Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.

Identification of axon growth promoters in the secretome of the deer antler velvet.

Every spring, deer cast their old antlers and initiate a regeneration process, which yields a new set of antlers of up to 1m in length. Over the course of three months, branches of the trigeminal nerve, originating from the frontal skull, innervate velvet, a modified skin that covers the regenerating antler. The rate of growth of these axons reaches up to 2cm per day making them the fastest regenerating axons in adult mammals. Here, we aim to identify the factors secreted by velvet that promote such high speed axon growth. Our experiments with cultures of adult rat trigeminal neurons demonstrate that conditioned medium harvested from velvet organotypic cultures has greater axon growth-promoting properties than a medium conditioned by normal skin. The axon growth-promoting effects of velvet act synergistically with the extracellular matrix (ECM) protein laminin, a component of the basal lamina present in the deer antler. Our proteomic analyses identified several axon growth promoters in the velvet-conditioned medium (VCM), including soluble proteins such as nerve growth factor (NGF) and apolipoprotein A-1, as well as matrix extracellular proteins, such as periostin and SPARC. Additional in vitro analyses allowed us to determine that a synergic relationship between periostin and NGF may contribute to neurite growth-promoting effects of velvet secretome. A combinatorial approach using these factors may promote regeneration at high speeds in patients with peripheral neuropathies.

Atorvastatin modulates the profile of proteins released by human atherosclerotic plaques.

The mechanisms by which hydroxymethylglutaryl CoenzymeA reductase inhibitors (statins) reduce atherosclerotic cardiovascular morbidity and mortality remain poorly understood. Statins have been shown to modulate the levels of different inflammatory proteins both in carotid atherosclerotic plaques and in the blood of patients with atherosclerosis. In this work, we hypothesize that statins could also modulate the levels of the proteins secreted by cultured atherosclerotic plaques. Thus, the secretomes obtained from complicated atherosclerotic plaques incubated in the presence/absence of atorvastatin (10 micromol/l, 24 h) were analysed and compared by two-dimensional electrophoresis, considering the fibrous adjacent areas as controls. In total, 54 proteins (83 protein isoforms) were identified by Mass Spectrometry (MS): 24 proteins were increased and 20 proteins decreased in atheroma plaque supernatants compared to controls. Some of these proteins, like Cathepsin D, could play a significant role in plaque instability, becoming a potential target for therapeutical treatment. Interestingly, 66% of the proteins differentially released by atherosclerotic plaques reverted to control values after administration of atorvastatin, among them, Cathepsin D. Moreover, plaques obtained from patients who received atorvastatin treatment prior to carotid endarterectomy showed decreased Cathepsin D expression relative to plaques from non-treated patients. In conclusion, this proteomic approach has shown that statins are able to modulate the secretome of atherosclerotic plaques, and new therapeutical targets for statins have been characterised.

Ca2+-binding allergens from olive pollen exhibit biochemical and immunological activity when expressed in stable transgenic Arabidopsis.

Employing transgenic plants as alternative systems to the conventional Escherichia coli, Pichia pastoris or baculovirus hosts to produce recombinant allergens may offer the possibility of having available edible vaccines in the near future. In this study, two EF-hand-type Ca2+-binding allergens from olive pollen, Ole e 3 and Ole e 8, were produced in transgenic Arabidopsis thaliana plants. The corresponding cDNAs, under the control of the constitutive CaMV 35S promoter, were stably incorporated into the Arabidopsis genome and encoded recombinant proteins, AtOle e 3 and AtOle e 8, which exhibited the molecular properties (i.e. MS analyses and CD spectra) of their olive and/or E. coli counterparts. Calcium-binding assays, which were carried out to assess the biochemical activity of AtOle e 3 and AtOle e 8, gave positive results. In addition, their mobilities on SDS/PAGE were according to the conformational changes derived from their Ca2+-binding capability. The immunological behaviour of Arabidopsis-expressed proteins was equivalent to that of the natural- and/or E. coli-derived allergens, as shown by their ability to bind allergen-specific rabbit IgG antiserum and IgE from sensitized patients. These results indicate that transgenic plants constitute a valid alternative to obtain allergens with structural and immunological integrity not only for scaling up production, but also to develop new kind of vaccines for human utilization.

Modulación mediante tratamiento con atorvastatina y/o amlodipino del perfil de proteínas liberadas por placas ateroscleróticas humanas / Modulation of the profile of proteins released by human atherosclerotic plaques through

Introducción. Entre las diversas estrategias seguidas para analizar la compleja estructura de la placa de ateroma, nuestro grupo se ha centrado en el estudio de las proteínas secretadas por la pared vascular, que podrían ser potenciales marcadores plasmáticos. Las estatinas o los agentes bloqueadores de los canales de calcio (BCC) pueden modular los valores de las proteínas circulantes en sujetos con diversas afecciones cardiovasculares. Métodos. La electroforesis bidimensional y la espectrometría de masas (EM) se han aplicado al estudio de proteínas diferencialmente secretadas por placas de ateroma incubadas ex vivo respecto a zonas fibrosas adyacentes. Asimismo, hemos estudiado el efecto modulador de la atorvastatina (10­5 M), el amlodipino (10­6 M) o la combinación de ambos fármacos. Resultados. De una media de 620 proteínas detectadas por gel, se identificaron en total 83 proteínas secretadas por las placas ateromatosas mediante EM: 34 proteínas incrementadas en los sobrenadantes de las placas cultivadas ex vivo respecto a las zonas control y 31 con valores de secreción inferiores en la zona ateromatosa. La adición in vitro de fármacos a las placas de ateroma produjo diferentes efectos en los valores de secreción proteicos, dependiendo del tipo de fármaco y de la proteína considerada, si bien la mayoría de las proteínas identificadas revertía a valores control independientemente del tratamiento. Conclusión. Mediante este análisis proteómico hemos caracterizado nuevas proteínas potencialmente involucradas en la formación e inestabilidad de la placa de ateroma. La modulación por distintos tratamientos farmacológicos de dichos marcadores puede ayudar a comprender nuevos mecanismos de acción de dichos fármacos (AU)

Introduction. Analysis of the complex structure of the atheroma plaque is a classical object of cardiovascular research. We studied proteins secreted by the vascular wall, which could be potential plasma markers. Statins or calcium channel blockers modulate the levels of different circulating proteins in patients with diverse cardiovascular diseases. Methods. Two-dimensional electrophoresis and mass spectrometry were applied to identify and characterize proteins differentially secreted by atheroma plaque samples incubated ex vivo versus adjacent fibrous segments considered as controls. We also analyzed the modulatory effect of atorvastatin (10­5 M), amlodipine (10­6 M) and the combination of both drugs. Results. From an average of 620 proteins detected per gel, a total of 83 proteins secreted by atheroma plaque samples were identified by mass spectrometry: 34 proteins were increased in atheroma plaque supernatants versus control segments while 31 showed decreased secretion levels in atheroma plaques. Different effects were detected after in vitro drug administration to complicated atheroma plaques, depending on the kind of drug and protein considered, although the majority of the proteins identified reverted to normal levels independently of the treatment administered. Conclusion. Proteomic analysis characterized a significant number of novel proteins potentially involved in the formation and instability of complicated atherosclerotic plaques. Modulation of these markers by different drugs may help us to understand new potential mechanisms through which these drugs exert their beneficial effects on atherothrombosis (AU)