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

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

Human CD6 down-modulation following T-Cell activation compromises lymphocyte survival and proliferative responses

Available evidence indicates that the CD6 lymphocyte surface receptor is involved in T-cell developmental and activation processes, by facilitating cell-to-cell adhesive contacts with antigen-presenting cells and likely modulating T-cell receptor (TCR) signaling. Here, we show that in vitro activation of human T cells under different TCR-ligation conditions leads to surface downregulation of CD6 expression. This phenomenon was (i) concomitant to increased levels of soluble CD6 (sCD6) in culture supernatants, (ii) partially reverted by protease inhibitors, (iii) not associated to CD6 mRNA down-regulation, and (iv) reversible by stimulus removal. CD6 down-modulation inversely correlated with the upregulation of CD25 in both FoxP3- (Tact) and FoxP3+ (Treg) T-cell subsets. Furthermore, ex vivo analysis of peripheral CD4+ and CD8+ T cells with activated (CD25+) or effector memory (effector memory T cell, CD45RA-CCR7-) phenotype present lower CD6 levels than their naïve or central memory (central memory T cell, CD45RA-CCR7+) counterparts. CD6lo/- T cells resulting from in vitro T-cell activation show higher apoptosis and lower proliferation levels than CD6hi T cells, supporting the relevance of CD6 in the induction of proper T-cell proliferative responses and resistance to apoptosis. Accordingly, CD6 transfectants also showed higher viability when exposed to TCR-independent apoptosis-inducing conditions in comparison with untransfected cells. Taken together, these results provide insight into the origin of sCD6 and the previously reported circulating CD6-negative T-cell subset in humans, as well as into the functional consequences of CD6 down-modulation on ongoing T-cell responses, which includes sensitization to apoptotic events and attenuation of T-cell proliferative responses

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma

Human CD6 down-modulation following T-Cell activation compromises lymphocyte survival and proliferative responses

Available evidence indicates that the CD6 lymphocyte surface receptor is involved in T-cell developmental and activation processes, by facilitating cell-to-cell adhesive contacts with antigen-presenting cells and likely modulating T-cell receptor (TCR) signaling. Here, we show that in vitro activation of human T cells under different TCR-ligation conditions leads to surface downregulation of CD6 expression. This phenomenon was (i) concomitant to increased levels of soluble CD6 (sCD6) in culture supernatants, (ii) partially reverted by protease inhibitors, (iii) not associated to CD6 mRNA down-regulation, and (iv) reversible by stimulus removal. CD6 down-modulation inversely correlated with the upregulation of CD25 in both FoxP3- (Tact) and FoxP3+ (Treg) T-cell subsets. Furthermore, ex vivo analysis of peripheral CD4+ and CD8+ T cells with activated (CD25+) or effector memory (effector memory T cell, CD45RA-CCR7-) phenotype present lower CD6 levels than their naïve or central memory (central memory T cell, CD45RA-CCR7+) counterparts. CD6lo/- T cells resulting from in vitro T-cell activation show higher apoptosis and lower proliferation levels than CD6hi T cells, supporting the relevance of CD6 in the induction of proper T-cell proliferative responses and resistance to apoptosis. Accordingly, CD6 transfectants also showed higher viability when exposed to TCR-independent apoptosis-inducing conditions in comparison with untransfected cells. Taken together, these results provide insight into the origin of sCD6 and the previously reported circulating CD6-negative T-cell subset in humans, as well as into the functional consequences of CD6 down-modulation on ongoing T-cell responses, which includes sensitization to apoptotic events and attenuation of T-cell proliferative responses

CD6 modulates thymocyte selection and peripheral T cell homeostasis

The CD6 glycoprotein is a lymphocyte surface receptor putatively involved in T cell development and activation. CD6 facilitates adhesion between T cells and antigen-presenting cells through its interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), and physically associates with the T cell receptor (TCR) at the center of the immunological synapse. However, its precise role during thymocyte development and peripheral T cell immune responses remains to be defined. Here, we analyze the in vivo consequences of CD6 deficiency. CD6(-/-) thymi showed a reduction in both CD4(+) and CD8(+) single-positive subsets, and double-positive thymocytes exhibited increased Ca(2+) mobilization to TCR cross-linking in vitro. Bone marrow chimera experiments revealed a T cell-autonomous selective disadvantage of CD6(-/-) T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T cell selection events occur in the absence of CD6. CD6(-/-) mice displayed increased frequencies of antigen-experienced peripheral T cells generated under certain levels of TCR signal strength or co-stimulation, such as effector/memory (CD4(+)TEM and CD8(+)TCM) and regulatory (T reg) T cells. The suppressive activity of CD6(-/-) T reg cells was diminished, and CD6(-/-) mice presented an exacerbated autoimmune response to collagen. Collectively, these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T cell subpopulations, including T reg cells

Multifaceted Effects of Soluble Human CD6 in Experimental Cancer Models

Background: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. Methods: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEμTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. Results: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. Conclusions: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma

Whole-genome sequencing reveals host factors underlying critical COVID-19

Altres ajuts: Department of Health and Social Care (DHSC); Illumina; LifeArc; Medical Research Council (MRC); UKRI; Sepsis Research (the Fiona Elizabeth Agnew Trust); the Intensive Care Society, Wellcome Trust Senior Research Fellowship (223164/Z/21/Z); BBSRC Institute Program Support Grant to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/10002070, BBS/E/D/30002275); UKRI grants (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1); UK Research and Innovation (MC_PC_20029); the Wellcome PhD training fellowship for clinicians (204979/Z/16/Z); the Edinburgh Clinical Academic Track (ECAT) programme; the National Institute for Health Research, the Wellcome Trust; the MRC; Cancer Research UK; the DHSC; NHS England; the Smilow family; the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA award number UL1TR001878); the Perelman School of Medicine at the University of Pennsylvania; National Institute on Aging (NIA U01AG009740); the National Institute on Aging (RC2 AG036495, RC4 AG039029); the Common Fund of the Office of the Director of the National Institutes of Health; NCI; NHGRI; NHLBI; NIDA; NIMH; NINDS.Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care or hospitalization after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease