1β2. DSCs and FDCs, however, exhibited characteristics not observed in MSCs: DSCs expressed FDC-associated antigens CD14, CD21 and CD23, B cell-activating factor and secreted C-X-C motif chemokine 13. Moreover, DSC lines but not MSC lines inhibited the spontaneous apoptosis of B lymphocytes, a typical functional attribute of FDC. During culture with progesterone and cAMP, FDCs, like DSCs but in contrast to MSCs, changed their morphology from a fibroblastic to a rounder shape, and cells secreted prolactin. CONCLUSIONS Our results suggest that DSCs and FDCs share a common precursor in MSCs but this precursor acquires new capacities when it homes to peripheral tissues. We discuss these shared properties in the context of immuneendocrine regulation during pregnancy. © The Author 2012.


Palomino-Morales R.,Institute Parasitologia Y Biomedicina | Gonzalez-Juanatey C.,Hospital Xeral Calde | Vazquez-Rodriguez T.R.,Hospital Xeral Calde | Torres O.,Institute Parasitologia Y Biomedicina | And 4 more authors.
Clinical and Experimental Rheumatology | Year: 2010

Objective: To assess whether the polymorphism of the macrophage migration inhibitory factor (MIF) gene at the position -173 is implicated in the disease susceptibility, risk of cardiovascular (CV) events and presence of subclinical atherosclerosis in patients with rheumatoid arthritis (RA). Patients and methods: A series of 293 unselected patients fulfilling the 1987 American College of Rheumatology classification criteria for RA seen at the rheumatology outpatient clinic of Hospital Xeral-Calde, Lugo, Spain and 526 matched controls were studied for differences in the MIF-173 G/C gene biallelic polymorphism. A total of 182 consecutive patients that had been periodically followed between March 1996 and September 1996 until patient's death or January 1, 2008 were assessed for the presence of CV events. Moreover, between March and December 2007, a subgroup of unselected RA patients with no history of CV events was studied for the presence of subclinical atherosclerosis by the assessment of the endothelial function (n=107) and the carotid artery intima-media thickness (IMT) (n=91) by ultrasonography studies. Patients and controls were genotyped for the MIF-173 G/C gene polymorphism using a PCR system with pre-developed TaqMan allelic discrimination assay. Results: No significant differences in allele or genotype frequencies for the MIF-173 gene polymorphism between RA patients and controls were found. Forty-four of the 182 patients followed between 1996 and January 2008 experienced CV events. Although the frequency of MIF-173 GG homozygous was increased in those who had CV events (88.6%) compared to those who did not suffer these complication (73.2%), the difference was not statistically significant. It was also the case when we analyzed the potential influence of MIF-173 genotypes in the presence of endothelial dysfunction or increased carotid IMT of patients with RA. Conclusions: Our results do not show that MIF-173 gene polymorphism may infer a direct risk for disease susceptibility or CV disease in patients with RA. © Copyright Clinical and Experimental Rheumatology 2010.


Parolini O.,Instituto Ospedaliero | Souza-Moreira L.,Institute Parasitologia y Biomedicina | O'Valle F.,University of Granada | Magatti M.,Instituto Ospedaliero | And 3 more authors.
Arthritis and Rheumatology | Year: 2014

Objective. Rheumatoid arthritis (RA) is an autoimmune disease caused by loss of immunologic self tolerance and characterized by chronic joint inflammation. Cells isolated from human amniotic membrane (HAMCs) were recently found to display immunosuppressive properties. The aim of this study was to characterize the effect of HAMCs on antigen-specific T cell responses in RA patients and to evaluate their therapeutic potential in a preclinical experimental model of RA. Methods. We investigated the effects of HAMCs on collagen-reactive T cell proliferation and cytokine production, on the production of mediators of inflammation by synoviocytes, and on the generation of Treg cells in peripheral blood mononuclear cells and synovial membrane cells isolated from RA patients. Mice with collagen-induced arthritis (CIA) were treated with HAMCs after disease onset, and clinical scores and joint levels of mediators of inflammation were evaluated. We determined Th1/Th17-mediated autoreactive responses in the mice by measuring the proliferation and the cytokine profile of lymph node cells restimulated with collagen. Results. Treatment with HAMCs suppressed synovial inflammatory responses and antigen-specific Th1/ Th17 activation in cells isolated from RA patients. Moreover, HAMCs stimulated the generation of human CD4+CD25+FoxP3+ Treg cells with a capacity to suppress collagen-specific T cell responses. Systemic infusion of HAMCs significantly reduced the incidence and severity of CIA by down-regulating the 2 deleterious components of disease: Th1-driven autoimmunity and inflammation. In mice with CIA, HAMC treatment decreased the production of various inflammatory cytokines and chemokines in the joints, impaired antigenspecific Th1/Th17 cell expansion in the lymph nodes, and generated peripheral antigen-specific Treg cells. HAMCs also protected the mice from experimental sepsis, inflammatory bowel disease, and autoimmune encephalomyelitis. Conclusion. HAMCs have emerged as attractive candidates for a cell-based therapy for RA. © 2014, American College of Rheumatology.


PubMed | Institute Parasitologia y Biomedicina and University of Granada
Type: Journal Article | Journal: Immunology and cell biology | Year: 2014

Follicular dendritic cells (FDCs) present antigens to B cells in the lymphoid follicle and inhibit B-cell apoptosis. In previous work, we obtained human FDC lines that allowed us to study the antigen phenotype and functions of these cells, finding that they expressed -smooth muscle (SM) actin (a protein involved in cell contraction) and were able to contract collagen gel matrixes in gel contraction assays. Actin polymerization associated with cell contractility is essential for many cellular functions. We report here that interleukin (IL)-2 and interferon (IFN)- increased FDC contractility, and IL-10 reduced contractility, whereas IL-4 had no effect. Tumor necrosis factor (TNF) and lymphotoxin (LT)-12, cytokines involved in FDC differentiation, also increased FDC contractility. In different cell systems, cell contraction is related with the incorporation of -SM actin into stress fibers. By confocal microscopy, we showed that cytochalasin D, an inhibitor of actin polymerization, inhibited -SM actin incorporation and relaxed FDCs. Likewise, IL-10 significantly decreased the proportion of FDCs with -SM actin-positive stress fibers, whereas cytokines that increased FDC contractility also increased this proportion. However, none of the cytokines tested significantly affected -SM actin expression as determined by flow cytometry. IL-10, in addition to decreasing FDC contractility, increased the inhibitory activity of FDC in spontaneous B-cell apoptosis (P<0.05), but the other cytokines did not affect this activity. We conclude that cytokines related with FDC physiology regulate the contractility of these cells, and IL-10 also regulates the effect of FDC on B-cell apoptosis.


Delgado M.,Institute Parasitologia y Biomedicina | Ganea D.,Temple University
Amino Acids | Year: 2013

Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide/ neurotransmitter, is widely distributed in both the central and peripheral nervous system. VIP is released by both neurons and immune cells. Various cell types, including immune cells, express VIP receptors. VIP has pleiotropic effects as a neurotransmitter, immune regulator, vasodilator and secretagogue. This review is focused on VIP production and effects on immune cells, VIP receptor signaling as related to immune functions, and the involvement of VIP in inflammatory and autoimmune disorders. The review addresses present clinical use of VIP and future therapeutic directions. © 2011 Springer-Verlag.

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