Biomedical subjects

José Yélamos

Publications and source records attributed to José Yélamos.

9 recordsLinked to original sources

PubMed2006

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes.

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.

Animals↗

PubMed2005

A role for human Sp alpha as a pattern recognition receptor.

Human Sp alpha is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, which suggests that it may play an important role in the regulation of the innate and adaptive immune systems. In the present study we show that recombinant human Sp alpha (rSp alpha) binds to the surface of several gram-positive and gram-negative bacterial strains. Competition studies indicated that such binding is mediated by the recognition of lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, through nonoverlapping sites on the Sp alpha molecule. The most conserved part of LPS (2-keto-3-deoxyoctulosonic acid and lipid A) was shown to be involved in the recognition by Sp alpha. Bacterial binding studies using the SRCR domain 1 of Sp alpha showed that this domain retains both the LPS and LTA binding activities, indicating that both bacterial interacting sites are retained in a single SRCR domain. Furthermore, rSp alpha induced aggregation of gram-positive and gram-negative bacteria strains. On the other hand, rSp alpha inhibited tumor necrosis factor-alpha secretion by human monocytes stimulated with LPS or LTA. Binding of Sp alpha to conserved components of bacterial surfaces and modulation of the monocyte response indicate that this molecule is an active constituent of the innate immune response of the host.

Amino Acid Sequence↗

PubMed2005

Establishment of an immortalized PARP-1-/- murine endothelial cell line: a new tool to study PARP-1 mediated endothelial cell dysfunction.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a critical role in endothelial cell dysfunction associated with various pathophysiological conditions. To elucidate PARP-1 pathways involved in endothelial cell dysfunction, it is essential to establish "in vitro" experimental models using isolated endothelial cells. So far, two approaches have been used: primary endothelial cells from PARP-1-/- mice which have a limited life-span, being a major handicap if large quantities of cells are required; and pharmacological inhibition of PARP in PARP-1+/+ endothelial cell lines, which is not specific for PARP-1 and would have biological effects different that genetic inhibition. To overcome these limitations, we have established an immortalized PARP-1-/- endothelial cell line (HYKO6) by transfection of primary cells with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. The HYKO6 cell line exhibited endothelial characteristics, such as constitutive expression of CD105, CD31, ICAM-2, VCAM-1, and von Willebrand factor and formation of capillary-like structures (CLS) on Matrigel surface. However, expression of ICAM-1 antigen is lost in the HYKO6 cells. After TNF-alpha treatment, HYKO6 cells exhibited increased expression of E-selectin and VCAM-1. Likewise, NF-kappaB-dependent transcriptional activation was increased in the HYKO6 cell line in response to TNF-alpha at a level similar to that found for primary PARP-1-/- cells. This cell line should provide, for the first time, a valuable tool to study PARP-1 pathways in endothelial cell dysfunction.

Animals↗

PubMed2005

Role of lipopolysaccharide and cecal ligation and puncture on blood coagulation and inflammation in sensitive and resistant mice models.

The hemostatic system is severely disturbed during endotoxemia, leading to a hypercoagulable state. However, it remains uncertain to what extent hypercoagulability is the critical factor in determining the clinical course rather than just the consequence of a severe systemic inflammatory response. To answer this question, we evaluated the evolution of hemostatic and inflammatory markers, as well as histological features, in mice sensitive and resistant to two models of endotoxemia: lipopolysaccharide-injection and cecal ligation puncture. Genetic (knockout mice) and pharmacological (PJ34) blockade of the nuclear enzyme PARP-1 was used to achieve resistance to the endotoxemia. In both models, endotoxemia resulted in antithrombin deficiency, decreased platelets, and fibrin deposition in organs, which were similar in all groups of mice. By contrast, proinflammatory mediators, inflammatory cell infiltration (especially that mediated by mononuclear cells), and organ degeneration were more intense in sensitive animals. Further studies supported a negative role for the triggering of the coagulation cascade in the mortality associated with the endotoxic shock. Hirudin had a minor effect on cell infiltration and organ damage, despite causing a potent inhibition of fibrin deposition. On the other hand, a sublethal dose of lipopolysaccharide yielded significant fibrin deposition but weak activation of the inflammatory response. Our results suggest that activation of coagulation by endotoxemia is severe and independent of the inflammatory response. However, such activation may act with fibrin deposition to have a minor influence on survival in sepsis.

Animals↗

PubMed2005

Inhibition of poly(ADP-ribose) polymerase attenuates the severity of acute pancreatitis and associated lung injury.

The severity of acute pancreatitis results from the transmigration and activation of leukocytes within the pancreas and the local synthesis and release of proinflammatory-soluble mediators that transform a local injury into a systemic inflammatory response. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether the genetic deletion of PARP-1 or PARP-2 (a new member of the PARP family) genes, or pharmacological inhibition of PARP activity might affect the development and severity of acute pancreatitis and pancreatitis-associated lung injury. Secretagogue-induced acute pancreatitis was achieved by 12 hourly intraperitoneal injections of cerulein in mice deficient in PARP-1 or PARP-2 genes, and wild-type (WT) littermate mice untreated or treated with PARP activity inhibitors. The severity of pancreatitis was assessed by measurements of serum amylase, lipase, interleukin-1beta and IL-6, pancreatic water content, histologic grading and pancreas myeloperoxidase (MPO) activity. Lung injury was evaluated by quantifying MPO activity and morphological changes. We found that the severity of acute pancreatitis and pancreatitis-associated lung injury was significantly attenuated in mice lacking PARP-1, but not PARP-2, compared with WT mice. Interestingly, administration of PARP inhibitors, 3-aminobenzamide or PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyacetamide HCl), in WT mice markedly decreased acute pancreatitis severity and pulmonary-associated injury in a larger extension than genetic deletion of PARP-1. Our results support the potential therapeutic application of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury.

Acute Disease↗

PubMed2004

Genetic and pharmacological inhibition of poly(ADP-ribose) polymerase-1 interferes in the chlamydial life cycle.

Chlamydiaceae are intracellular bacteria responsible for a variety of infections, ranging from asymptomatic to very severe, in humans and animals. We have investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in Chlamydophila abortus infection using PARP-1-/- and their littermates PARP-1+/+ mice. Infection was resolved more efficiently by PARP-1-/- than PARP-1+/+ mice. However, the inflammatory response was similar in both strains, suggesting a potential role for PARP-1 in the cross-talk between this microorganism and the host cells. PARP-1-/- fibroblasts showed a 10-fold lower rate of chlamydiae production than PARP-1+/+. Moreover, a strong inhibition of bacterial production was also observed after pharmacological inhibition of PARP-1 activity in McCoy cells. Likewise, PARP-1 inhibition induced a higher level of cell death of infected cells, interfering in this way with the normal bacterial cell cycle. Overall, we identify PARP-1 as a new molecule involved in chlamydial developmental cycle, although the intrinsic mechanisms deserve further studies.

Animals↗

PubMed2004

Transcription regulation of TNF-alpha-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) has been involved in endothelial cell dysfunction associated with various pathophysiological conditions. The intrinsic mechanism of PARP-1-mediated endothelial cell dysfunction could be related to PARP-1 overactivation, NAD(+) consumption and ATP depletion. An alternative way could involve transcription regulation. By using high-density microarrays, we examined early tumor necrosis factor alpha (TNF-alpha)-stimulated gene expression profiles in PARP-1(+/+) and PARP-1(-/-) murine heart endothelial cells. TNF-alpha modulated a significant number of genes in both cell types. We have identified a set of genes whose expression in response to TNF-alpha is modulated by PARP-1, whereas the expression of others is PARP-1-independent. Up-regulation of several genes involved in the inflammatory response is hampered in the absence of PARP-1. Moreover, NF-kappaB-dependent transcriptional activation is partially inhibited in PARP-1(-/-) compared to PARP-1(+/+) cells. However, we found that PARP-1 might also silence transcription of several NF-kappaB target genes. Overall, our results show that PARP-1 is regulating the expression of genes by the endothelial cells both in a positive and a negative fashion, with the final effects depending on the gene. Individual studies of these genes are now necessary to clarify the intrinsic mechanism by which PARP-1 is controlling transcription and thereby finding out different therapeutic approaches involving PARP-1.

Animals↗

PubMed2003

Endothelial cell chimerism does not influence allograft tolerance in liver transplant patients after withdrawal of immunosuppression.

BACKGROUND: Human liver allografts sometimes show self-induced permanent tolerance without immunosuppression. It has recently been proposed that the replacement of liver donor endothelial cells by recipient cells could confer a survival advantage. The aim of this study was to analyze liver endothelial cell replacement in relation to the response (tolerance or rejection) after withdrawal of immunosuppression in liver transplant patients. METHODS: Nine liver recipient patients were entered into a program of immunosuppressive drug withdrawal. The authors studied liver endothelial cell chimerism in five of these patients who received a liver from a donor of the opposite sex by in situ hybridization for X and Y chromosomes. RESULTS: Three patients (33%) achieved complete withdrawal of immunosuppression. The authors' data show similar endothelial cell chimerism in both the tolerant and nontolerant patients analyzed. CONCLUSIONS: Endothelial cell chimerism has nothing to do with the induction of clinical tolerance in liver transplant patients after withdrawal of immunosuppression.

Adult↗

PubMed2002

Isolation and characterization of immortalized porcine aortic endothelial cell lines.

Primary porcine endothelial cells have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (AOCs) to facilitate in vitro studies of different pathological process involving the endothelium. Primary porcine aortic endothelial cells (PAECs) were transfected with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. Flow cytometry analysis demonstrated uptake of acetylated low density lipoproteins (Ac-LDL) and constitutive expression of SLA class I, CD29, CD31, CD41/61, CD80/86, CD46, SWC3, and LAMP-1 antigens by all analyzed lines and showed little differences to primary cells. The functional similarity between primary and immortalized endothelial cells was demonstrated in a cytotoxicity assay using a human natural killer cell line (NKL) as effector. The AOCs cell lines should be valuable tools for in vitro study of the human immune response against pig endothelial cells. In addition, they would be very useful to gain insight in the pathogenesis of some viral haemorrhagic diseases of pig such as African swine fever (ASF) or classical swine fever (CSF).

Animals↗