José Yélamos

Life-supporting human complement regulator decay accelerating factor transgenic pig liver xenograft maintains the metabolic function and coagulation in the nonhuman primate for up to 8 days

Background. It is not known whether the pig liver is capable of functioning efficiently when transplanted into a primate, neither is there experience in transplanting a liver from a transgenic pigs expressing the human complement regulator human complement regulator decay accelerating factor (h-DAF) into a baboon. The objective of this study was to determine whether the porcine liver would support the metabolic functions of non-human primates and to establish the effect of hDAF expression in the prevention of hyperacute rejection of porcine livers transplanted into primates. Methods. Five orthotopic liver xenotransplants from pig to baboon were carried out: three from unmodified pigs and two using livers from h-DAF transgenic pigs. Findings. The three control animals transplanted with livers from unmodified pigs survived for less than 12 hr. Baboons transplanted with livers from h-DAF transgenic pigs survived for 4 and 8 days. Hyperacute rejection was not detected in the baboons transplanted with hDAF transgenic pig livers; however, it was demonstrated in the three transplants from unmodified pigs. Baboons transplanted with livers from h-DAF transgenic pigs were extubated at postoperative day 1 and were awake and able to eat and drink. In the recipients of hDAF transgenic pig livers the clotting parameters reached nearly normal levels at day 2 after transplantation and remained normal up to the end of the experiments. In these hDAF liver recipients, porcine fibrinogen was first detected in the baboon plasma 2 hr postreperfusion, and was present up to the end of the experiments. One animal was euthanized at day 8 after development of sepsis and coagulopathy, the other animal arrested at day 4, after an episode of vomiting and aspiration. The postmortem examination of the hDAF transgenic liver xenografts did not demonstrate rejection. Interpretation. The livers from h-DAF transgenic pigs did not undergo hyperacute rejection after orthotopic xenotransplantation in baboons. When HAR is abrogated, the porcine liver maintains sufficient coagulation and protein levels in the baboon up to 8 days after OLT.

The CD5 ectodomain interacts with conserved fungal cell wall components and protects from zymosan-induced septic shock-like syndrome

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spα, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by β-glucan but not by mannan. The Kd of the rshCD5/(1→3)-β-d-glucan phosphate interaction is 3.7 ± 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1→3)-β-d-glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock

CD6 is a lymphocyte receptor that belongs to the scavenger receptor cysteine-rich superfamily. Because some members of the scavenger receptor cysteine-rich superfamily act as pattern recognition receptors for microbial components, we studied whether CD6 shares this function. We produced a recombinant form of the ectodomain of CD6 (rsCD6), which was indistinguishable (in apparent molecular mass, antibody reactivity, and cell binding properties) from a circulating form of CD6 affinity-purified from human serum. rsCD6 bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of lipoteichoic acid and LPS, respectively. The Kd of the LPS–rsCD6 interaction was 2.69 ± 0.32 × 10−8 M, which is similar to that reported for the LPS–CD14 interaction. Further experiments showed that membrane CD6 also retains the LPS-binding ability, and it results in activation of the MAPK signaling cascade. In vivo experiments demonstrated that i.p. administration of rsCD6 before lethal LPS challenge significantly improved mice survival, and this was concomitant with reduced serum levels of the proinflammatory cytokines TNF-α, IL6, and IL-1β. In conclusion, our results illustrate the unprecedented bacterial binding properties of rsCD6 and support its therapeutic potential for the intervention of septic shock syndrome or other inflammatory diseases of infectious origin.

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)α and a skewed repertoire of TCRα toward the 5′ Jα segments. Our results show that in the absence of PARP‐2, the survival of DP thymocytes undergoing TCRα 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 TCRα rearrangements preceding a positively selected TCR.

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-1β 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.

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).

Loss of poly(ADP-ribose) polymerase-2 leads to rapid development of spontaneous T-cell lymphomas in p53-deficient mice

Poly(ADP-ribose) polymerase-2 (Parp-2) belongs to a family of enzymes that catalyse poly(ADP-ribosyl)ation of proteins. Parp-2 deficiency in mice (Parp-2−/−) results in reduced thymic cellularity associated with increased apoptosis in thymocytes, defining Parp-2 as an important mediator of T-cell survival during thymopoiesis. To determine whether there is a link between Parp-2 and the p53 DNA-damage-dependent apoptotic response, we have generated Parp-2/p53-double-null mutant mice. We found that p53−/− backgrounds completely restored the survival and development of Parp-2−/− thymocytes. However, Parp-2-deficient thymocytes accumulated high levels of DNA double-strand breaks (DSB), independently of the p53 status, in line with a function of Parp-2 as a caretaker promoting genomic stability during thymocytes development. Although Parp-2−/− mice do not have spontaneous tumours, Parp-2 deficiency accelerated spontaneous tumour development in p53-null mice, mainly T-cell lymphomas. These data suggest a synergistic interaction between Parp-2 and p53 in tumour suppression through the role of Parp-2 in DNA-damage response and genome integrity surveillance, and point to the potential importance of examining human tumours for the status of both genes.

Therapeutic treatment with poly(ADP-ribose) polymerase inhibitors attenuates the severity of acute pancreatitis and associated liver and lung injury

The mortality associated with acute pancreatitis (AP) is largely attributable to abnormalities that occur in distant organs and supportive care remains the only treatment for patients with these complications. Recently, prophylactic pharmacological blockade of poly(ADP‐ribose) polymerase (PARP) enzymes has been shown to attenuate the severity of the disease. However, the clinical relevance of PARP inhibitors administered after the onset of AP remains uncertain. The aim of the present study was to investigate the therapeutic effects of PARP inhibitors in established AP.

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.

Parp-2 is required to maintain hematopoiesis following sublethal γ-irradiation in mice

Hematopoietic stem cells self-renew for life to guarantee the continuous supply of all blood cell lineages. Here we show that Poly(ADP-ribose) polymerase-2 (Parp-2) plays an essential role in hematopoietic stem/progenitor cells (HSPC) survival under steady-state conditions and in response to stress. Increased levels of cell death were observed in HSPC from untreated Parp-2-/- mice, but this deficit was compensated by increased rates of self-renewal, associated with impaired reconstitution of hematopoiesis upon serial bone marrow transplantation. Cell death after γ-irradiation correlated with an impaired capacity to repair DNA damage in the absence of Parp-2. Upon exposure to sublethal doses of γ-irradiation, Parp-2-/- mice exhibited bone marrow failure that correlated with reduced long-term repopulation potential of irradiated Parp-2-/- HSPC under competitive conditions. In line with a protective role of Parp-2 against irradiation-induced apoptosis, loss of p53 or the pro-apoptotic BH3-only protein Puma restored survival of irradiated Parp-2-/- mice, whereas loss of Noxa had no such effect. Our results show that Parp-2 plays essential roles in the surveillance of genome integrity of HSPC by orchestrating DNA repair and restraining p53-induced and Puma-mediated apoptosis. The data may affect the design of drugs targeting Parp proteins and the improvement of radiotherapy-based therapeutic strategies.