El análisis proteómico mediante 2D-DIGE y MS/MS permite discriminar en un mismo experimento entre ratones silvestres (WT) o deficientes CD38 (CD38ko) y entre ratones afectados o no por la artritis inducida por colágeno

Comunicaciones a congreso

Possible role of highly activated mucosal NK cells against viral respiratory infections in children undergoing haematopoietic stem cell transplantation

Infection is the leading cause of non-relapse-related mortality after allogeneic haematopoietic stem cell transplantation (HSCT). Altered functions of immune cells in nasal secretions may influence post HSCT susceptibility to viral respiratory infections. In this prospective study, we determined T and NK cell numbers together with NK activation status in nasopharyngeal aspirates (NPA) in HSCT recipients and healthy controls using multiparametric flow cytometry. We also determined by polymerase chain reaction (PCR) the presence of 16 respiratory viruses. Samples were collected pre-HSCT, at day 0, +10, +20 and +30 after HSCT. Peripheral blood (PB) was also analyzed to determine T and NK cell numbers. A total of 27 pediatric HSCT recipients were enrolled and 16 of them had at least one viral detection (60%). Rhinovirus was the most frequent pathogen (84% of positive NPAs). NPAs of patients contained fewer T and NK cells compared to healthy controls (p = 0.0132 and p = 0.120, respectively). Viral PCR + patients showed higher NK cell number in their NPAs. The activating receptors repertoire expressed by NK cells was also higher in NPA samples, especially NKp44 and NKp46. Our study supports NK cells relevance for the immune defense against respiratory viruses in HSCT recipients.This work was supported in part by the National Health Service of Spain, Instituto de Salud Carlos III (ISCIII), FONDOS FEDER grant (FIS) PI18/01301, CRIS Foundation to Beat Cancer, Patients’ Support Associations Fundación Mari Paz Jiménez Casado and La Sonrisa de Álex and a Small Grant Award from the European Society for Paediatric Infectious Diseases.S

REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI

Proteomic analysis of sera from rheumatoid arthritis B6 WT and CD38KO mice by using proteominer fractionation and two-dimensional difference gel electrophoresis (DIGE)

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Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment

AKT activation seems to be associated with apoptotic signals and not with pro-survival signals in a pristane-induced lupus model.

Several studies have shown that in addition to its role as a survival factor and tumor promoting agent, AKT is also able to exhibit pro-apoptotic effects under diverse conditions, including oxidative stress, cytokine stimulation and exposure to cytotoxic chemicals like staurosporine, methotrexate, docetaxel and etoposide. Moreover, phosphorylation of second mitochondria-derived activator of caspases (SMAC) by AKT promotes caspase-3 activation during etoposide-induced apoptosis in HeLa cells. Our data show that injection of pristane into the peritoneum induces apoptosis-mediated cell death of peritoneal exudate cells (PECs), as evidenced by the increased number of annexin V+ peritoneal cells and their increased levels of cleaved/active caspase-3. Indeed, the higher levels of activated caspase-3 protein in WT PECs, particularly at 2-weeks post pristane treatment, are indicative of a higher rate of apoptosis compared to Cd38¿/¿ cells. In contrast, no differences were observed in the levels of MCL-1, an anti-apoptotic protein and member of the BCL2 family. Furthermore, kinases ERK1/2 and AKT showed distinct activation kinetics in pristane-elicited PECs. Interestingly, caspase-3 activation followed similar kinetics to AKT activation in both WT and Cd38¿/¿ PECs, while ERK activation correlated with increased levels of MCL-1. In summary our data strongly suggest that in the pristane-induced lupus model AKT activation is associated with apoptotic signals and not with survival signals. Further studies, however, are required to identify specific pro- and anti-apoptotic target proteins that are phosphorylated by ERK or AKT following pristane treatment, and that regulate the apoptotic process

Mice deficient in CD38 develop an attenuated form of collagen type II-induced arthritis

CD38, a type II transmembrane glycoprotein expressed in many cells of the immune system, is involved in cell signaling, migration and differentiation. Studies in CD38 deficient mice (CD38 KO mice) indicate that this molecule controls inflammatory immune responses, although its involvement in these responses depends on the disease model analyzed. Here, we explored the role of CD38 in the control of autoimmune responses using chicken collagen type II (col II) immunized C57BL/6-CD38 KO mice as a model of collagen-induced arthritis (CIA). We demonstrate that CD38 KO mice develop an attenuated CIA that is accompanied by a limited joint induction of IL-1β and IL-6 expression, by the lack of induction of IFNγ expression in the joints and by a reduction in the percentages of invariant NKT (iNKT) cells in the spleen. Immunized CD38 KO mice produce high levels of circulating IgG1 and low of IgG2a anti-col II antibodies in association with reduced percentages of Th1 cells in the draining lymph nodes. Altogether, our results show that CD38 participates in the pathogenesis of CIA controlling the number of iNKT cells and promoting Th1 inflammatory responses

Contribución de CD38 al desarrollo de la artritis autoinmune inducida por colágeno en un modelo murino: estudios proteómicos y funcionales

Tesis Univ. Granada. Programa Oficial de doctorado: Inmunología Molecular y Celula

Utilización de ProteoMiner para el análisis proteómico de muestras de líquido sinovial (LS) de pacientes con osteoartritis

2 páginas.ProteoMiner o Combinatorial Peptide Ligand Libraries (CPLL) [1] actualmente están compuestas de una gran variedad de hexapéptidos sintetizados, que actúan de ligandos para las proteínas de las muestras. Y están siendo actualizadas como herramientas de gran utilidad para el mapeo de proteomas de distintos organismos. Esta herramienta permite el objetivo de reducir del rango dinámico de concentraciones en diversos tipos de muestras, como plasma y suero, disminuyendo la concentración de las proteínas más abundantes y el enriquecimiento en proteínas menos abundantes, de cara a una identificación de estas últimas, como paso previo a un posterior análisis proteómico [2]. Proteínas menos abundantes en las muestras biológicas son potencialmente de gran interés para el estudio de nuevo biomarcadores de enfermedad.Ministerio Sanidad y Consumo-ISCIII-FIS, Proyecto: FIS06/1502 (M.Z.). CSIC; Proyecto: PI-200820I216 (M.Z.). Junta de Andalucía (J.A.), Consejería Innovación Ciencia y Empresa y Consejería Educación y Ciencia. Proyecto: PC08-CTS-04046 (J.S. y M.Z.). Ministerio Educación y Ciencia (MEC)- Ministerio Ciencia e Innovación (MICINN) Proyecto: SAF-2008-03685 (J.S. y M.Z.). CSIC Beca JAE Intro a M.D.P.-M.Peer reviewe