Identificació immunològica i caracterització de les propietats biològiques de la proteïna catiònica d'eosinòfil

Consultable des del TDXTítol obtingut de la portada digitalitzadaLa proteïna catiònica d'eosinòfil (ECP) és una ribonucleasa que es troba als grànuls secundaris dels eosinòfils. Els nivells d'ECP en fluids corporals s'utilitza com a indicador del número i activació dels eosinòfils. L'ECP és tòxica per diversos patògens com són bacteris, paràsits, virus i per cèl·lules de mamífer. Per identificar immunològicament l'ECP s'ha seleccionat una regió específica de la proteïna (D112-P123) com a possible epítop antigènic. La regió D112-P123 no es troba en l'RNasa A, 1, 4 i 5 i és on s'observa una màxima desviació de l'esquelet de la cadena principal de l'ECP i la neurotoxina derivada d'eosinòfil (EDN) amb la qual comparteix un 67% d'identitat de seqüència. S'han immunitzat conills amb un pèptid sintètic (D112-P123) conjugat a una proteïna transportadora i s'han purificat els anticossos policlonals amb una columna d'afinitat amb l'ECP immobilitzada. L'anticòs D112-P123 reconeix específicament l'ECP en condicions reductores i no reductores i no presenta reactivitat creuada amb l'EDN, l'RNasa A i altres proteïnes control. La immunodetecció per transferència Western amb l'anticòs D112-P123 ha mostrat que hi ha una relació lineal entre la quantitat de proteïna (1-75 ng) i el senyal obtingut. S'ha assajat la reactivitat de l'anticòs D112-P123 en fluids corporals i granulòcits. L'anticòs D112-P123 detecta la forma nadiua no glicosilada i les formes glicosilades de l'ECP en granulòcits, esput i plasma. S'ha obtingut una bona correlació entre els nivells d'ECP determinants amb l'anticòs D112-P123 i amb un anticòs que comercialitza Pharmacia & Upjohn (Uppsala, Suècia). Per estudiar la relació entre l'estructura i les activitats biològiques de l'ECP s'han obtingut variants de la proteïna en residus específics de l'ECP. S'han escollit residus catiònics i aromàtics exposats en la superfície de la proteïna (W10K, W35A/R36A, R75A/F76A, R101A/R104A) i de la regió del loop D115-Y122 (R121A, R121A/Y122A, (_ 115-122) ECP i (115-122 EDN) ECP . S'ha estudiat l'efecte de l'ECP i les seves variants en l'activitat ribonucleasa, la disrupció de membranes, la citotoxicitat per bacteris gramnegatius i grampositius i l'inhibició del creixement de cèl·lules de mamífer. També s'ha realitzat una predicció de l'estructura tridimensional dels mutants per modelatge molecular. Les variants de l'ECP no modifiquen substancialment ni l'estructura tridimensional de la proteïna ni l'activitat ribonucleasa. L'activitat sobre membranes de l'ECP i les seves variants s'ha analitzat utilitzant vesícules sintètiques. L'ECP mostra una clara preferència per desestabilitzar vesícules acídiques resultat que suggereix que les càrregues positives de la proteïna són importants per la interacció amb les membranes. L'estudi de l'efecte de les mutacions en l'activitat sobre les membranes ha mostrat que aminoàcids catiònics específics i els triptòfans de la proteïna (W10, W35R36 i R101R104) estan relacionats amb la desestabilitazació de la membrana. Aquests resultats correlacionen bé amb l'efecte dels mutants en la inhibició del creixement de cèl·lules de mamífer. Les regions W35R36 i W10 són essencials per la inhibició de la proliferació. Altres residus catiònics i aromàtics R75F76, R101R104, Y122 tenen un paper secundari en l'inhibició del creixement que es pot explicar per la possible interacció d'aquests residus amb els carbohidrats de la superfície de les cèl·lules de mamífer. La regió W35R36 té un paper essencial en l'activitat bactericida per grampositius i gramnegatius. Els altres residus estudiats tenen efectes diferents en l'activitat bactericida de l'ECP depenent es tracti d' E. coli o S. aureus. Mentre els residus R101R104 i R75F76 són importants per l'activitat bactericida sobre E. coli, els residus W10 i la regió del bucle D115-Y122 són necessaris per l'activitat bactericida sobre S. aureus. Podem concloure que les regions catiòniques i hidrofòbiques estudiades participen en la capacitat de l'ECP per desestabilitzar membranes biològiques i/o en la interacció de la proteïna amb components de la paret bacteriana o amb els carbohidrats de la superfície de cèl·lules de mamífer.Eosinophil cationic protein (ECP) is a ribonuclease located in the secondary granules of eosinophils. ECP levels in body fluids are used as an indicator of number and activation of eosionophils. ECP is toxic for many pathogens including bacteria, parasites, virus and for mammalian cells. To identify ECP we have selected a specific loop region of the protein (D112-P123) as a putative antigenic epitope. This sequence is absent in RNase A, 1, 4 and 5 and the polypeptide main chain adopts a specific conformation in ECP and also in eosinophil-derived neurotoxin (EDN) which shares 67 % sequence homology with ECP. A synthetic peptide containing the sequence and linked to a carrier protein was used to obtain rabbit polyclonal antibodies which were further purified by an affinity column with ECP as a ligand. D112-P123 antibody specifically recognizes ECP in reducing and nonreducing conditions and does not cross-react with EDN, RNase A or other control proteins. The immunodetection of recombinant ECP by Western blot has shown a lineal relationship between the quantity of protein (1-75 ng) and the signal obtained. The reactivity of the antibody was assayed in different body fluids and granulocytes. D112-P123 antibody detects the glycosylated and nonglycosylated forms of the native ECP in granulocytes, plasma and sputum. A good correlation has been obtained between ECP levels determined by the antibody D112-P123 and by an antibody obtained from a commercial source. To study the relationship between the structure and biological activities of ECP we have obtained variants of the protein in specific amino acids residues. We have chosen cationic and aromatic amino acids located at the protein surface (W10K, W35A/R36A, R75A/F76A and R101A/R104A) and in the loop region D115-Y122 (R121A, R121A/Y122A, (_ 115-122) ECP and (115-122 EDN) ECP. For the wild type form and mutants we have determined the ribonuclease and membrane-lytic activity, the cytotoxicity for gram negative and gram positive bacteria and the mammalian cell growth inhibition. The three dimensional structure of ECP mutants has been predicted by molecular modelling. ECP variants do not show important changes neither on the three dimensional folding nor on the ribonuclease activity of the protein. The lytic-activity of ECP and mutants on cell membranes has been analysed using synthetic lipid vesicles. ECP shows a notable preference to destabilize acidic liposomes which suggests that the electrostatic interaction between membrane and the protein are important for protein binding. The study of the effect of the mutations in the membrane-lytic activity has shown that specific arginine residues and the two tryptophan residues of the protein (W10, W35R36 and R101R104) are related to the membrane destabilization. This results correlate well with the effect of the mutants in the mammalian cell growth inhibition. W35R36 and W10 are essentials for the inhibition of proliferation while other cationic and aromatic residues, R75F76, R101R104 and Y122, play a secondary role which might be explained for the possible interactions of these residues with the carbohydrates on the surface of mammalian cells. W35R36 are essentials for the bactericidal activity for gram positive and negative bacteria. Other amino acid residues have different effects depending on the bacterial strain E. coli or S. aureus. While R101R104 and R75F76 are necessary for the bactericidal activity for E. coli the W10 residue and the loop region D115-Y122 are necessaries for the cytotoxic activity for S. aureus. We can conclude from the studied ECP mutants that specific cationic and hydrophobic residues studied participate on the ability of the protein to destabilize biological membranes and/or in the interaction of the protein with components of the bacterial cell wall or the surface of mammalian cells

Importancia de la comunicación de una paciente en UCI: plan de cuidados estandarizado

Cartel presentado en la Segunda Conferencia Internacional de Comunicación en Salud, celebrada el 23 de octubre de 2015 en la Universidad Carlos III de MadridIntroducción: paciente de 44 años de edad que ingresa en UCI tras una intervención quirúrgica la cual se complicó sufriendo un shock hemorrágico precisando politransfusión y clampaje de la vena cava. Objetivos: establecer un plan de cuidados para mejorar la comunicación, privada en este momento por la intubación orotraqueal (ITO) y por la sedación. Un segundo objetivo es la recuperación de la movilidad de la paciente. Metodología: la metodología empleada en este plan de cuidados es la valoración por necesidades básicas de Virginia Henderson constando de 14 necesidades. Resultados: Tras la valoración, se han identificado los diagnósticos deterioro de la comunicación verbal y deterioro de la movilidad física con sus correspondientes objetivos e intervenciones. Conclusiones: de los dos diagnósticos enfermeros de la NANDA, sólo se ha solucionado el deterioro de la comunicación verbal, quedando sin resolver el deterioro de la movilidad física, aportando recomendaciones para su posterior recuperación domiciliaria

Análisis de las distintas formas de tratamientos utilizados en el síndrome de Tourette en la infancia

Cartel presentado en el 3er Congreso Internacional de Comunicación en Salud (3ICHC), celebrado los días 19 y 20 de octubre de 2017 en la Universidad Carlos III de Madrid.El objetivo de este trabajo es exponer y analizar las distintas formas de tratamientos utilizados en la actualidad acerca del síndrome de Tourette

Polymorphism FXII 46C>T and cardiovascular risk: additional data from Spanish and Tunisian patients

<p>Abstract</p> <p>Background</p> <p>Previous studies showed an association between Coagulation Factor XII 46C>T polymorphism and variation in FXII plasma levels, as 46C>T seems to affect the translation efficiency. Case-control studies in Spanish samples indicated that genotype T/T is an independent risk factor for venous thrombosis, ischemic stroke and acute coronary artery disease. In this study, we tried to reaffirm the importance of 46C>T in two samples from Spain and Tunisia.</p> <p>Findings</p> <p>A Transmission Disequilibrium Test (TDT) based on 101 family trios from Barcelona with one offspring affected by ischemic heart disease and a classical case-control study based on 76 patients with IHD and 118 healthy individuals from North and Centre-South Tunisia were conducted. Subjects were genotyped for 46C>T and data were analyzed accordingly, revealing no association in any of the two samples (TDT: P = 0.16, relative risk 1.17; case-control study: P = 0.59, odds ratio 1.36).</p> <p>Conclusion</p> <p>The results suggest that 46C>T is not a risk factor for ischemic heart disease in any of the two analyzed samples and therefore the polymorphism seems not to be a universal risk factor for cardiovascular diseases.</p

Potential signals of natural selection in the top risk loci for coronary artery disease: 9p21 and 10q11

Background: Coronary artery disease (CAD) is a complex disease and the leading cause of death in the world. Populations of different ancestry do not always share the same risk markers. Natural selective processes may be the cause of some of the population differences detected for specific risk mutations. Objective: In this study, 384 single nucleotide polymorphisms (SNPs) located in four genomic regions associated with CAD (1p13, 1q41, 9p21 and 10q11) are analysed in a set of 19 populations from Europe, Middle East and North Africa and also in Asian and African samples from the 1000 Genomes Project. The aim of this survey is to explore for the first time whether the genetic variability in these genomic regions is better explained by demography or by natural selection. Results: The results indicate significant differences in the structure of genetic variation and in the LD patterns among populations that probably explain the population disparities found in markers of susceptibility to CAD. Conclusions: The results are consistent with potential signature of positive selection in the 9p21 region and of balancing selection in the 9p21 and 10q11. Specifically, in Europe three CAD risk markers in the 9p21 region (rs9632884, rs1537371 and rs1333042) show consistent signals of positive selection. The results of this study are consistent with a potential selective role of CAD in the configuration of genetic diversity in current human populations

A Conquista romana da Hispânia Citerior

A conquista romana da Península Ibérica, e mais precisamente do território que viria a ser a província da Hispânia Citerior, tem suscitado um interesse particular nos últimos anos. Intervenções arqueológicas preencheram uma lacuna no conhecimento da atividade militar dos séculos II-I a.C., que basicamente só era conhecida através de fontes antigas. Graças ao trabalho contínuo de várias equipas de investigação, temos atualmente um panorama completamente diferente. Nestas novas investigações sobre o primeiro século da conquista romana da Península, o século II a.C., que há poucas décadas era um período completamente desconhecido, surge como um período crucial para propor novas hipóteses sobre o modelo romano de conquista deste território do norte peninsular. Este modelo de conquista foi seguramente aplicado, com algumas nuanças, aos outros territórios que viriam a constituir o futuro Império Romano

Transplantation of Human-Fetal-Spinal-Cord-Derived NPCs Primed with a Polyglutamate-Conjugated Rho/Rock Inhibitor in Acute Spinal Cord Injury

NPC transplantation; Cell priming; Human fetal neural precursorTrasplante de NPC; Cebado celular; Precursor neuronal fetal humanoTrasplantament de NPC; Cebament cel·lular; Precursor neuronal fetal humàNeural precursor cell (NPC) transplantation represents a promising therapy for treating spinal cord injuries (SCIs); however, despite successful results obtained in preclinical models, the clinical translation of this approach remains challenging due, in part, to the lack of consensus on an optimal cell source for human neuronal cells. Depending on the cell source, additional limitations to NPC-based therapies include high tumorigenic potential, alongside poor graft survival and engraftment into host spinal tissue. We previously demonstrated that NPCs derived from rat fetal spinal cords primed with a polyglutamate (PGA)-conjugated form of the Rho/Rock inhibitor fasudil (PGA-SS-FAS) displayed enhanced neuronal differentiation and graft survival when compared to non-primed NPCs. We now conducted a similar study of human-fetal-spinal-cord-derived NPCs (hfNPCs) from legal gestational interruptions at the late gestational stage, at 19–21.6 weeks. In vitro, expanded hfNPCs retained neural features, multipotency, and self-renewal, which supported the development of a cell banking strategy. Before transplantation, we established a simple procedure to prime hfNPCs by overnight incubation with PGA-SS-FAS (at 50 μM FAS equiv.), which improved neuronal differentiation and overcame neurite-like retraction after lysophosphatidic-acid-induced Rho/Rock activation. The transplantation of primed hfNPCs into immune-deficient mice (NU(NCr)-Foxn1nu) immediately after the eighth thoracic segment compression prompted enhanced migration of grafted cells from the dorsal to the ventral spinal cord, increased preservation of GABAergic inhibitory Lbx1-expressing and glutamatergic excitatory Tlx3-expressing somatosensory interneurons, and elevated the numbers of preserved, c-Fos-expressing, activated neurons surrounding the injury epicenter, all in a low percentage. Overall, the priming procedure using PGA-SS-FAS could represent an alternative methodology to improve the capabilities of the hfNPC lines for a translational approach for acute SCI treatment.This research was funded by Fundació Marató TV3 2017/refs.20172230, 20172231, Agencia Valenciana de Innovación (AVI) (INNVAL10/19/047 and Grants RTI2018-095872-B-C21 and PDI2021-1243590B-I00/ERDF funded by MCIN/AEI//10.13039/501100011033 and by ERDF A way of making Europe). This project was also funded by Project 964562 (RISEUP), H2020 FetOpen program

Puig Castellar de Biosca (Lleida). Una fortificación romana del siglo II a. C. En el noreste de la Hispania Citerior

The recent archaeological excavations undertaken at the hill of Puig Castellar (Biosca, Lleida) have revealed the remains of singular Roman military settlement for its typological features and early dating. A solid defence wall with towers and visual control of the nearby territory surrounds the site. Inside, Romans built a large building at the highest point, which records a number of auxiliary rooms around a central courtyard. The courtyard included a rock cut cistern. Besides, there are other buildings close to the main building and others accommodated to the inner face of the surrounding defence wall as well as an area with great iron metallurgical activity. The large amount of Italian pottery with a well-dated horizon between 180-120 BCE as well as constructive and decorative techniques identified in the buildings reveal a possible military and administrative complex of the earlier decades of Roman conquest in Hispania.Las recientes excavaciones arqueológicas efectuadas en el cerro de Puig Castellar de Biosca (Lleida) han dejado al descubierto los vestigios de un establecimiento militar romano, tipo castellum, singular por sus características tipológicas y su temprana cronología en el marco hispánico. El asentamiento muestra un amplio dominio visual sobre el territorio inmediato; está delimitado por una muralla defensiva con torres. En su interior destaca un gran edificio, erigido en el punto más elevado del recinto, que muestra varias dependencias distribuidas en torno a un patio central que a su vez dispone de una gran cisterna excavada en la roca. También se han localizado algunas dependencias auxiliares cerca del edificio principal y otras adosadas a la cara interna de la muralla, así como una zona con gran actividad metalúrgica de hierro. La gran cantidad de material cerámico recuperado, sobre todo de importación, con un horizonte cronológico bien definido entre el 180 y el 120 a. C., así como las técnicas constructivas y decorativas identificadas en los edificios, nos permiten afirmar que se trata de un complejo militar y administrativo de los primeros decenios de la conquista romana de Hispania

Human diversity in Jordan: polymorphic alu insertions in general jordanian and bedouin groups

Jordan, located in the Levant region, is an area crucial for the investigation of human migration between Africa and Eurasia. However, the genetic history of Jordanians has yet to be clarified, including the origin of the Bedouins today resident in Jordan. Here, we provide new genetic data on autosomal independent markers in two Jordanian population samples (Bedouins and the general population) to begin to examine the genetic diversity inside this country and to provide new information about the genetic position of these populations in the context of the Mediterranean and Middle East area. The markers analyzed were 18 Alu polymorphic insertions characterized by their identity by descent, known ancestral state (lack of insertion), and apparent selective neutrality. The results indicate significant genetic differences between Bedouins and general Jordanians (p = 0.038). Whereas Bedouins show a close genetic proximity to North Africans, general Jordanians appear genetically more similar to other Middle East populations. In general, these data are consistent with the hypothesis that Bedouins had an important role in the peopling of Jordan and constitute the original substrate of the current population. However, migration into Jordan in recent years likely has contributed to the diversity among current Jordanian population groups

MRI and CFS oligoclonal bands after autologus hematopoietic stem cell transplantation in MS

To analyze the MRI and CSF oligoclonal bands (OB) changes in patients with MS who underwent an autologous hematopoietic stem cell transplantation (AHSCT). Background: AHSCT is evaluated as an alternative therapy in severe MS. In previous series of AHSCT for MS, data on MRI or OB outcome were limited or not provided. Methods: five patients with a median Kurtzke's EDSS score of 6.5, more than two attacks, and confirmed worsening of the EDSS in the previous year received an AHSCT. Hematopoietic stem cells were mobilized with cyclophosphamide (3 g/m2) and granulocyte colony-stimulating factor (5 microg/kg/d). The graft was T cell depleted by positive CD 34+ selection. Conditioning regimen included BCNU (300 mg/m(2)), cyclophosphamide (150 mg/kg in 3 days), and antithymocyte globulin (60 mg/kg in 4 days). MRI scans were scheduled at baseline and 1, 3, 6, and 12 months and OB analysis at baseline and 3 and 12 months post-AHSCT. Results: four patients had a stable or improved EDSS after a median follow-up of 18 months (range, 12 to 24 months). The fifth patient's condition deteriorated during AHSCT. She partially improved and remained stable after month 3 after AHSCT. The baseline CSF OB persisted 1 year after AHSCT. MRI studies after AHSCT showed no enhanced T1 lesions and no new or enlarging T2 lesions. The median percentage change of T2 lesion load was -11.8% (range, -26.6 to -4.0%). All patients had a decrease of corpus callosum area at 1 year (median, 12.4%; range, 7.8% to 20.5%) that did not progress in the two patients evaluated at 2 years after AHSCT. Conclusions: although the persistence of CSF OB suggests the lymphocytes were not eliminated from the CNS, the follow-up MRI studies showed no enhanced T1 brain lesions and a reduction in the T2 lesion load that correlated with the clinical stabilization of MS after AHSCT