immunitat innata immunodeficiènciesgrupo español de inmunodeficiencias primarias (geidp) y...

VIIè CONGRÉS Societat Catalana d’Immunologia (SCI)

XIè SIMPOSI adjunt

Grupo Español de Inmunodeficiencias Primarias (GEIDP) y Registro Español de Inmunodeficiencias Primarias (REDIP)

Programa Final

Barcelona, 14 i 15 de novembre de 2013

Immunitat Innata

Immunodeficiències

2 www.sci.cat

Organization Committee (SCI Board): Welcome to the Congress,

On behalf of the organising committee, we would like to warmly welcome you to the

VIIth Societat Catalana d’Immunologia (SCI) Congress and also to the XIth Grupo Español

de Inmunodeficiencias Primarias (GEIDP) y Registro Español de Inmunodeficiencias

Primarias (REDIP) Symposium. We believe that our meeting will present high level

scientific knowledge with the contribution of immunologists and specialists who are

experts in these fields.

Dr. Cándido Juárez

SCI President

President: Dr. Cándido Juárez

Secretary: Dra. Mercè Martí

Treasurer: Dra. María José Amengual

Vice President: Dr. Jorge Lloberas

Member: Dra. Irene Puga

Member: Dr. Daniel Benítez

Member: Dr. Carles Morte

Member: Dra. Aura Muntasell

Congress Office: Sra. Eva Palacios ([email protected])

www. sci.cat 3

This Congress is sponsored by:

Gold Sponsor

Silver Sponsors

Sponsor awards for best oral and poster

communications

http://www.sci.cat/

4 www.sci.cat

Content

This Congress is sponsored by: ............................................................................................... 3

Content .................................................................................................................................... 4

Scheme first day ...................................................................................................................... 5

Scheme second day................................................................................................................. 6

Abstracts .................................................................................................................................. 8

Oral Communications Session I Clinical Immunology 1-5 ............................................... 8

Oral Communications Session II Innate Response 6-10 ............................................... 13

Oral Communications Session III From Innate to Adaptative Immunity 11-15 .............. 18

E-poster List ........................................................................................................................... 23

Posters From Innate to Adaptative Immunity 1-5 ............................................................... 23

Posters Clinical Immunology 6-14 ...................................................................................... 28

Posters Methodology and Techniques 15-22 ..................................................................... 37

Posters Innate Response 23-26 ......................................................................................... 45

2014 Events ........................................................................................................................... 49

Lifelong Learning SCI Program .......................................................................................... 49

New members ........................................................................................................................ 50

Registration form to SCI ..................................................................................................... 50

Participant information ........................................................................................................... 50

Useful information............................................................................................................... 51

List of participants............................................................................................................... 52

Other useful information and notes .................................................................................... 55

www.sci.cat 5

Scheme first day

Thursday, November 14th

15:30 17:30

Arrival, Registration and Documentation delivery

16:20 16:30

Welcome to the VIIth CONGRESS of SCI and XIth GEIDP-REDIP Dr. Cándido Juárez (President of SCI) Dr. Manuel Hernández (President of the Organizing Committee GEIDP-REDIP)

16:30 17:30

Chair: Dr. Margarita López Trascasa and Dr. Cándido Juárez

DR. PETER GARRED Department of Clinical Medicine, Section of Surgery and Internal Medicine, Rigshospitalet (Copenhagen)

“Complement Deficiency”

17:30 17:45

Poster viewing – Coffee Break Posters can be viewed, since 16:00h, on the 4 electronic panels located in the Hall

17:45 18:45


DR. FRANCISCO LOZANO Unitat Immunitat Innata, Hospital Clínic – CDB Centre de Diagnòstic Biomèdic–

Immunologia

“Myths and realities of mannose-binding lectin deficiency”

18:45 19:45


DR. ANNE PUEL Laboratoire de Génétique Humaine des Maladies Infectieuses. Inserm, Paris.

“Immunodeficiencies: From the Clinic to the Bench”

19:50 End of session

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Scheme second day

Friday, November 15th

08:30 09:00

Arrival, Registration and Documentation delivery

09:00 10:00

Chair: Dr. María José Amengual

DR. PABLO PELEGRÍN Inflammation and Experimental Surgery Research Unit. Murcia Biomedical Research Institute (IMIB).

“Innate immune regulation by the Inflammasome”

10:00 11:00

Oral Communications Session I: Clinical Immunology

Chair: Dr. Ramón Gimeno and Dr. María José Amengual

10:00h C5 inherited deficiency characterization in two non-related African families. Clara Franco-

Jarava et al.(oral presentation 1)

10:12h Diverse lymphocyte phenotype in DiGeorge Syndrome. Lozano-Ramos S. et al.(oral

presentation 2)

10:24h Determinació d´adenosin deaminasa en sang de taló. Una prova de concepte pel

diagnòstic neonatal universal del dèficit d´ADA a Catalunya. M.Pilar Llobet Agulló (oral

presentation 3)

10:36h Mutaciones Somáticas en K-Ras en un paciente con Síndrome Linfoproliferativo

Autoinmune. Laura Martínez-Martinez et al.(oral presentation 4)

10:48h Identification and characterization of a novel splice site mutation in the SERPING1 gene in

a family with hereditary angioedema. R. Colobran et al. (oral presentation 5)

11:00 11:30


11:30 12:30

Chair: Dr. Jorge Lloberas

DR. ANGEL CORBÍ Centro de Investigaciones Biológicas, CSIC, Madrid.

“Novel markers and regulators of human macrophage polarization”

12:30 13:30

Assemblea General Ordinària SOCIETAT CATALANA d’IMMUNOLOGIA (12.30h – First Call) Us hi esperem a tots: els socis i no-socis!!

13:35 15:00

Poster viewing – LUNCH Posters can be viewed, since 09:00h, on the 4 electronic panels located in the Hall

www.sci.cat 7

15:00 16:00

Chair: Dr. Mercè Martí

DR. ANNE PUEL Laboratoire de Génétique Humaine des Maladies Infectieuses. Inserm, Paris.

“Inborn errors of the TLR-NF-kB signaling pathway and infectious diseases”

16:00 16:55

Oral Communications Session II: Innate Response

Chair: Dr. Aura Muntasell and Dr. Eva Martínez-Cáceres

16:00h Reciprocal negative cross-talk between the nuclear receptor LXR and STAT1 affects IFN-γ-

induced neuroinflammation and LXR-mediated lipid homeòstasis. Monica Pascual-García et

al. (oral presentation 6)

16:12h The exonuclease Trex1 restrains the macrophage pro-inflammatory activation. Selma

Pereira-Lopes et al. (oral presentation 7)

16:24h STAT1 as a repressor. Lorena Valverde et al. (oral presentation 8)

16:36h Role of inflammation in Influenza A H1N1 pandemic virus. Pamela Martínez Orellana et al.

(oral presentation 9)

16:48h NKG2C zygosity influences CD94/NKG2C receptor function and the NK cell compartment

redistribution in response to human citomegalovirus. María López et al. (oral presentation

10)

16:55 17:20


17:20 18:15

Oral Communications Session III: From Innate to Adaptative Immunity

Chair: Dr. Daniel Benítez and Dr. Mercè Martí

17:20h Phagocytic B cells: from fish to mammals. David Parra et al. (oral presentation 11)

17:32h Identification of IL-10 producing B cells in the human gut mucosa. Elisabeth Calderón

Gómez et al. (oral presentation 12)

17:44h Conseqüències funcionals de la unió de plaquetes als limfòcits T en la inflamació. Carlos

Zamora et al (oral presentation 13)

18:56h Redundancy of CDR3 sequences and public TCR in T1D. Carlos Martínez-Torró et al. (oral

presentation 14)

18:08h Innate immune signatures in malaria-naïve adults infected with Plasmodium falciparum

sporozoites by needle injection with different doses and routes. Gemma Moncunill et al.

(oral presentation 15)

18:15 19:15

Chair: Dr. María Montoya

DR. ALBERTO MANTOVANI Professor at Università degli Studi di Milano. Istituto Clinico Humanitas

“Interactions between the cellular and the humoral arm of innate immunity”

19:15 19:30

Prize to the best communication and poster in the Congress. Dr. Cándido Juárez (President of SCI) Funded by MILTENY Biotec End of Congress

8 www.sci.cat

Abstracts

Oral Communications Session I Clinical Immunology 1-5

1 C5 inherited deficiency characterization in two non-related African families

Clara Franco-Jarava (1); Andrea Martín-Nalda (2); Roger Colobran (1,3); Laura Minguell (2); Hernández-González, Manuel (1); and Pere Soler-Palacín (2)

(1) Immunology Division. Hospital Universitari Vall d’Hebron (HUVH). Vall d’Hebron Research Institute (VHIR). (2) Pediatric Infectious Diseases and Immunodeficiencies Unit. Hospital Universitari Vall d´Hebron. Vall d’Hebron Research Institute (VHIR). (3) Clinical and Molecular Genetic Division. Hospital Universitari Vall d’Hebron (HUVH). Vall d’Hebron Research Institute (VHIR

Complement C5 deficiency (C5D; MIM#120900) is a rare autosomical recessive genetic defect associated to recurrent Gram-negative bacterial infections, especially caused by Neisseria spp. Despite the fact that more than 50 cases have been reported, the molecular basis has only been elucidated in a few of them. The aim of the present study is to characterize the complement in two patients, a girl of 3 years (P1) and a male 5 years old (P2) that had an episode of meningococcal septicaemia due to uncommon serotypes (E29 and Y) respectively. Both patients were of North African ancestry but unrelated. The study showed normal C3 and C4 serum levels. Complement activity through both the classical and alternative pathways was almost absent by a liposome based immunoassay (Wako Pure Chemical Industries, Japan) and by haemolytic assays (The Binding Site, UK), respectively. Total absence of C5 protein in patients´ sera was assessed through Ouchterlony radial immunodiffusion (The Binding Site,UK) and a commercial ELISA kit (Quidel Corporation, USA) revealed the absence of sC5b9. Haemolytic activity could be restored by adding purified C5 molecule (Quidel Corporation, USA) to deficient patients´ sera, result that confirmed the defect for C5 molecule in both family patients. Afterwards, the asymptomatic young brother of case 1

was also diagnosed of C5 deficiency. In one of the families (P2), cDNA sequencing of the gene encoding for C5 showed that the patient was homozygous for a deletion of 3 nucleotides in exon 9 (c.960_962del) leading to a deletion of Asn320 (new mutation). The presence of the mutation was confirmed by gDNA sequencing. The genetic study of the second family (P1) is still to be performed. Current characterization of this new mutation through in silico modelling will enable us to elucidate the relationship between structure and function of this protein. In conclusion, we have described one non-previously characterized mutation causing C5 deficiency. These results are in accordance with the high heterogeneity that characterizes this disorder. Further systematic studies will be required to know the real prevalence of this deficiency in the population and to deepen knowledge of the mechanisms underlying its clinical implications.

www.sci.cat 9

Oral Communications Session I Clinical Immunology

2 Diverse lymphocyte phenotype in DiGeorge Syndrome

Lozano-Ramos S.I. (1), Rus-Merchán, Amanda (1), Teniente Serra, Aina (2), Pintos-Morell G. (2), Rodrigo C. (1), Martínez-Cáceres, Eva M. (1)

(1) Department of Immunology. Germans Trias i Pujol University Hospital; (2) Department of Pediatrics. Germans Trias i Pujol University Hospital. Universitat Autonoma de Barcelona

DiGeorge syndrome (DGS) is secondary to a 22q11.2 microdeletion that causes cardiac

abnormalities and an abnormal embryonic development of the third branchial arch, which is

involved in thymus and parathyroid gland formation. It may be associated with a primary combined

immunodeficiency. DGS patients are highly heterogeneous in their clinical and laboratory features.

Patients usually present lymphopenia in infancy that may resolve later in childhood. Research has

been focused mainly on lymphocyte homeostasis and humoral immunity. No extensive

multiparametric phenotypical analysis of peripheral blood lymphocytes has been reported.

The aim of this study is to describe characteristic features in the lymphocyte subpopulations from

four patients diagnosed of DSG in our centre.

Material and methods: Peripheral blood from four pediatric patients diagnosed of DGS with

22q11.2 microdeletion was analyzed by multiparametric flow cytometry. With the aim to analyze LT

maturation, CD3+, CD4+CD8- or CD4-CD8+ subsets were selected and identifying in them the

following subpopulations Recent thymic emigrants (RTE) (CCR7+CD45RA+CD27+CD31+PTK7+),

naive (CCR7-CD45RA+CD27+), central memory (CCR7-CD45RA-CD27+), early effector memory

(CCR7-CD45RA-CD27+), late effector memory (CCR7-CD45RA-CD27-) and reverted effector

memory (TEMRA) (CCR7-CD45RA+CD27-). Furthermore, naive (CD19+CD27-IgD+IgM+),

unswitched memory (CD19+CD27+IgD+/-IgM+) and switched memory (CD19+CD27+IgD-IgM-) B-

cells were analyzed.

Results: Patients did not show leucopenia. No abnormalities in B-cell subpopulations were found.

However, in all patients polyclonal hypergammaglobulinemia was detected in childhood.

Patients 2, 3 and 4 showed a mild TCD4 lymphopenia (400-600 cell/ µL)

Patient 1 (8 years) showed normal lymphocyte count with normal percentages of TCD4 and TCD8,

with an important increase of LT CD8 naïve (61% VN: 6-54%)

Patient 2 (15 years) had not alterations in the percentages of TCD4 or TCD8 subpopulations.

However, the maturation analysis revealed a low percentage of LTCD4 naïve subpopulation (8.9%

VN: 15-45%) and a predominance of TCD4 late effector memory subsets (51.2% VN: 12-36%).

Despite the mild lymphopenia detected in patient 3, no maturation alterations were detected.

In the analysis of patient 4 (20years), TCD4 effector memory and TEMRA subsets were absent.

(0% VN: 2-14% and 0% VN: 1-40% respectively). Whereas an increased percentage of TCD8

central memory subset was detected (28.6% VN: 1.5-16%).

Conclusion: In spite of sharing 22q11.2 microdeletion, our patients were heterogeneous in their

lymphocyte phenotype.

The application of this analysis together with the clinical features may be a useful tool in the

diagnosis and sub-classification of DGS patients.

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3 Determinació dádenosin deaminasa en sang de taló. Una prova de concepte pel diagnòstic neonatal universal del dèficit dÁDA a Catalunya.

M. Pilar Llobet Agulló

Grup de Treball dÍmmunodeficiències de la Societat Catalana de Pediatria i la Societat Catalana dÍmmunologia.

Introducció i objectius: El dèficit dádenosin deaminasa (ADA) és una de les formes més

freqüents dímmunodeficiència combinada greu (IDCG). Es pot diagnosticar en sang seca de taló

mitjançant la determinació del nivells de deoxiadenosina per espectometria de masses en tàndem

(TMS). Dades europees recents en demostren la utilitat, el baix cost i la repercussió en el

pronòstic vital dels pacients diagnosticats. El GTI ha presentat oficialment la sol•licitud per a la

inclusió dáquesta entitat en el programa de cribratge neonatal universal al nostre país.

Presentem dos pacients amb dèficit dÁDA de dos centres de Barcelona en els que demostrem

que el diagnòstic s´hagués pogut dur a terme al naixement mitjançant aquesta metodologia.

Cas1: Pacient de sexe femení debuta als 17 dies de vida amb un quadre clínic compatible amb

una bronquiolitis infecciosa, que evoluciona a una pneumopatia intersticial. Es detecta una

limfopènia greu i es completa léstudi immunitari, que diagnostica al mes de vida una IDCG T-B-,

que posteriorment sídentifica genèticament com a dèficit ADA als 12 mesos dédat (mutació

homozigota c.362+1_+4del4bp). Se sotmet al pacient a trasplantament de progenitors

hematopoètics (TPH) de donant no emparentat als 3 mesos de vida. Actualment, un any post-

trasplantament, la pacient presenta una correcta reconstitució immunològica i hematològica i un

quimerisme 100% de donant.

Cas 2: Pacient de sexe femení de 10 mesos dédat remesa a un dels centres des de Galícia per

quadre de pneumonitis per citomegalovirus y Pneumocystis jirovecii associat a limfopènia greu

que es cataloga de IDCG T-B-. Posteriorment es confirma genèticament el diagnòstic de dèficit

dÁDA als 11 mesos dédat (dues mutacions en heterozigosi c.466C>T / p.Arg156Cys i c.646G>A

/ p.Gly216Arg). Se sotmet a la pacient a un TPH de donant no emparentat. Actualment en dia + 60

postrasplant es troba estable clínicament amb bona recuperació hematològica i un quimerisme

100%.

En tots dos casos es procedeix a la recuperació de mostra de sang de taló i determinació dels

valors de adenosina i 2-deoxiadenosina mitjançant TMS obtenint uns valors de 17,26 i 37 µmol/L

(VN<1,5), 24,71 i 2,88 µmol/L (VN<0,07) respectivament, fet que demostra que el diagnòstic

s´hagués pogut realitzar durant el període neonatal.

Conclusió: la presentació dáquests dos casos pretén aportar més dades sobre la possibilitat

dún diagnòstic precoç del dèficit dÁDA mitjançant léstudi de la prova de taló. Atès que un

diagnòstic precoç i lábsència de complicacions infeccioses s´han demostrat associades a un

millor pronòstic vital en aquests pacients, des del GTI considerem que s´ha de procedir a incloure

aquesta entitat al programa de detecció neonatal del nostre país.

www.sci.cat 11


4 Mutaciones Somáticas en K-Ras en un paciente con Síndrome Linfoproliferativo Autoinmune

Laura Martínez-Martinez, Isabel Jimenez Bernal, Maria Victoria Rubiales, Isabel Badell, Oscar de la Calle-Martín

Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona.

Introduction: Autoimmune lymphoproliferative syndrome (ALPS) is a genetic disorder of

lymphocyte apoptosis characterized by early onset of lymphoproliferation and autoimmune

cytopenias. ALPS is related to several genetic defects, but most patients (>70%) had

dominant mutations affecting the FAS protein. A few patients present similar clinical

manifestations without elevated levels of double negative T cells (DNT:

CD3+TCRαβ+CD4-CD8-), the hallmark in patients with ALPS-FAS.

Aims: To identify the genetic alteration in a patient clinically compatible with ALPS

(lymphoproliferation and autoimmuninty).

Methods: The patient, a 4-years-old boy of non-consanguineous parents, presented large

splenomegaly, multiples adenopathies and autoimmune pancytopenia (anemia,

neutropenia and thrombocytopenia) since 2 years-old. The patient also suffered several

episodes of fever and infections. Immunosuppression and IVIG treatment were established

achieving a general health improvement, including a reduction of spleen. Lymphocyte

subpopulations were determined by flow cytometry. Analysis of FAS, FASL, CASP-8 and

CASP-10 genes were performed in DNA from peripheral blood, whereas N-RAS and K-

RAS genes were analyzed both in DNA from peripheral blood and buccal swab cells.

Results: The patient presented normal levels of DNT cells (<1%), but B cell lymphocytosis

and substantial monocytosis. Analysis of different ALPS-related genes showed a

heterozygous substitution in the K-RAS gene (p.G13D) in DNA from peripheral blood. The

mutation was not present in DNA from non-hematopoietic cells, indicating that the patient

presented a somatic mutation. This mutation has been previously described also in ALPS

like patients.

Conclusions: We report a novel patient with a somatic mutation in the K-RAS gene

(p.G13D).

12 www.sci.cat


5 Identification and characterization of a novel splice site mutation in the SERPING1 gene in a family with hereditary angioedema.

Roger Colobran; Sergio Lois; Xavier De la Cruz; Ricardo Pujol-Borrell; Manuel Hernández-González; Mar Guilarte

Vall d´Hebron Institut de Recerca (VHIR).

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-

dominant disease caused by mutations in SERPING1 gene. The main clinical feature of

C1INH deficiency is the spontaneous edema of the submucosal layers. More than 280

different mutations scattering the entire SERPING1 gene have been reported. We

identified and characterized a new mutation in SERPING1 gene in a Spanish familily with

hereditary angioedema. The mutation (c.685+2T>A) disrupts the donor splice site of intron

4 leading to the loss of exon 4 in mutant mRNA. We demonstrated that mutant mRNA is

mostly degraded, probably by the surveillance pathway no-go mRNA decay. Bioinformatic

analysis showed that the mutant protein, if produced, would be non-functional since the

protein lacks a stretch of 45 amino acids affecting the functional RCL loop. Finally, we

found a reduction of the wild-type mRNA expression in c.685+2T>A carriers.

www.sci.cat 13

Oral Communications Session II Innate Response 6-10

6 Reciprocal negative cross-talk between the nuclear receptor LXR and STAT1 affects IFN-γ-induced neuroinflammation and LXR-mediated lipid homeostasis

Monica Pascual-García (1); Laura Rué (2); Theresa León (1); Josep Julve (3); Jose Maria Carbó (1); Jonathan Matalonga (1); Herbert Auer (4); Antonio Celada (1); Joan Carles Escolà-Gil (3); Knutt R. Steffensen (5); Esther Pérez-Navarro (2); Annabel Valledor (1)

(1) Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona (UB) (2) Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, UB (3) Institut de Investigació Biomedica, Hospital de la Santa Creu I Sant Pau. (4) Core Facilities-Institut de Recerca Biomèdica. (5)Karolinska Institute, Sweden.

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in cont rol of

inflammation. In this study we have explored the impact of LXR activation on the

macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional

profiling studies demonstrate that ∼38% of the IFN-γ-induced transcriptional response is

repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on

selected IFN-γ-induced genes in primary microglia and in a model of IFN-γ-induced

neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the

promoters tested in this study without affecting STAT1 phosphorylation. A closer look into

the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear

receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We

have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets.

Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent

upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol

response element binding protein 1c. Downregulation of ABCA1 expression correlated

with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with

IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of

LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression

of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on

the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute

to STAT1-dependent downregulation of LXR targets. The results from this study suggest

an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with

implications both in the control of IFN-γ-mediated immune responses and in the regulation

of lipid metabolism.

14 www.sci.cat

Oral Communications Session II Innate Response

7 The exonuclease Trex1 restrains the macrophage pro-inflammatory activation

Selma Pereira-Lopes (1), Teja Celhar (2), Gloria Sans-Fons (1), Maria Serra (1), Anna-Marie Fairhurst (2), Antonio Celada (1) and Jorge Lloberas (1)

(1) Departament de Fisiologia i Immunologia, Parc Cientific de Barcelona, Universitat de Barcelona. (2) Singapore Immunology Network, Immunos, Singapore.

The three prime repair exonuclease 1 (TREX1) is the most abundant exonuclease in

mammalian cells. Mutations in Trex1 gene are being linked to the development of Aicardi-

Goutières syndrome, an inflammatory disease of the brain, and Systemic Lupus

Erythematosus. In clinical cases and in a Trex1-deficient murine model, chronic production

of type I IFN plays a pathogenic role. In this study we demonstrate that Trex1-/- mice

present inflammatory signatures in many different organs, including the brain. Trex1 is

highly induced in macrophages in response to pro-inflammatory stimuli, including TLR7

and TLR9 ligands. Our findings show that in the absence of Trex1, macrophages

displayed an exacerbate pro-inflammatory response. More specifically, following pro-

inflammatory stimulation Trex1-/- macrophages exhibited an increased TNF-α and IFN-y

production, higher levels of CD86 and increased antigen presentation to CD4+ T cells, as

well as an impaired apoptotic T cell clearance. These results evidence an unrevealed

function of the Trex1 as a negative regulator of macrophage inflammatory activation and

demonstrate that macrophages play a major role in diseases associated with Trex1

mutations which contributes to the understanding of inflammatory signature in these

diseases.

www.sci.cat 15


8 STAT1 as a repressor

Lorena Valverde-Estrella; Consol Farrera; Antonio Celada; Jorge Lloberas

Department de Fisiologia i Immunologia, Parc Cientific de Barcelona, Universitat de Barcelona.

IFN-γ is the major activator of macrophages. This cytokine interacts with the specific

receptor and triggers the sequential activation of the JAK-STAT-1 pathway, which results

in transcriptional up-regulation of more than 300 genes.

Macrophage proliferation induced by M-CSF requires ERK-1/2 phosphorylation during a

short period of 5 minutes. The dephosphorylation is carried out by the MKP-1 that is

induced by M-CSF. The addition of IFN-γ blocks proliferation that is due to an elongation

of ERK phosphorylation. This is due to the inhibition of MKP-1 expression.

Here we show that IFN-γ modulates inhibition of MKP-1 induction by M-CSF through the

binding of STAT1 to a particular region in the mkp-1 promoter. Using a gene array we

have found that 36 genes have similar expression patterns and in 33 of them we have

found the sequence where STAT1 is binding. We have controlled these results by

quantitative PCR. To further study this repressive effect of STAT1, we have cloned the

promoter of cxcl2 which is repressed by IFN-γ stimuli and contains the specific region of

STAT1 binding.

In conclusion we have found a new sequence where STAT1 binds and acts as a

repressor.

16 www.sci.cat


9 Role of inflammation in Influenza A H1N1 pandemic virus

Pamela Martínez Orellana; Raquel Almansa; Beatriz Vidaña; Lorena Córdoba; Jorge Martínez; Jesús Bermejo-Martin; María Montoya

Centre de Recerca en Sanitat Animal (CRESA).

Influenza A (H1N1) 2009 pandemic virus (pdmH1N1) caused an outbreak that affected

human population spreading illness and death among worldwide. Nowadays

pdmH1N1virus continues to be a public health threat. Mainly, the reported cases have

been characterized by mild and/or self-limiting infection. Nevertheless, a subset of patients

required hospitalization and mechanical ventilation (MV) support. Previous studies

revealed an implication of IL-6 together with IL-8, IL-10 y G-CSF in pdmH1N1 infection

when a significant increase of these cytokines was shown in clinically severe patients.

To evaluate how an exacerbated inflammation could affect the pathogenesis of the

pdmH1N1 influenza infection, C57/Bl6 (C57) mice were infected with a

A/CATALONIA/63/2009 strain (CAT09) and inoculated with LPS to stimulated the release

of general proimmflamatory cytokines. The specific role IL-6 was also investigated by

inoculating pcDNA3.1+-mIL6 -plasmid (pIL-6) in CAT09 infected or uninfected mice. In all

experiments, groups of animals consisted on: control mice, LPS or pIL6 treated mice,

CAT09-infected mice and CAT09+LPS or pIL6 treated mice. All animals were daily

observed and weighed. At necropsy, anesthetized animals were bled and samples of lung

and trachea were collected.

Analysis of weight loss of CAT09+LPS mice showed a peak of reduction at 2pi. Animals

do not recover their body weight until day 10 pi. These animals show a profound decrease

on body weight from 2 to 5 dpi when compare with the rest of the groups. On the other

hand, CAT09+pIL-6 mice had a very sharp decrease on weight loss at 1 to 5dpi when

compare with the rest of the groups and they started to recover body weight from 8 to 10

dpi, similarly as the CAT09 infected mice. Virus replication on lung of infected mice was

determined and similar viral titres were observed when CAT09+LPS –group were compare

to CAT09-infected group. Lungs of CAT09+pIL-6 –mice showed lower titres than CAT09-

infected mice. Finally, histopathology analysis and IL-6 levels of serum and lung were

measure by ELISA. Results so far indicated that the presence of LPS and/or IL-6

overproduction is detrimental for pdmH1N1 2009 infection.

www.sci.cat 17


10 NKG2C zygosity influences CD94/NKG2C receptor function and the NK cell compartment redistribution in response to human cytomegalovirus

María López (2); Aura Muntasell (1); Andrea Vera (1); Gemma Heredia (2); Neus Romo (2); Judith Peñafiel (1); Manuela Moraru (3); Joan Vila (1); Carlos Vilches (3); Miguel López-Botet (1,2)

(1) IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain; (2) Immunology Unit, Univertsity Pompeu Fabra, Barcelona, Spain; (3) Immunogenetics-HLA, Immunology Department, Hospital Universitario Puerta de Hierro, Majadahonda, Spain.

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a

functionally competent NK-cell subset expressing the activating CD94/NKG2C receptor.

Factors underlying the wide variability of this effect observed in HCMV-seropositive

healthy individuals and exacerbated in immunocompromized patients are uncertain. A

deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C

genotype with circulating NKG2C+ NK-cell numbers was observed in HCMV+ children. We

have assessed the influence of NKG2C gene dose on the NK-cell repertoire in a cohort of

young healthy adults (N = 130, median age 19 years). Our results revealed a relation of

NKG2C copy number with surface receptor levels and with NKG2C+ NK-cell numbers in

HCMV+ subjects, independently of HLA-E dimorphism. Functional studies showed

quantitative differences in signaling (i.e. iCa2+ influx), degranulation, and IL-15-dependent

proliferation, in response to NKG2C engagement, between NK cells from NKG2C+/+ and

hemizygous subjects. These observations provide a mechanistic interpretation on the way

the NKG2C genotype influences steady-state NKG2C+ NK-cell numbers, further

supporting an active involvement of the receptor in the HCMV-induced reconfiguration of

the NK-cell compartment. The putative implications of NKG2C zygosity over viral control

and other clinical variables deserve attention.

18 www.sci.cat

Oral Communications Session III From Innate to Adaptative Immunity 11-15

11 Phagocytic B cells: from fish to mammals

David Parra; Oriol Sunyer

Departamento de Biologia Cel·lular, Fisiologia i Immunologia. Universitat Autonoma de Barcelona.

Until recently, it was believed that primary B cells were not capable of performing

phagocytosis. In 2006 we broke this paradigm by showing for the first time that teleost and

amphibian primary IgM+ B cells contained phagocytic capacity. Here we present the

evolutionary conservation of this ability in mammals. Thus, we report a previously

unrecognized ability of peritoneal cavity (PerC) B-l B lymphocytes to phagocytose latex

beads and bacteria. Both B-1a and B-1b cell subsets in PerC, are able to mature their

phagosomes into phagolysosomes and have the ability to kill internalized bacteria.

Nevertheless, their phagocytic and bactericidal capacities were significantly lower than

those found for PerC macrophages, probably because of the higher level of lysosomal

proteases in macrophages compared to B cells. In addition to taking up large particles,

PerC B-1a and B-1b B cells are capable to present antigen from internalized particles to

CD4+ T cells. Notably, when antigen is particulate, B-1 B cells require 100 times less

antigen to prime CD4+ T cells than when antigen is soluble. The novel phagocytic and

microbicidal abilities identified in B-1 B lymphocytes strengthen the innate nature that has

long been attributed to these cells and position these cells at the crossroads that link

innate with adaptive immune processes. Furthermore, this findings support further the

hypothesis proposed by us and others that B cells evolved from a phagocytic predecessor.

www.sci.cat 19

Oral Communications Session III From Innate to Adaptative Immunity

12 Identification of IL-10 producing B cells in the human gut mucosa

Elisabeth Calderón ; Raquel Cabezón; Rut Mora; Julià Panés; Azucena Salas

Institut d´Investigacions Biomèdiques August Pi i Sunyer. Barcelona.

B cells are classically considered for their unique capacity to produce antibodies. Besides

this, B cells can also present antigens, co-stimulate T cells and produce cytokines. In

recent years, the ability of B cells to negatively regulate cellular immune responses by IL-

10 production has been described in different immunological and pathological settings. In

humans, IL-10 producing regulatory B cells have been identified in peripheral blood.

However, the ability to produce IL-10 by human gut mucosal B cells remains unknown. To

explore this, we isolated two main subsets of intestinal lamina propria B cells:

CD19+CD20+HLA-DR+CD38- (B cells) and CD19+CD20-HLA-DR-CD38+ (plasma cells)

and determined their IL-10 production in response to different stimuli. TLR9 combined with

BCR stimulation induced the highest IL-10 production in both subsets as measured in

culture supernatants by ELISA; the percentage of IL-10 producing cells was higher within

the plasma cell population. Our results provide evidence that gut mucosal B cells are able

to produce IL-10, suggesting that they could contribute to regulate intestinal immunity.

20 www.sci.cat


13 Conseqüències funcionals de la unió de plaquetes als limfòcits T en la inflamació

Carlos Zamora, Elisabet Cantó, Juan C. Nieto, M. Angels Ortiz, Cesar Diaz-Torné, Cesar Diaz-Lopez, Josep M. Llobet, Cándido Juárez, Sílvia Vidal

Institut dÍnvestigacions Biomèdiques Hospital de la Santa Creu i Sant Pau (IBB Sant Pau).

Introducció: El CD36 es un receptor scavenger que es troba expressat en cèl•lules del

sistema immune innat com els monòcits i les plaquetes. En els darrers experiments, hem

observat que una subpoblació de limfòcits T són també CD36+.

Objectius: Hem analitzat si lórigen de léxpressió del CD36 es extrínsec o intrínsec als

limfòcits. Després, utilitzant CD36 com a marcador, hem investigat la capacitat funcional

dáquesta subpoblació i quin paper pot tenir en lártritis reumatoide (AR).

Mètodes: A partir de cèl•lules mononuclears perifèriques de donants sans, es va avaluar

lórigen del CD36 present en una subpoblació de limfòcits T per citometria de fluxe i

microscòpia de fluorescència. Es va comparar la capacitat proliferativa i la producció de

citoquines dels limfòcits T CD36+ i els T CD36-, estimulant-los amb anti- CD3, CD2 i

CD28. Finalment, es va comparar els nivells de limfòcits T CD36+ entre donants sans i

pacients dÁR i els nivells de citocines que produeixen.

Resultats: Lánàlisi per citometria de fluxe i microscòpia de fluorescència mostra que la

subpoblació de limfòcits T CD36+ porta plaquetes unides i que són aquestes les que la

fan positiva per CD36. Vam observar que la proliferació després de 72h i 7 dies

déstimulació era menor en els limfòcits T CD4+ CD36+ que en els CD36-. La producció

de IL-17 i IFN-γ intracel•lular també estava reduïda en els limfòcits T CD36+. Un grup de

pacients dÁR presentava un percentatge de CD4+CD36+ comparable als donants sans i

un altre grup presentava nivells superiors. Els pacients dÁR amb elevats títols

dánticossos contra pèptids citrulinats (anti-CCP) i factor reumatoide (FR), nivells elevats

de IL-17 i IFN-γ en sèrum, i major risc cardicovascular presentaven menor percentatge de

limfòcits T CD4+ amb plaquetes unides.

Conclusions: La unió de plaquetes modifica la funcionalitat dels limfòcits T, suggerint que

es un mecanisme regulatori que opera en aquells pacients de AR amb fenotip menys

sever.

www.sci.cat 21


14 Redundancy of CDR3 sequences and public TCR in T1D

Carlos Martínez-Torró; Carme Garcia-Martín; Cristina Xufré; Eduardo Pizarro; Lorena Usero; Manuela Costa; Carme Roura-Mir; Dolores Jaraquemada; Mercè Martí

Institut Biotecnologia i Biomedicina; Universitat Autònoma de Barcelona.

TCR repertoires are more redundant than previously proposed, i.e. there are TCRs that occur more frequently than expected or that are shared by different individuals. The idea that public TCRs could be involved in T1D comes from the evidence that a the CDR3 sequence of a pancreas-expanded Vβ7 TCR (CASSQVAGAGTGELFF) described by our group (Codina-Busqueta et al. J. Immunol 2011) had been previously found by Conrad et al. (Nature 1994) in the purified islets from a diabetic donor. The two patients shared HLA-A2, HLA-Cw3 and HLA-DR4. Moreover, results from Garcia-Martin (unpublished) have shown other CDR3 sequences from the pancreas infiltrates of different pancreas from diabetic donors. These results have prompted us to hypothesize that autoreactive public TCR could be involved in the development of the autoimmune process and that if so, they should be detected in PBLs from T1D donors. M&M. High throughput sequencing was perfomed using MySeq sequencer from Illumina to analyze the TCR repertoire of TRBV4 (Vβ7), TRBV20 (Vβ22) and TRBV25 (Vβ11) families. PBMCs were isolated from two diabetic patients at the onset (D1, D2), and two controls (C1, C2). Samples were chosen by their HLA-typing as all of them shared the mentioned alleles. mRNA from 6x106 cells were retrotranscribed and two rounds of PCR were performed: the first to amplify the family and to add the sequence-adaptors and the second to extend the amplimer with the identfyer sequence of the sample (all PCR products are mixed and run at the same time) and the adaptor-sequence allowing the binding to the flow-cell. Results. A total of 5,135,286 bona-fide sequences were obtained from which 125845 sequences were unique, representing an average of 2.45%, an indirect mesure of the high redundancy of sequences obtained. As a preliminary approach, CDR3 sequences from all

families and samples with a relative abundancy over a 0.1% were selected and its presence in the other samples´ sequences was determined. Similarity between samples varies depending on the family studied. A 60% of the TRBV25 sequences were shared between diabetics (D1-D2) compared with 14 and 22% for TRBV20 and TRBV4, respectively. One of the shared sequences in the TRBV4 family was CASSQVAGAGTGELFF, indicating that monoclonal expansions in the pancreas are found in peripheral blood of diabetics, but not in controls. Conclusions. High throughput sequencing is a powerful technique that allows the analysis of the TCR repertoire from T1D patients. Our interest is to identify new expanded intraislet sequences in more samples (from nPOD), and to search these sequences in a larger panel of PBMC from patients and controls, in order to establish a TCR-signature that could be used as a biomarker for T1D.

22 www.sci.cat


15 Innate immune signatures in malaria-naïve adults infected with Plasmodium falciparum sporozoites by needle injection with different doses and routes

Gemma Moncunill, Patricia Gómez-Pérez, Alfons Jimenez, Pau Cisteró, Diana Barrios, Kim Lee Sim, Almudena Legarda, José Muñoz, Rosa M. Antonijoan, Maria Rosa Ballester, Stephen Hoffman, Pedro L. Alonso, Carlota Dobaño

Centre de Recerca en Salut Internacional de Barcelona (CRESIB).

Factors from the innate immune response activated upon a first infection in a malaria-

naive adult volunteer are not well characterized. In particular, it is not known what are the

determinants and kinetics of the innate immune response and how they affect the

induction of adaptive immunity to malaria. Dendritic cells (DCs) are highly potent antigen

presenting cells uniquely able to initiate primary immune responses, including tolerogenic

responses. In addition, cytokines and chemokines are key effectors in the induction and

regulation of immune responses.

The objective of our study is to determine the effect of different doses, routes and volumes

of PfSPZ inoculations on the induction of innate immune responses. Eventually, we aim to

define how the innate responses during the challenge determine the acquired immune

responses.

In the context of a controlled human malaria infection trial in 36 naïve hosts we analysed

immune responses before challenge, at days 1, 7, 21 or malaria diagnosis, and during the

treatment period and follow up visits at day 35 and 90. Whole blood was used for

phenotyping of DCs and human RNA extraction with a QIAamp kit, followed by

transcriptional analysis with Affymetrix microarrays. Plasma samples were used for

cytokine and chemokine profiling by Luminex multiplex assays.

We characterized the kinetics of different subsets of peripheral blood DCs, CD123+

(plasmacytoid DC), CD11c+ (myeloid DC) and BCDA3+ using a combination of multiple

markers including a lineage cocktail and HLA-DR. Preliminary results show that at time of

malaria diagnosis there is an increase of the percentage of CD40+, CD86+ and PD-L1

myeloid DCs, and a remarkable decrease of BDCA3+ DCs, while no differences were

detected in the frequencies of plasmaytoid DCs. In addition a pro-inflammatory

cytokine/chemokine profile in plasma was observed at the time of parasitemia detection.

Transcriptional signatures will be correlated with plasma cytokines/chemokines and DC

subset composition, and later to acquired cellular immune responses to elucidate how

innate immune responses induce adaptive responses.

Comprehensive characterization of innate immune responses, particularly the DCs

activation to Plasmodium infection in vivo will help understand the acquisition of immunity

to malaria.

www.sci.cat 23

E-poster List

Posters From Innate to Adaptative Immunity 1-5 The authors will attend the poster on 14/11/2013, 17:30h

1 Oxidative stress effect on progesterone-induced blocking factor (pibf) binding to pibf-receptor in lymphocytes

Carlos de la Haba; José R. Palacio; Tamas Palkovics; Júlia Szekeres-Barthó; Antoni Morros; Paz Martínez

Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular i Centre d’Estudis en Biofísica (CEB), Facultat de Medicina, Universitat Autònoma de Barcelona (UAB) i Unitat d´Immunologia, Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain

Receptor-ligand binding is an essential interaction for biological function. Oxidative stress

can modify receptors and/or membrane lipid dynamics, thus altering cell physiological

functions. The aim of this study is to analyze how oxidative stress may alter receptor-

ligand binding and lipid domain distribution in the case of progesterone-induced blocking

factor (PIBF)/PIBF-R. For membrane fluidity analysis of MEC-1 lymphocytes, two-photon

microscopy was used in individual living cells. Lymphocytes were also double stained with

AlexaFluor647/PIBF and Laurdan to evaluate PIBF/PIBF-R distribution in the different

membrane domains, under oxidative stress. A new procedure has been developed which

quantitatively analyzes the regionalization of a membrane receptor among the lipid

domains of different fluidity in the plasma membrane. We have been able to establish a

new tool which detects and evaluates lipid raft clustering from two-photon microscopy

images of individual living cells. We show that binding of PIBF to PIBF-R causes a

rigidification of plasma membrane which is related to an increase of lipid raft clustering.

However, this clustering is inhibited under oxidative stress conditions. In conclusion,

oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces

lipid raft clustering.

24 www.sci.cat

Poster From Innate to Adaptative Immunity The authors will attend the poster on 14/11/2013, 17:30h

2 Dexamethasone regulates PGE2 receptors controlling tolerogenic dendritic cells function

Georgina Flórez-Grau; Raquel Cabezón; Carolina España; Julià Panés; Daniel Benítez-Ribas

Department of Gastroenterology, Hospital Clínic de Barcelona, IDIBAPS.

Introduction: Dendritic cells (DCs) are considered to be one of the most important antigen-

presenting cells (APCs) that have a crucial role in linking innate and adaptive immune responses.

In the last decade, these cells have been used in medicine to modify the immune response in

diseases that range from cancer to autoimmunity. Tolerance induction is required to treat

inflammatory diseases. Dexamethasone has been used as tolerogenic agent, due to its capacity to

generate tolerogenic dendritic cells (tol-DCs) in vitro.

Several reports have characterized dexamethasone-induced tolerogenic DCs (tol-DCs). However

these tol-DCs need further molecular characterization that would help us to understand the

molecules involved in tolerance induction and identify specific markers.

Based on previous microarray data from our lab, CD72 was selected as candidate molecule

specifically expressed on tol-DCs comparing to mature dendritic cells (mDCs).

CD72 is an inhibitory co-receptor whose regulation is dependent of the presence of a

glucocorticoid and Prostaglandin E2 (PGE2 ), used as a part of maturation cocktail (MC, composed

by IL-6, IL-1b, TNF-a and PGE2) to generate tol-DCs. PGE2 exerts its functions through EP

receptors (1-4) and can mediate inflammatory or anti-inflammatory responses on DCs, depending

on the context.

Here we describe how these PGE2 receptors are differently regulated by Dexamethasone using

CD72 as a marker for tol-DCs.

Results: We demonstrated that when PGE2 was removed from MC, CD72 expression was

completely inhibited. Furthermore, adding PGE2 exogenously was sufficient to up-regulate CD72

at the same expression level as tol-DCs generated using all MC components. Suggesting that

PGE2 has an essential role in CD72 modulation as well as, it can have a crucial role in tol-DCs. In

order to investigate the differential effect of PGE2 in tol-DCs versus mDCs the expression of EP

receptors was analyzed. EP2,3,4 receptors were up-regulated in tol-DCs at both mRNA and

protein levels relative to immature dendritic cells. Experiments with specific EP2 and EP4 receptors

agonists showed that only EP2 agonist was able to induce CD72 expression opposite to EP4

agonist. Supernatants of tol-DCs treated with EP agonists and gram-negative bacteria were test for

IL-10 production showing that EP2 agonist was able to keep the same high levels of IL-10

respecting to PGE2 –treated tol-DCs

Conclusions: Our results shown that dexamethasone induces changes in the expression pattern

of PGE2 receptors in human DCs, showing that EP2,3,4 were up-regulated in tol-DCs. CD72 was

essential to determine all this characterization, pointing it as a good marker to study the role of

PGE2 and its receptors. Interestingly, we describe that PGE2 acts in tol-DCs through EP2, which is

related with the anti-inflammatory role of PGE2 and in concordance with the anti-inflammatory

environment around tol-DCs created by dexamethasone.

Tol-DCs treated with EP2 agonist expressed CD72 and produced high amounts of IL-10;

consequently we can conclude that EP2 is involved in immune-regulatory processes.

Further studies are required to identify the role of CD72 in tol-DCs.

www.sci.cat 25


3 Analysis of B and T regulatory cell populations in Papua New Guinean pregnant women exposed to malaria

Pilar Requena, Joseph J. Campo, Alex Umbers, Maria Ome, Regina Wangnapi, Diana Barrios, Anna Rosanas, Michela Menegon, Alfredo Mayor, Elisa de Lazzari, Sergi Sanz, Carlo Severini, Chetan Chitnis, Myriam Arevalo-Herrera, Peter M. Siba, Azucena Bardají, Ivo Mueller, Stephen Rogerson, Clara Menéndez, Carlota Dobaño


Immunomodulation associated with gestation might contribute to the burden of malaria in

pregnancy. Immune effector mechanisms to protect against Plasmodium infection during

pregnancy include antibody and cellular responses. On one hand, memory B cells (MBC)

are required for long-lasting antibody immunity, however exposure to Plasmodium

falciparum parasites has been associated with perturbations of B cell subpopulations.

CD27- “atypical” cells with similar expression to hyporesponsive “exhausted” cells in HIV,

are expanded in exposed individuals, but their role is yet clear as they have can produce

regular amounts of functional antibodies. On the other hand, T regulatory cells are

expanded in pregnancy, particularly in the placenta, and elevated Treg, IL-10 and TGF-β

levels have been associated with malaria. However, it is not known whether Tregs

contribute to the onset of the disease or to the control of immune-mediated pathology

associated with malaria.

In the context of the PregVax study, we characterized the regulatory T cell and B cell

phenotype of peripheral blood mononuclear cells in pregnant and non-pregnant women

from a malaria endemic area of Papua New Guinea where P. vivax and P. falciparum

infections co-exist and compared them with pregnant and non-pregnant naïve women from

Spain. We analysed the relationship between T and B cell phenotypes and immune

mediators, including multiple IgG antibodies to Plasmodium antigens and plasma

cytokines, and whether potential changes in T and B cell populations during pregnancy are

associated with morbidity outcomes, including susceptibility to Plasmodium infection.

We found that malaria-exposed pregnant women had higher levels of active and resting

atypical MBC, and these positively correlate with malaria-specific antibodies, including P.

vivax. Atypical MBC levels positively correlated with pro-inflammatory, TH1 and TH2

cytokines and inversely with eotaxin.

Pregnant women had increased levels of MBCs in peripheral blood and decreased levels

of MZ-like MBC. This trend was not observed in malaria-exposed women. In addition,

malaria-exposed women had lower levels of IL-10+ T cells but this effect did not seem to

be malaria-driven. Tregs were decreased in pregnant women, but no effect of malaria

exposure or association with malaria infection or antibodies was observed.

26 www.sci.cat


4 Human thymocytes express functional receptors for thyroid-stimulating hormone

Mireia Giménez-Barcons, Roger Colobran, Ana Gómez Pau and Ricardo Pujol-Borrell

Vall d´Hebron-Institut de Recerca.

Graves´ disease (GD) is a common autoimmune disease in which the thyroid stimulating

hormone receptor (TSHR) represents the primary auto-antigen. Auto-antibodies bind to the

THSR mimicking its ligand, thyroid stimulating hormone (TSH), causing the characteristic

clinical disease symptoms, such as goitre, ophtalmopathy and thymic hyperplasia. We

have previously shown that the TSHR belongs to the category of tissue-restricted antigens

and that loss of central tolerance to TSHR is due, in part, to a polymorphism of THSR

gene that determines its lower expression in the thymus thus increasing susceptibility to

GD. In order to further explore the association between thymic TSHR expression and GD,

we analysed TSHR expression in thymic glands from non-autoimmune donors. Our results

show that, unexpectedly, TSH receptor (TSHR) is expressed in thymocytes. Further

analysis of thymic populations revealed that TSHR expression was found in thymocytes of

the earliest stages, as defined by CD34 and CD1A expression, with the highest expression

found in the double positive CD4+CD8+ thymocytes. In addition, TSHR expression was

residual in the single positive stage CD4+CD8- and CD4+CD8+, and absent in peripheral

and cord blood T cells. TSHR expression in thymocytes is confirmed by

immunofluorescent staining, western blotting and reverse transcriptase-PCR of full-length

transcripts. Noticeably, thymic TSHR is functional as evidenced by the ability of TSH, long

recognized as the major functional stimulus for thyroid cells, to induce the formation of

intrathymic cyclic AMP. Remarkably, monoclonal stimulating antibodies, but not blocking

antibodies, both derived from patients with thyroid disease, stimulated the production of

cAMP in thymocytes. Taken together, these data support the notion that the action of

TSHR antibodies is not confined to the thyroid but that it contributes to maintaining a flow

of T cells from the thymus, some TSHR specific, resulting in the amplification of the

autoimmune response. Additionally, our results have broad clinical implications in that they

may help to clarify the controversial cause of the thymic hyperplasia long observed in

patients with GD.

www.sci.cat 27


5 Decreased CD4 and CD8 T effector eemory cells in patients with Graves´ Disease. An analysis of T- and B- peripheral blood compartment

Aina Teniente Serra (1,4); Berta Soldevila (2); Bibiana Quirant (1); Marco A. Fernández (3); Manel Puig-Domingo (2); Eva M. Martínez-Cáceres (1,4)

(1) Immunology Department. (2) Endocrinology and Nutrition Department. Hospital Universitari Germans Trias i Pujol. (3) Cytometry Unit. Institut d´Investigació Germans Trias i Pujol. (4) Universitat Autònoma de Barcelona.

Introduction: Graves´ disease (GD) is an organ-specific autoimmune disorder

characterized by the production of autoantibodies against thyroid-stimulating hormone

receptor (TSHRAb) and lymphocyte infiltration in the thyroid gland, which consist

predominantly of CD4 T cells although there is also infiltration of CD8 T cells. Moreover,

disturbances in naïve/memory CD4 and CD8 T-lymphocytes, regulatory T cells and B-

lymphocyte subpopulations in peripheral blood of GD patients have been pointed, but a

comprehensive multiparametric flow cytometric analysis is still lacking.

Aim: To perform an extensive analysis of the T and B peripheral blood cell compartments

in GD patients in order to identify those subpopulations that are relevant in the

pathogenesis of the disease.

Material and methods: CD4 and CD8 T cells (including naïve, central memory, effector

memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells

subsets (naïve, unswitched memory, switched memory and transitional B cells) were

analyzed in peripheral blood of stable GD patients (n=26) and healthy donors (HD; n=40)

using multiparametric flow cytometry.

Results: We found a decrease of effector memory CD4 and CD8 T cells (p=0,036 and

p=0,0023, respectively) as well as a decrease in regulatory T (p=0,020) and B cells

(p=0,030) in peripheral blood of GD patients compared with HD. In contrast, an increase of

double positive CD4+CD8+ (DP) lymphocytes with memory phenotype was observed

(p=0,049) compared with HD.

Conclusion: In GD patients, even in stable clinical situation, there is an increase of

effector T cells and a decrease of regulatory lymphocyte subpopulations, suggesting their

involvement in the pathogenesis of the disease.

28 www.sci.cat

Posters Clinical Immunology 6-14 The authors will attend the poster on 15/11/2013, 11:00h

6 Estudi del patró dánticossos "rods and rings"

Joan Climent; Francisco Morandeira; Mariona Mestre; Jordi Bas

Servei dÍmmunologia. Hospital Universitari de Bellvitge.

INTRODUCCIÓ: El patró dímmunofluorescència dÁNA anomenat “rods and rings”

(bastons i anells) ha estat descrit en cèl•lules HEp-2 i s´ha considerat específic de

pacients infectats pel virus de l´hepatitis C (VHC), on es relaciona amb el tractament i

lévolució dels pacients. Lóbjectiu dáquest estudi és identificar aquest patró entre totes

les mostres dáutoanticossos rebudes al laboratori i valorar la seva relació amb el VHC i el

tractament, així com estudiar la seva aparició en altres patologies.

PACIENTS I MÈTODES : S´han inclòs les mostres positives (N=78) des del 2010: 55

homes i 23 dones, promig dédat de 57 anys (26 - 79). El diagnòstic ha estat realitzat

mitjançant tècnica estàndard dÍFI en substrat comercial d´HEp2. El patró “bastons i

anells” consisteix en una estructura característica fibril•lar de localització citoplasmàtica,

de forma allargada o circular.

RESULTATS: El present treball comprèn la sèrie més nombrosa publicada fins ara en un

centre. Dels 78 pacients positius, 68 casos eren positius per VHC i 10 casos presentaven

altres patologies aparentment no relacionades amb el virus C (asma bronquial, Wilson,

nefropatia, AR, SAF, psoriasi, ictus, colangitis limfoplasmacitària i poliartràlgies).

Del pacients amb VHC, 7 no rebien tractament antiviral en el moment de la determinació

dels ANA. En 43 casos, dels que es disposava seguiment, s´ha pot comprovar la seva

positivització en 12, mentre que en 8 pacients mostra un curs fluctuant. Aquests canvis no

semblen correlacionar-se amb cap esdeveniment evolutiu.

CONCLUSSIONS: El patró “rods and rings” apareix principalment en VHC, però també i

de forma significativa, en altres patologies no relacionades.

Lánàlisi preliminar realitzat no permet associar lánticòs amb el tractament ni amb

lévolució dels pacients.

www.sci.cat 29

Poster Clinical Immunology The authors will attend the poster on 15/11/2013, 11:00h

7 PROTOCOL DÉSTUDI DE DEFECTES PRIMARIS EN LA VIA IL12-IFN-γ

Ana Esteve-Sole (1,2); Azucena González (3); Mireia Antón (3); Montserrat Masó (3); Eva González (3); Mónica Piquer (1,2); Ana Mª Plaza (1,2); Juan Ignacio Arostegui (2,3); Manel Juan (2,3); Laia Alsina (1,2)

(1) Secció dÁl·lèrgia i Immunologia Clínica, Hospital Sant Joan de Déu, Universitat de Barcelona (2) Unitat Funcional Hospital Clínic – Hospital Sant Joan de Déu, (3) Servei dÍmmunologia, Hospital Clíınic, IDIBAPS, Universitat de Barcelona.

Les Malalties Mendelianes amb Susceptibilitat a Micobacteris (MSMD) es troben dins del

grup dÍmmunodeficiències Primàries (IDPs). Consisteixen en defectes congènits en la via

de IL12-IFN-γ i cursen amb una elevada susceptibilitat a les infeccions disseminades per

micobacteris no patogènics i altres patògens intracel·lulars. Les MSMDs requereixen

estudis específics per al seu diagnòstic, doncs els estudis habituals en cas de sospita de

IDP (quantificacions fenotípiques de poblacions limfocitàries, capacitat proliferativa

limfocitària, nivell dímmunoglobulines o la resposta vacunal) no mostren variacions

indicatives.

Lóbjectiu dáquest treball és implementar al nostre laboratori les tècniques

diagnòstiques per MSMD conegudes, així com desenvolupar noves estratègies per donar-

hi una resposta.

Els procediments pel diagnòstic dels pacients amb MSMD que hem optimitzat són: i)

mesura del nivell basal de IFN-γ en plasma, ii) detecció dels receptors de IFN-γ (IFNGR1 i

IFNGR2) i de IL12 en membrana i iii) cultiu cel·lular amb BCG amb i sense coestimulació

per IL12 o IFN-γ i mesurant els nivells de IFN-γ i altres citocines en els sobrenedants dels

cultius. Dáquesta manera es restringeix la part de la via afectada (defecte de producció

de IFN-γ o defecte de resposta de IFN-γ), per a orientar el posterior estudi genètic (IL12B,

IL12RB1 i TYK2; IFNGR1 IFNGR2, STAT1, IRF8, IKBKG CYBB; respectivament). Per a

millorar i complementar aquestes tècniques diagnòstiques estem posant a punt la detecció

de la fosforilació d´STAT1 (una molècula clau en aquesta via) en resposta a IFN-γ

mitjançant citometria de flux.

En láctualitat hem analitzat tres pacients amb sospita de MSMD amb resultats

satisfactoris. Estem segurs que gràcies a aquests estudis serem capaços de diagnosticar

i, secundàriament, tractar dúna manera més òptima aquests pacients que poden trobar-

se infradiagnosticats en el nostre entorn.

30 www.sci.cat


8 Alteracions de les cèl·lules sensorials en el ratolí NOD. Possibles implicacions en el desenvolupament de la diabetis mellitus tipus 1

Arpa B. (1); Rosell E. (1); Sábado J. (2); Carrascal J. (1); Autonell I. (3); Casanovas A. (2); Mora C. (1); Vives-Pi M. (3); Verdaguer J. (1)

(1) Immunology Unit, Department of Experimental Medicine, University of Lleida, Spain. (2) Cellular Neurobiology Unit, Department of Experimental Medicine, University of Lleida, Spain. (3) Laboratory of Immunobiology for Research and Diagnostic Applications, Germans Trias i Pujol University Hospital, Barcelona, Spain.

La Diabetis Mellitus Tipus 1 (T1D) és una malaltia complexa que es caracteritza per la

destrucció selectiva de les cèl·lules β pancreàtiques productores dínsulina. Malgrat que la

diana principal són els illots, les cèl·lules del sistema nerviós que les innerven també

pateixen látac autoimmunitari. Recentment hem descrit que en els illots pancreàtics del

model de ratolí non-obese diabetic (NOD) prediabètic existeix un grup predominant de

limfòcits B infiltrants amb reactivitat contra elements del sistema nerviós perifèric. No

obstant, el sistema nerviós no és un actor passiu en el decurs de la malaltia, ja que també

s´ha descrit el paper decisiu que tenen un subgrup de neurones en el control de léstrès

de la cèl·lula beta i la inflamació de líllot en la diabetis autoimmune.

Lóbjectiu dáquest estudi es analitzar els cossos neuronals de les neurones que innerven

el pàncrees, situats als ganglis de lárrel dorsal, i observar si aquestes neurones també

pateixen látac autoimmunitari. En una primera aproximació, es va realitzar un anàlisis

histològic de les neurones en animals de diferents edats i de diferents soques: NOD;

NOD.Rag-/- i C57.

Es va observar la presència dínfiltrat limfocitàri en un dels animal de 32 setmanes dédat,

no obstant, la majoria dels animals NOD i NOD.Rag-/- estudiats presentaven cèl·lules

amb vacuoles al seu citoplasma. Aquesta afectació repesenta un 1-2% de les cèl·lules del

gangli de lárrel dorsal en aquestes soques.

Els resultats obtinguts apunten que els animals amb fons genètic NOD, de forma

independentment del sistema immunitari, presenten danys fisiopatològics a algunes de les

neurones que innerven el pàncrees. Aquest fenòmen podria estar relacionat amb léstrès

de la cèl·lula beta i látac autoimmunitari que pateixen els ratolins NOD. Atès que només

es tracten de resultats preliminars, més experiments seran necessaris per descriure

aquest fenòmen en detall.

www.sci.cat 31

Poster Clinical Immunology The authors will attend the poster on 15/11/2013, 11:00

9 Prevalença i rellevància clínica dels nivells d’IgE específica a Pru p 3 inferiors al cut off en pacients sensibilitzats a préssec.

Anna Mensa (1); Jordi Milà (1); Lucía Millán (1); Ramon Vilella (1); Manel Juan (1); Joan Bartra (2); Mariona Pascal (1)

(1) Servei d’Immunologia de l’Hospital Clínic de Barcelona; 2 Unitat d’Al·lèrgia, Servei de Pneumologia, ICT, Hospital Clínic de Barcelona.

Introducció: Lál·lèrgia al préssec és una de les principals causes dál·lèrgia a aliments vegetals

a l´àrea mediterrània. La proteïna de transferència de lípids (LTP) del préssec, Pru p 3, és

lál·lergen majoritari dáquest fruit i acostuma a ser el sensibilitzador primari. Les proteïnes LTP

son panal·lèrgens presents en múltiples fonts al·lergèniques (aliments vegetals i pol·lens),

taxonòmicament relacionades o no, que poden produir reaccions al·lèrgiques sistèmiques severes

com ara anafilaxis. En el diagnòstic dál·lèrgia al préssec i/o Síndrome LTP (quan participa més

dúna proteïna LTP dáliments vegetals no taxonòmicament relacionats) és freqüent la

determinació dÍgE específica (sIgE) a Pru p 3 pel mètode ImmunoCAP, immunoassaig amb un

límit de detecció de 0,1 kU/L i punt de tall (cut off) establert en 0,35 kU/L.

Objectiu: Analitzar la prevalença i la rellevància clínica dels nivells de sIgE a Pru p 3 inferiors al

cut off en pacients sensibilitzats a préssec.

Mètodes: Estudi observacional retrospectiu de pacients sensibilitzats a préssec. Els pacients es

van agrupar en funció dels nivells de sIgE a Pru p 3 (f420, ImmunoCAP, Phadia Thermofisher

Scientific). Es van revisar les històries clíniques de dos grups de pacients: inferiors a 0,35 kU/L

(n=44, Grup A) i superiors a 17,5 kU/L (n=33, Grup B), recollint dades sobre la simptomatologia

relacionada amb la ingesta de préssec, la implicació de cofactors i la sensibilització a altres

aliments vegetals.

Resultats: Dels 284 pacients amb nivells de sIgE a Pru p 3 superior a 0,1 kU/L, 48 (16,9%) tenien

valors entre 0,1-0,35 kU/L, 28 (9,9%) entre 0,36-0,75 kU/L, 83 (29,2%) entre 0,76-3,5 kU/L, 90

(31,7%) entre 3,6-17 kU/L i 35 (12,3%) entre 17,5-100 kU/L. Per història clínica [A vs. B en %]:

52,3 vs. 30,3% toleren la polpa de préssec i 31,8 vs. 21,2% manifesten símptomes dúrticària de

contacte amb la pell. Dels pacients amb símptomes post-ingesta (A: n=21 i B: n=23), 10 pacients

(47,6%) del A i 7 (30,4%) del B van presentar síndrome dál·lèrgia oral (SAO) (p=0,534). Pel que

fa a reaccions sistèmiques, 2 pacients (9,5%) del A i 12 (52,2%) del B (p=0,001) amb angioedema

i/o urticària, 4 pacients (19%) del A i 9 (39,1%) del B (p=0,022) símptomes gastrointestinals, i en 8

pacients (38,1%) del A i 6 (26,1%) del B anafilaxi (p=0,506). Léspectre de sensibilitzacions a

altres aliments vegetals és major en B que en A. La implicació de cofactors (AINE i/o exercici) s´ha

vist en 8 pacients (18,2%) del A i en 16 (48,5%) del B (p=0,006).

Conclusions: Els nivells de sIgE a Pru p 3 inferiors al cut off (0,35) són rellevants en els pacients

amb sospita clínica dál·lèrgia al préssec i/o Síndrome LTP. Els nivells de sIgE no correlacionen

amb la severitat dels símptomes però sí en léspectre de sensibilitzacions a altres aliments

vegetals. Els cofactors tindrien un paper important en desencadenar símptomes dál·lèrgia a

proteïnes LTP i estarien relacionats amb els nivells de sIgE.

32 www.sci.cat


10 T cell repertoire and T-cell receptor excision circles (TRECs) in the study of Combined Immunodeficiency.

Mónica Martínez-Gallo (1), Roger Colobran (1), Laia Gifre (1), Ana Sanchis (1), Pere Soler (2), Andrea Martín (2).

(1) Hospital Universitari Vall d´Hebron, (2) Pediatric Infectious Disases and Immunodeficiencies Unit of Vall d´Hebron Hospital.

Among primary immunodeficiencies, patients with combined immunodeficiency represent

a diagnostic challenge. An early evaluation of T-cell impairment is key for medical

intervention, if needed bone marrow transplantation. The T-cell receptor excision circles

(TRECs) content of T cell populations revealing recent thymic output of naïve T cell and T-

cell receptor (TCR) repertoire diversity reflecting broader responses to multiple antigens,

are both important in resisting infections.

Fifteen patients, followed in the Pediatric Infectious Disases and Immunodeficiencies Unit

of Vall d´Hebron Hospital, were included in the study. All patients have clinical phenotypes

suggestive of combined immunodeficiency, with clinical history of recurrent or persistent

infections, low numbers of circulating T cells. TRECs values were evaluated by

quantitavire PCR (measured as TRECs / 100ng gDNA) and TCR repertoire diversity was

analysed by flow citometry and molecular biology, spectratyping from peripheral blood

mononuclear.

By analyzing the TCR repertoire by flow cytometry and spectratyping was studied a group

of patients with suspected combined immunodeficiency. The two techniques have

comparable results differentiating between polyclonal and restricted repertoires. Of the

fifteen patients studied, five have shown clearly TCR-Vbeta polyclonal repertoire. The rest

showed restricted TCR patterns. TRECs quantification was significantly reduced in

patients with restricted repertoire identifying T cell impairment.

Assessment of thymus capabilities by TRECs value and TCR repertoire diversity can help

in the early diagnosis of T cell immunodeficiency.

www.sci.cat 33


11 Novel compound heterozygous mutation in the ATM gene in a patient with ataxia telangiectasia showing absence of TRECs and polyclonal TCR vbeta diversity.

Roger Colobran; Mónica Martínez-Gallo; Andrea Martín-Nalda; Pere Soler.

Immunology Service, and Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d´Hebron.

Ataxia-telangiectasia (A-T) is a rare autosomal recessive disorder characterized by

progressive cerebellar ataxia, oculocutaneous telangiectasias, variable degrees of

immunodeficiency and predisposition to malignancies. The incidence of the disease is

approximately 1 in 100,000 live births, and is caused by mutations in the ataxia-

telangiectasia mutated (ATM) gene. The ATM gene product is a large (∼350 kDa, 3056

amino acids), evolutionarily conserved serine/threonine protein kinase that has a central

role in the cellular response to DNA damage.

Here we report a novel compound heterozygous ATM mutation in a Spanish patient with

A-T. Complete sequencing of ATM cDNA revealed two major alterations: (1) A

heterozygous nucleotide change in the exon 16 (c.2413C>T) causing a stop codon at

amino acid 805 (p.Arg805X) and leading to a truncated protein of 804 aa. (2) A

heterozygous nucleotide change affecting the third position of the donor splice site in

intron 23 (c.3402+3A>C), leading to the loss of exon 23, causing a frameshift and,

consequently, a truncated protein of 1115 aa (p.Arg1095SerfsX22).

Beyond the progressive neurological impairment chraracterized of AT, the patient showed

T lymphocytopenia. We quantified the T-cell Receptor Excision Circles (TRECs) founding

a complete absence and TCR Vbeta diversity was analysed by flow cytometry showing a

policlonal distribution of the repertoire.

34 www.sci.cat


12 Detection of Phospholipase A2 receptor (PLA2R) antibodies and its clinical correlation in Primary Membranous Nephropathy

B. Patricia Villarroel (1); Nuria Pérez (2); Luis Quintana (2); Milagros García (1); E. Azucena González (1); Odette Viñas (1).

(1) Immunology Department. Hospital Clínic of Barcelona; (2) Nephrology Department Hospital Clínic of Barcelona.

Membranous nephropathy (MN) is the leading cause of a nephrotic syndrome in adults.

The disease is characterized by immune complexes in the subepithelial space of the

glomerular filtration barrier. An in situ immune complex formation is considered to be the

pathogenetic mechanism, which means that circulating antibodies bind to an antigen

expressed on glomerular podocytes. In approximately 25% of patients, MN is associated

with an underlying disease such as systemic lupus erythematodes, malignant tumors,

exposure to certain drugs or infections. When no secondary cause is apparent, the

disease is classified as primary.

Different podocyte proteins have been reported as the target of the immune response in

IMN. Among them anti-PLA2r antibodies seem to play a major role, being present in 52-

78% of cases of primary MN.

Methods: We measured anti-PLA2RABs in serum samples from 34 patients with NM

biopsy proven, (32 primary and 2 secundary) and 6 patients with another renal disease by

an indirect immunofluorescence test performed on human embryonic kidney cells

(HEK293) expressing the PLA2R1 protein (Euroimmun) and by ELISA (EUROIMMUN), a

quantitative monospecific assay using polystyrene microplate strips coated with human

purified PLA2R antigen. And we analysed clinical features (age, genre, serum creatinine

and proteinuria).

Results: We detected anti-PLA2r antibodies in 46.87% (n=32) of the patients with primary

MN by IFI and 42.85% (n=21) by ELISA. None of the 8 patients with renal disease other

than idiopathic MN were positive to PLA2R

We do not detect statistically significant differences between the age, genre or serum

creatinine. It exists a strong correlation between antibodies levels and disease activity

(proteinuria) (P= 0,0009).

Conclusions: This test could be very useful for diagnosis and monitoring the patients with

primary MN. This is an easy, cheap and fast test that could help us to individualize the

immunosupresor treatment.

www.sci.cat 35


13 Mecanismes antiinflamatoris en la mucosa intestinal de pacients amb colitis ulcerosa en remissió: paper del receptor decoy de la IL-1 (IL1R2)

Rut Mora; Núria Planell; Isabella Dotti; Azucena Salas; Elisabeth Calderón

Institut dÍnvestigacions Biomèdiques Agust Pi i Sunyer (IDIBAPS).

La colitis ulcerosa (CU) és una malaltia inflamatòria intestinal crònica détiologia

desconeguda, que cursa amb períodes de remissió alternats amb brots dáctivitat

inflamatòria.

Lóbjectiu del nostre estudi va ser identificar potencials mecanismes antiinflamatoris en la

remissió de la malaltia inflamatòria intestinal, ja que els processos que afavoreixen el

manteniment de la quiescència de la CU en la remissió son desconeguts.

A partir de lánàlisi transcripcional de la mucosa de pacients amb CU activa, en remissió, i

mucosa control, vàrem identificar el gen IL1R2 sobre-expressat en la mucosa intestinal de

pacients en remissió, comparat amb els pacients amb CU activa i controls. LÍL1R2

codifica per el receptor tipus 2 de la IL-1β (IL-1R2) que forma part un conjunt de proteïnes

de la família de la IL-1 que inhibeixen lácció de la IL-1β. La IL-1β és una citosina

proinflamatòria que recentment s´ha descrit el seu paper en la renovació de les cèl•lules

mare epitelials. Láugment en la transcripció de IL1R2 es va correlacionar amb un

increment en la secreció del receptor soluble, IL-1sR2, mesurat en cultius déxplants de

mucosa intestinal. Mitjançant immunohistofluorescència vam poder identificar les cèl•lules

epitelials així com les cèl•lules plasmàtiques IgA+ de la mucosa colònica, com les

principals productores de IL-1R2. Quantificant per citometria de flux, lépiteli EpCAM1+

presentava un percentatge més alt de cèl•lules que expressen IL-1R2 intracel•lular a la

mucosa intestinal en remissió. Principalment vam identificar les cèl•lules epitelials

diferenciades més allunyades de les bases de les criptes intestinals, com a importants

productores dáquesta proteïna. Estudis in vitro, utilitzant tècniques de cultiu de cèl•lules

mare de lépiteli intestinal, també demostraven que léxpressió de IL1R2 augmenta quan

séliminen els senyals activadors de la via de la β-catenina, induint el fenotip més

diferenciat de les cèl•lules epitelials.

En resum els nostres estudis demostren, que la remissió a la CU es caracteritza no

només per una baixada dels senyals proinflamatoris, sinó també per un augment en

léxpressió de IL-1R2 com a mecanisme regulador de la inflamació i potencial controlador

de la regeneració epitelial.

36 www.sci.cat


14 GPI-anchor and GPI-anchored protein expression on leukocytes from PMM2-CDG patients.

De la Morena-Barrio M.E.; Hernández-Caselles T.; Corral J.; García-López R.; Martínez-Martínez I.; Pérez-Dueñas B.; Altisent C.; Sevivas T.; Kristensen S.R.; Guillén E.; Miñano A.; Vicente V.; Jaeken J.; Lozano M.L.

Facultad de Medicina, Universidad de Murcia.

Background. Mutations in PMM2, by impairing the phosphomannomutase-2 activity, lead

to the most frequent congenital disorder of glycosylation PMM2-CDG that is presented

with multiple organs affected causing immunological dysfunction among other features.

Defective mannose-1-phosphate, is responsible for abnormal N-glycosylation, and is also

implicated in the biosynthesis of glycosylphosphatidyl inositol (GPI) anchor in these

patients.

Objective. We evaluated putative GPI-anchor and GPI-anchored proteins defects in

peripheral blood cells from PMM2-CDG patients.

Methods. The expression of GPI-anchor and seven GPI-anchored proteins was evaluated

by flow cytometry in different cell types from twelve PMM2-CDG patients. Additionally,

expression of CD16 in neutrophils was studied by Western blot, and plasma proteins of

hepatic synthesis also by HPLC.

Results. Patients and controls had similar surface expression of GPI-anchor and most

GPI-anchored proteins. Nevertheless, patients displayed a significant diminished binding

of two anti-CD16 antibodies (3G8 and KD1) to neutrophils and also of anti-CD14 (61D3) to

monocytes. Interestingly, CD16 immunostaining and asialotransferrin levels significantly

correlated with patients´ age. Analysis by flow cytometric of CD14 with MP9, and CD16

expression in neutrophils by Western blot using H-80 ruled out deficiencies of these

antigens.

Conclusions. PMM2 mutations do not impair GPI-anchor or GPI-anchored protein

expression. However, the glycosylation anomalies caused by PMM2 mutations might

affect the immunoreactivity of monoclonal antibodies that could lead to incorrect

conclusions about the expression of different proteins, including GPI-anchored proteins.

Neutrophils and monocytes are sensitive to PMM2 mutations, leading to abnormal

glycosylation in immune receptors, which might potentially affect their affinity to their

ligand, and contribute to infection. This study also confirms less severe hypoglycosylation

defects in older PMM2-CDG patients.

www.sci.cat 37

Posters Methodology and Techniques 15-22

The authors will attend the poster on 15/11/2013, 13:30h

15 New tool to calculate cPRA and how different considerations affect cPRA value

Elisabet Rigol-Monzó; Europa A. González; Patricia Villarroel; Montserrat Digón; Carles Serra-Pagès; Guadalupe Ercilla; Jaume Martorell

Hospital Clínic i Provincial de Barcelona.

In almost all transplantation protocols a negative Crossmatch (XM) based on Complement

Dependent Cytotoxicity (CDC) is mandatory, frequently negative detection of Donor

Specific Antibodies (DSA) by Single Antigen Beads (SAB) is also recommended. The

Calculated Panel Reactive Antidody (cPRA) is a parameter that estimates the percentage

of donors that would be incompatible for a recipient that have DSA based on the results of

SAB test.

In this study a cPRA calculator tool has been developed with a spreadsheet application

that directly uploads the Luminex® software output and we have assessed if different

criteria such as Mean Fluorescence Intensity (MFI) cut-off value, consideration of HLA-C*

and HLA-DQB1* or the use of high resolution typing can significantly modify the cPRA

value.

The mean of cPRA values obtained by our automatic spreadsheet differs in less than 1 %

(0.4 % & 0.6 %) from the ones calculated by Eurotransplant and UNOS online tools.

Modification of MFI cut-off values from 1500 to 3000 changes cPRA 2.4 % and to 6000

11.7 %. cPRA-class-II mean increases 20% when DQB1* and DRB1* data are both

considered in the calculation algorithm. C* addition to loci A* and *B to calculate cPRA-

class-I increases the mean 1%. The mean of the differences between cPRA using high

instead of low resolution data is 3.2 %.

In conclusion, changes in MFI cut-off values have only slight incidence on cPRA. The

inclusion of locus DQB1* increases significantly cPRA while C* has minor effect. No major

differences are found using high instead of low resolution data. The designed tool permits

to have a precise estimation of the probability to find a donor with a negative virtual XM,

thus it could be helpful in the clinical practice to give the adequate priority to a recipient.

38 www.sci.cat

Poster Methodology and Techniques The authors will attend the poster on 15/11/2013, 13:30h

16 Inducció dún model dímmunització oral amb ovoalbúmina en rates Lewis

Mariona Camps-Bossacoma (1), Mar Abril-Gil (1), Filipa Vicente (1), Malen Massot-Cladera (1), Mònica Comalada (2), Francisco J. Pérez-Cano (1), Àngels Franch (1), Margarida Castell (1).

(1) Departament de Fisiologia; Facultat de Farmàcia; Universitat de Barcelona.(2) IRB Barcelona - Institut de Recerca en Biomedicina de Barcelona.

Lóbjectiu dáquest estudi ha estat desenvolupar un model de sensibilització oral amb

ovoalbúmina (OVA) en rates de la soca Lewis. S´ha disposat de rates de 4 setmanes dédat,

mascles i femelles, que han seguit diferents protocols dímmunització. Concretament s´ha

administrat OVA per via oral (p.o.) (50 o 100 mg/rata) juntament amb toxina colèrica (30 g/rata)

seguint una pauta de 1, 2 o 3 vegades a la setmana. També s´ha disposat dún grup dánimals

que només han rebut OVA p.o. i dún grup no tractat. Després de 3 setmanes dádministració

dÓVA i una setmana de descans, s´ha induït un xoc anafilàctic administrant una dosi elevada

dÓVA p.o. (200 mg/rata). Seguidament s´ha procedit a lóbtenció de limfòcits de melsa i ganglis

limfàtics mesentèrics (GLM) per estudiar el patró de secreció de citocines Th1 i Th2 (Luminex) en

resposta a una estimulació amb OVA. S´ha avaluat la resposta en anticossos sèrics anti-OVA

(totals, IgG, IgM, IgA i IgE) mitjançant tècniques dÉLISA i s´ha determinat la concentració dÍgA

fecal (total i específica) en mostres obtingudes al llarg de léstudi.

Totes les rates Lewis femelles que han rebut 50 mg dÓVA i 30 ug de toxina colèrica 3

vegades/setmana han desenvolupat anticossos anti-OVA que es detecten després de 14 dies des

de línici de ládministració. La concentració dánticossos anti-OVA augmenta progressivament al

llarg de léstudi. Aquests anticossos són fonamentalment dísotip IgG i IgM. No es detecta IgE

anti-OVA, fet que coincideix amb lábsència de manifestacions després de la provocació del xoc

anafilàctic. En el cas de la IgA fecal, no es detecten anticossos específics i no es produeixen

canvis significatius en la concentració dÍgA total.

La quantitat dÍL-4, IL-6 i IL-10 alliberada per limfòcits de GLM dels animals que han desenvolupat

anticossos tendeix a ser superior a la del grup de referència, mentre que disminueix lálliberament

dÍFN-γ, IL-1β i TNF-α. Els esplenòcits procedents de les rates sensibilitzades, a diferència dels

del grup de referència, produeixen quantitats detectables de les citocines anteriorment

esmentades.

Els animals que han rebut una dosi superior dÓVA, amb protocols dúna o dues vegades per

setmana amb o sense toxina colèrica, no han desenvolupat anticossos anti-OVA o la incidència

dánimals responedors ha estat < 50%.

En conclusió, s´ha aconseguit un model de sensibilització oral en rates Lewis femelles mitjançant

ládministració oral dÓVA juntament amb toxina colèrica. Els animals presenten un increment en

la síntesis dánticossos específics anti-OVA, i modifiquen el patró de secreció de citocines en GLM

desplaçant-se cap a la formació de citocines Th2.

www.sci.cat 39


17 Valoració de la resposta anafilàctica en rates Brown Norway

Mar Abril-Gil, Alba Garcia-Just, Trinitat Cambras, Malen Massot-Cladera, Francisco J. Pérez-Cano, Franch, À., Castell, M.

Departament de Fisiologia; Facultat de Farmàcia; Universitat de Barcelona.

El xoc anafilàctic és la manifestació més greu dúna resposta al·lèrgica que compromet de

forma ràpida i intensa la funció cardiovascular i respiratòria, entre dáltres. En models

experimentals dál·lèrgia resulta difícil disposar díndicadors objectius i quantificables

dáquesta resposta. El propòsit dáquest treball ha estat establir diferents variables

indicatives de xoc anafilàctic després de la inducció dál·lèrgia a ovoalbúmina (OVA) en

rates de la soca Brown Norway.

Es van emprar rates Brown Norway femelles de 4 setmanes dédat que van ser

immunitzades per via intraperitoneal amb OVA juntament amb hidròxid dálumini i toxina

de Bordetella pertussis. Es va disposar dún grup control no sensibilitzat. Periòdicament

es va determinar la concentració IgE anti-OVA per tècniques dÉLISA. A les 6 setmanes

de la immunització, els animals van rebre una dosi dÓVA per via oral per tal de provocar

un xoc anafilàctic. Immediatament després i durant 20 minuts, es va procedir a

monitoritzar láctivitat motora (mitjançant actímetres). Es van recollir mostres sèriques

cada 30 minuts des de la provocació oral fins a les 2 hores i també es va mesurar la

temperatura rectal dels animals. En les mostres sèriques obtingudes després del xoc

anafilàctic es va quantificar la proteasa II de mastòcits (RMCP-II) mitjançant tècniques

dÉLISA.

Els animals van desenvolupar anticossos IgE anti-OVA després de 2 setmanes de la

immunització. En referència a la valoració de la resposta anafilàctica els animals

immunitzats amb OVA van presentar un menor nombre de moviments en el registre de

láctivitat motora que és significativa durant els primers 10 minuts (p<0,05). Així mateix, la

temperatura corporal dáquests animals va disminuir 2ºC durant les dues hores posteriors

al xoc. En les mostres sèriques obtingudes durant aquest període de temps, la

concentració sèrica de RMPC-II va augmentar significativament en els animals

immunitzats. Els canvis en láctivitat motora, temperatura corporal i concentració sèrica de

RMPC-II es correlacionen entre si.

En conclusió, les tres variables estudiades són bons indicadors de la resposta anafilàctica

en aquest model dál·lèrgia desenvolupat en rates Brown Norway.

40 www.sci.cat


18 Study of immune anti-rotavirus response in a double infection rat model

Rigo-Adrover M., Vilardell N., Franch A., Castellote C., Castell M., Knipping K., Pérez-Cano Francisco J.

Departament de Fisiologia, Factultat de Farmàcia, Universitat de Barcelona. Nutricia Research, Utrecht, The Netherlands.

Group A rotavirus (RV) is the leading cause of diarrhoeal disease among young children

worldwide. The symptoms range from asymptomatic shedding to severe dehydration and

even death. After RV infection, immunity is not complete and re-infections usually occur

although they tend to be less severe than the first one. Although two oral vaccines are

available, the diarrhoea process can be modulated by dietary interventions with bioactive

components. Several animal models, mainly using mice and rats, have been used to

explore single RV infection and its pathology, clinical and immune response, and to test

vaccine efficacy or nutritional modulation of the process. However, a multiple infection

model is needed in order to study how immune response behaves in repeated infections.

The objective of the study was to develop an experimental model of a double RV infection

in rat. Therefore, Lewis neonatal rats were orally inoculated with SA-11 (simian rotavirus)

and the development of clinical parameters such as diarrhea, body temperature and faecal

pH were assessed daily. The level of immune protection induced by SA-11 inoculation was

determined by re-infection with EDIM (mouse rotavirus) later in life. Faecal-viral load,

rotavirus-specific antibody titers in serum, rotavirus-specific cell proliferation and rotavirus-

specific DTH were measured after EDIM inoculation. As a result of several designs we can

conclude that second infection with EDIM was not able to induce clinical symptoms but

modulated the immune response present due to first infection in terms of specific Ig levels

in systemic and intestinal compartments and DTH response.

This experimental model in sucking rats for rotavirus-induced diarrhea can be used to

assess several immune parameters including protection against re-infection and to study

modulation of (re)infection by nutritional interventions.

www.sci.cat 41


19 Multiparametric analysis by flow cytometry of leukocyte subpopulations in peripheral blood of healthy donors

Bibiana Quirant; Aina Teniente Serra; Rus-Merchán, Amanda; Marco A. Fernández Sanmartín; Ricardo Pujol-Borrell; Thomas Giese; Eva M. Martínez-Cáceres

Hospital Universitario German Trias i Pujol.

INTRODUCTION: The development of multiparametric flow cytometry has allowed the detailed analysis and characterization of lymphocyte subsets in peripheral blood being an important tool in diagnosis and following up of patients with immunomediated diseases. However a standardization of the immunophenotype of these subpopulations is still lacking. In this context, a consortium of laboratories was formed in 2009, led by the National Institutes of Health (NIH) and the Federation of Clinical Immunology Societies (FOCIS). The ENTIRE group established standardised whole blood panels based on their recommendations. Six FCE´s of ENTIRE agreed to analyze 50 healthy donors (HD). Here the results of the Barcelona group are presented. AIM: To analyse 50 HD following instrument set-up and antibodies panels proposed in the HIP-C protocol discussed in the COST-ENTIRE initiative. METHODS: Peripheral blood of 50 HD (BST, Catalonia) (28 F, 22M; aged 21-66) was analyzed by FC (LSR Fortessa®) using panels established in the ENTIRE-HIPC protocol for T cells (CD45, CD3, CD4, CD8, CCR7, CD45RA, CD38, HLA-DR, CD183, CD196), Treg cells (CD45, CD3, CD4, CD25, CD127, CCR4, CD45RO, HLA-DR), B cells (CD45, CD19, CD3, CD24, CD38, IgD, CD27, CD20) and NK, monocyte and dendritic cells (CD45, CD3, CD19, CD14, CD16, CD56, HLA-DR, CD123, CD11c, Slan). RESULTS: In this study are shown percentages and counts of 50 HD. Major subpopulations had less variability than minor subpopulations. In particular, functional T CD4 subpopulations as Th1, Th2 and Th17 showed elevated variability, such as did transitional and plasmablasts B cells. In CD8 T cells the major maturation subset was naive T cells (CCR7+CD45RA+; 39.89±17.43%), followed by terminally diferenciated effector T cells (TEMRA; CCR7-CD45RA+; 31.29±16.33%). Regarding CD4 T cells, the major subset was naive T cells (CD45RA+CCR7+; 41.53±13.74%) followed by Central Memory T (TCM; CD45RA-CCR7+; 39.03±10.17%). The percentage of Tregs cells were 5.21±1.54% of CD4 T cells. Of these, about a half of them were HLA-DR+ (2.02 ± 0.73%). In relation to B cells, 51.78±16.72% of CD19+ cells were naive B cells (CD27-IgD+) being the other subsets analysed (pre-switched memory, switched memory, exhausted memory, transitional B cells, plasmablasts) minor subpopulations in peripheral blood. When NK lymphocytes were analysed the major subpopulation was the CD56brCD16+ subset (87.30±8.48%) which has been reported as a cytotoxic subpopulation. Monocytes subpopulations were divided in classic monocytes (CD14+CD16-) (91.59±5.48%) and non-classic monocytes (CD14lowCD16+) (6.06±3.15%). Dendritic cell subpopulations were characterized by the expression of CD123 and CD11c, being classified as myeloid DCs those CD123lowCD11c+ (38.83±15.34%) and plasmacitoid DCs subset as CD123+CD11c-(11.25±15.30%). Moreover, in mDCs was analysed the expression of Slan, a marker to define a proinflammatory DCs subset, that represented a 58.96±14.45% of mDCs subset. CONCLUSIONS: These results together with results from other centres in this collaborative project will be relevant to get a representative cohort of healthy donors and standardize the immunophenotype. This will be essential in order to better utilize FC in multi-centric clinical trials, as well as in daily clinical immunological practice in the diagnosis, monitoring and follow up of treatments in immunomediated diseases.

42 www.sci.cat


20 Experiència inicial i comparativa dels ELISAS comercials emprats en la monitorització analítica de la resposta amb biològics.

Noemí De Moner(1); Rosa Rodríguez (1); Maria Antonia Romera (1); Belén Suárez (1); Julià Panés (2); Raimon Sanmartí (3); Alexis Sentís (4); Patricia Villarroel (1); Manel Juan (1); Jordi Yagüe (1).

(1) Servei dÍmmunologia - Centre de Diagnòstic Biomèdic. (2) Servei de Gastroenterologia; (3) Servei de Reumatologia; (4) Servei de Nefrologia. Hospital Clínic de Barcelona.

Introducció: Els tractaments amb fàrmacs biològics constitueixen opcions terapèutiques molt

efectives i en molts casos han canviat la manera i les opcions de tractament de diversos pacients.

Tot i que existeixen diversos tipus de biològics, els anticossos monoclonals són en láctualitat les

eines biològiques més emprades com a fàrmacs biològics (a excepció de lÉtanercept que és una

forma soluble recombinant del receptor del TNFα). Fins fa poc léficàcia dáquests tractaments

sávaluava només a través de léfectivitat en la milloria simptomàtica i no hi ha cap explicació per

la pèrdua progressiva de resposta que es dóna en un percentatge de pacients. Actualment, podem

mesurar la concentració del fàrmac i valorar els nivells terapèutics assolits en cada pacient. Al

mateix temps, la detecció dánticossos anti-fàrmac ens permet explicar les pèrdues de resposta

que es produeixen durant els tractaments amb aquests fàrmacs biològics. Existeixen diversos kits

comecials per determinar aquests nivells de fàrmacs i anticossos, i en el present treball es planteja

un estudi comparatiu entre les diverses opcions utilitzades al nostre laboratori.

Mètode: Sistema de detecció quantitativa:

- ELISA en sandwitx de primera i segona generació per anti-TNF-α (Infliximab i Adalimumab amb

mesura de nivells de fàrmac i anticossos anti-biològic); Promonitor

- ELISA en sandwitx per a anti-CD20 (Rituximab, amb mesura de nivells de fàrmac i anticossos

anti-biològic); Promonitor

- ELISA en sandwitx per a anti-IL6 (Tocilizumab, amb mesura de nivells de fàrmac i anticossos

anti-biològic); Theradiag

Mostres estudiades: 60 mostres de pacients amb malaltia inflamatòria intestinal (MII) tractats

amb Infliximab, 41 de pacients amb MII tractats amb Adalimumab, 19 pacients amb una patologia

renal dórigen immunològic tractats amb Rituximab i 39 pacients amb Artritis Reumatoide (AR)

tractats amb Tocilizumabamb. Totes les mostres corresponen a sèrums extrets en la “zona vall”,

just abans de la següent infusió prescrita.

Resultats/Conclusions: Tècnicament tots els ELISAs comercials analitzats mostren rangs

suficientment amplis per a definir tant les dosis presents com la presència dánticossos anti-

fàrmac. Els mètodes són senzills de dur a terme i permeten ser automatitzats. S´han definit nivells

terapèutics de Infliximab 2ug/ml i Adalimumab 4 ug/ml. La presència dánticossos en pacients no

responedors es detecta només en un 12-26% dels pacients tractats amb adalimumab i infliximab.

No es van detectar anticossos anti-Rituximab ni anti-Tocilizumab. Les dosis mesurades als

pacients no responedors van ser subteràpeutiques en 63-66% dels pacients. Pel contrari, les dosis

dels pacients responedors van ser terapèutiques en un 60-71% dels casos.

La detecció de nivells de fàrmac i anticòs s´ha incorporat al algorítimic terapèutic per a pendre

decisions clíniques. Ha tingut un impacte significatiu a la pràctica diaria.

www.sci.cat 43


21 Abordaje analítico en la búsqueda de una nueva mutación en el gen de la adenosina deaminasa.

Mireia Antón; Noemí De Moner; E. Azucena González; Montserrat Masó; Anna Mensa; Mariona Pascal; Eva González; Estibaliz Ruiz; Laia Alsina; Juan I. Arostegui; Manel Juan.

Servei d´immunologia, Hospital Clínic de Barcelona.

Introducción: Las mutaciones descritas en el gen Adenosina Deaminasa (ADA) son

responsables de formas autosómicas recesivas de inmunodeficiencia combinada grave

(AR-SCID) con un fenotipo linfocitario con déficit de linfocitos T, B y NK (T-B-NK-). En

general estas mutaciones se relacionan con cambios nucleotídicos que de manera directa

afectan a la secuencia proteica, por lo que la secuenciación de los exones suele ser

suficiente para su detección.

Objetivo: Definir el abordaje para el estudio familiar de un cuadro de déficit de ADA

atípico en una paciente con padres consanguineos.

Métodos: Fenotipo citofluorimétrico y estudio funcional linfocitario. Mapeo genético de la

pérdida de homocigosidad de la paciente y los familiares de primer grado sobre

microarrays Affimetrix GenomeWide. Secuenciación por sanger del gDNA de la paciente.

Resultados: El inmunofenotipo de los linfocitos circulantes muestra que la paciente

presenta un déficit de linfocitos T y B, siendo la población de células NK normal, junto con

la falta de activación celular y proliferación (T-B-NK+). No se detectaron mutaciones en los

genes RAG-1, RAG-2 y DCLRE1C, todos ellos causantes de AR-SCID T-B-NK+.

El estudio por hibridación de DNA para detectar la homocigosidad demostró 1356 zonas

de homozigosidad entre las cuales se encontraba el gen ADA.

La secuenciación por Sanger del gen ADA no detectó cambios en las regiones exónicas,

siendo necesario valorar las zonas adyacentes con el objetivo de descartar afectaciones

en las secuencias de splicing. De este modo se detectó una deleción homocigota de 4bp

en los cuatro primeros nucleótidos del intrón 4, en la zona responsable de la unión de la

maquinaria de splicing celular. Esta variante genética no ha sido descrita en la literatura,

de tal manera que puede excluirse razonablemente que se trate de un polimorfismo del

gen.

Conclusiones: Existen formas de alteración genética que probablemente afectando el

proceso de splicing del gen ADA definen un fenotipo analítico con células NK.

El estudio de la homocigosidad es útil para definir genes asociados a enfermedad en

familias consanguíneas.

De manera general se puede afirmar que es imprescindible valorar las regiones

adyacentes responsables de los mecanismos de splicing del mRNA y de manera general

las partes no codificantes de los genes, aunque este aspecto puede ser más

controvertido.

44 www.sci.cat


22 Proteome antibody screening methods for use in evaluation of naturally acquired immune responses to Plasmodium falciparum in vaccine trials

Joseph J. Campo, Jeff Skinner, Rie Nakajima-Sasaki, Douglas M. Molina, Li Liang, Jahit Sacarlal, Pedro L. Alonso, Peter D. Crompton, Philip L. Felgner, John J. Aponte, Carlota Dobaño


RTS,S is the leading vaccine candidate for Plasmodium falciparum malaria. An efficacious

pre-erythrocytic vaccine of short or medium duration may decrease subsequent exposure,

resulting in modified naturally acquired immunity. Proteomic screening methods are a

powerful tool for assessment of overall antibody response to pathogens.

Previous studies found a reduction of naturally acquired antibody responses to a number

of well-characterized P. falciparum blood stage antibodies after vaccination with RTS,S.

This study aims to examine the extent of the effect of RTS,S vaccination on naturally

acquired antibodies, identify cofactors of antibody responses and identify antibody

associations with protection.

We performed antibody screening of a broad selection of immunoreactive P. falciparum

antigens using protein microarrays. Samples were selected from Mozambican children of

1-4 years of age vaccinated with RTS,S/AS02. A total of 623 samples from the 8.5 months

cross-sectional were probed on a chip spotted with 824 highly immunoreactive P.

falciparum proteins. Records of clinical malaria cases prior to cross-sectional sampling and

parasitemia at time of sampling were used to classify antibody profiles by exposure status.

Post-sampling records of clinical malaria were used to model antibody associations with

susceptibility or protection.

The majority of P. falciparum antigenic targets were reactive in Mozambican vaccinees,

and of these proteins, the profile breadth and reactivity were similar between RTS,S and

comparator vaccinees. However, magnitude of response was higher to a subset of

antigens in the comparator vaccine group. Age, parasitemia and previous clinical malaria

episodes had large differential effects on magnitude. Antibody levels appeared to decay

rapidly after exposure. Cross-sectional antibody levels appeared to be mostly associated

with subsequent susceptibility to clinical malaria and not protection.

We found antibodies in these children to be short lived and probably the result of recent

exposure. Vaccination with RTS,S resulted in a moderate reduction in cross-sectional

antibody levels for a number of P. falciparum antigens, but no difference in profile breadth.

These data demonstrate the effects of an efficacious vaccine on parasite antibody

responses and the usefulness of the protein array platform in characterizing the impact of

malaria interventions on naturally acquired immunity.

www.sci.cat 45

Posters Innate Response 23-26

The authors will attend the poster on 15/11/2013, 17:20h

23 Role of ascitic fluid macrophages in spontaneous bacterial peritonitis with positive and negative bacterial culture

Juan C. Nieto, Elisabet Sánchez, Cristina Romero, Eva Román, Carlos Guarner, Cándido Juárez, Germán Soriano, Sílvia Vidal

IIB-Institut Recerca Hospital S. Pau, Barcelona.

Macrophages from ascitic fluid in cirrhotic patients with spontaneous bacterial peritonitis

(SBP) play a crucial role in resolving inflammation but their functional phenotype remains

unknown. We studied leukocyte markers involved in activation, adhesion, antigen

presentation, and scavenging on macrophages from the ascitic fluid of culture positive

SBP (CP-SBP), culture negative SBP (CN-SBP) and sterile ascites (SA) at diagnosis and

after antibiotic treatment. Percentage of lymphocytes, macrophages and neutrophils on

CP-SBP were significantly different than on CN-SBP and SA. SA macrophages showed

the highest expression of HLA-DR, CD11b and CD163, and the lowest expression of

CD64. Before treatment, CP-SBP macrophages expressed low levels of HLA-DR, CD16,

CD163, and CD206. CN-SBP macrophages had an intermediate phenotype between SA

and CP-SBP. Antibiotics significantly increased CD16+ percentage and upregulated HLA-

DR, CD11b and CD86 expression on CP-SBP macrophages. These changes were

comparable with monocytes from healthy donors cultured with LPS. IL-6 concentration in

ascitic fluid was significantly higher in CP-SBP than CN-SBP and SA, and was

downregulated after antibiotic treatment (p=0.03 and p=0.002). TNF-alpha correlated with

CD36 expression (r=0.56 and p=0.006). In conclusion, ascitic macrophages from CP-SBP

and CN-SBP have different functional profiles. Despite both conditions were resolved after

antibiotic treatment, only CP-SBP macrophage phenotype changed.

46 www.sci.cat

Poster Innate Response The authors will attend the poster on 15/11/2013, 17:20h

24 Estudi dels nivells déxpresió dels micro-RNAs 146, 346, 128 i 141 en la resposta inflamatòria via TLR2 i TLR4.

Germán Julià; Elisabet Cantó; Cándido Juárez; Sílvia Vidal

Institut de Recerca Hospital de la Santa Creu i Sant Pau.

Els microRNAs son RNA sc petits (dúns 22 nucleòtids) que formen part dún sistema de regulació

de léxpressió gènica a nivell post-transcripcional mitjançant el reconeixement específic de les

regions 3ÚTR ( 3´ no traduïdes) localitzades als mRNAs diana. Aquesta regulació te lloc per la

degradació del mRNA o la repressió de la seva traducció i es critica per al funcionament correcte

de moltes funcions com el cicle cel•lular, diferenciació, apoptosi, així com en processos de

regulació de la resposta immune.

Donada la importància de la regulació de la resposta immune per part de certs microRNAs, es van

analitzar previament a aquest treball léxpressió de 381 microRNA´s per microarrays en monòcits

humans estimulats durant 4 hores amb LTA identificant-se diferencies significatives en léxpressió

de 4 microRNAs: hsa-miR-146a, hsa-miR-346, hsa-miR-128 i hsa-miR-141.

Prenent aquests resultats previs com a punt de partida, i a banda de comprovar els resultats

obtinguts, es va analitzar: léxpressió dáquests 4 miRNAs per RT-qPCR de RNA total, de

monòcits en presència/absència dels estímuls inflamatoris LTA (via TLR-2) i també amb LPS (via

TLR-4). Alhora que es va estudiar la seva cinètica déxpressió durant diferents temps

déstimulació (3, 6 i 20 hores). Dígual forma que amb els monòcits humans, séstudià la línea

cel•lular monocítica THP-1, per avaluar el seu ús com model experimental en léstudi dels

microRNAs hsa-miR-146a i hsa-miR-346. Addicionalment séstudià léfecte dáltres estímuls

inflamatoris com son el TNFα, IL-1β i IL-10 en léxpressió dels hsa-miR-146a i hsa-miR-346 a la

línea cel•lular THP-1.

Els nostres resultats demostren que els nivells déxpressió del hsa-miR-146a augmenten, als

monòcits díndividus sans i a la línea cel·lular THP-1, després de 20 hores déstimulació tant amb

LTA com amb LPS. El TNF-α i la IL-1β indueixen la seva expressió a la línea celular THP-1,

mentre que la IL-10 no presenta cap influència. Pel que respecta al hsa-miR-346 sóbserva que

léstimulació durant 3 hores amb LTA o LPS incrementen la seva expressió als monòcits humans,

en canvi a la línea THP-1 únicament sóbserven canvis amb léstimulació durant 20 hores amb

LTA. Fet que restringeix l´ús de THP-1 com model experimental per léstudi dáquest microRNA.

Els nivells déxpressió del hsa-miR-128 als monòcits, síncrementen amb léstímul durant 20 hores

tant amb LTA com amb LPS. Léxpressió del hsa-miR-141 no es veu influenciada per léstímul

dels monòcits humans amb LTA, mentre que es troba disminuïda amb léstimulació durant 3 hores

amb LPS.

En tots els casos, es confirmen els resultats previs obtinguts per microarrays. Hem observat que

léstimulació amb LTA o LPS presenta diferències en la cinètica déxpressió de dos dels

microRNA analitzats. S´ha establert la concentració optima de LTA per léstímul de THP-1. Es

confirma la línea THP-1 com model experimental per léstudi hsa-miR-146a. Hem observat que el

TNF-α i la IL-1β incrementen léxpresió hsa-miR-146a a la línea cel·lular THP-1, mentre que la IL-

10 no te cap efecte.

www.sci.cat 47


25 Antigen-specific myeloid-derived suppressor cells generated during retroviral transduction of murine bone marrow ameliorate experimental autoimmune encephalomyelitis

Sílvia Casacuberta, Alba Gómez, Sergio López, Carme Costa, Mª José Mansilla, Herena Eixarch, Lluís Martorell, Xavier Montalban, Carmen Espejo, Jordi Barquinero Institut de Recerca Vall d´Hebron.

Our previous work showed that transferring bone marrow (BM) cells transduced with the

MOG40-55 autoantigen into non-myeloablated mice with experimental autoimmune

encephalomyelitis (EAE) improved the disease. We also reported that the vast majority of

cells that are generated in the standard retroviral transduction cultures of murine BM cells

consisted of myeloid cell populations (CD11b+ Gr-1low) and (CD11b+ Gr-1high), which

correspond to the phenotypes of the monocyte-like and the granulocyte-like myeloid-

derived suppressor cells (mMDSCs and gMDSCs). In vitro, these MDSCs expressed

arginase-1 and iNOS, produced reactive oxygen species and suppressed splenocyte

proliferation from EAE mice. Besides these well known suppressive mechanisms, both

MDSCs subtypes expressed PD-L1 (Programmed Death Ligand-1). With this background,

to further investigate the contribution of these cells to the observed therapeutic effect we

performed in vivo studies using either transduced unfractionated BM or purified MDSCs. In

a preventive approach, in the EAE model, a single i.v. infusion of 5x105 MOG-specific

MDSCs into non-myeloablated animals significantly ameliorated the clinical course of the

disease to a similar extent as total MOG-specific BM cells, indicating that MDSCs are

contributing to the therapeutic effect. Remarkably, this effect was absent in mice receiving

sham-transduced cells, indicating that antigen-specific mechanisms play a role in the

tolerogenic effect. Furthermore, the spleens of these mice contained significantly less

activated T cells (CD3+CD4+CD25+FoxP3-) and more B regulatory cells

(CD45+B220+CD5+CD1d+) than those of their counterparts. Adoptive cell therapy using

ex-vivo generated antigen-specific MDSCs constitute a useful approach to induce

tolerance in the context of autoimmune diseases.

48 www.sci.cat


26 Mechanisms of inhibition mediated by CD33 (siglec-3) in human NK cells.

Trinidad Hernández-Caselles, Rubén Corral-San Miguel, Antonio José Ruiz-Alcaraz and Pilar García-Peñarrubia.

Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia.

CD33 (siglec-3) is widely expressed in leukocytes of the myeloid lineage however it can

also be expressed on human NK cells where it could constitute a novel control mechanism

of the innate immune system. It exact role remains largely unknown. In NKL cells, we

previously found that CD33 displays specific modulation of cytotoxicity but not IFN

production triggered by NKG2D, contrasting with the best known CD94/NKG2A or ILT2 NK

inhibitory receptors that inhibit both cytotoxicity and cytokine secretion induced by different

activating receptors. In the present work we found that CD33 is expressed on a

CD56bright CD16- subset of primary human blood NK cells and on a subset of in vitro

activated NK cells. On activated primary NK cells, CD33 did not inhibit NKG2D function

however it co-precipitated with the SHP-2 phosphatase and was not recognized by the

HIM3-4 anti-CD33 mAb, indicating a CD33 basal inhibitory function probably under a

masked state. On NKL cells, CD33 mediated adhesion implicates cytoskeleton activation

whereas CD33 mediated inhibition on NKG2D induced cytotoxicity involves Vav1 as a

critical intermediate. Our results support the role of CD33 as inhibitory receptor that could

be involved in self tolerance, tumor and infected cells recognition by mature NK cells.

www.sci.cat 49

2014 Events

Lifelong Learning SCI Program

Data i hora: 6 de febrer de 2014, a les 18.30h. "Nuevas fronteras en la investigación en el VIH/SIDA". Conferència a càrrec del Dr. Javier Martínez-Picado (Professor de Recerca ICREA, Institut de Recerca de la SIDA IrsiCaixa i UAB, Barcelona); Lloc: Hospital de la Santa Creu i Sant Pau. Data i hora: 6 de març de 2014, a les 18.30h. "Immunoteràpia del càncer". Conferència a càrrec dels Dr. Ramón Vilella (Servei d'Immunologia Hospital Clínic i Provincial de Barcelona, Barcelona); Lloc: Parc de Recerca Biomèdica de Barcelona (PRBB), sala Xipre 1ª planta (c/Doctor Aiguader 88, Barcelona). Data i hora: 3 d'abril de 2014, a les 18.30h. "Aplicaciones clínicas de la inmunoterapia con células Natural Killer en el cáncer". Conferència a càrrec del Dr. Antonio Pérez Martínez (Universidad Autonoma de Madrid, Madrid); Lloc: Hospital Clínic. Data: 29 d'abril de 2014, dia de la IMMUNOLOGIA Tema: LES VACUNES Una iniciativa de la IUIS (International Union of Immunological Societies) amb la col·laboració directa de la EFIS (European Federation of Immunological Societies) Data i hora: 8 de maig de 2014, a les 18.30h. "Hematopoietic ontogeny in the fetal liver and Innate-like CD19+CD45Rlo B cell population". Conferència a càrrec de la Dra. Maria Luisa Gaspar (Centro Nacional de Microbiologia, ISCIII. Madrid); Lloc: Hospital de la Santa Creu i Sant Pau. Data i hora: 5 de juny de 2014, a les 18.30h. "Role of reticulocyte-derived exosomes in Plasmodium vivax malaria". Conferència a càrrec del Dr. Hernando del Portillo (CRESIB-Hospital Clínic, Barcelona); Lloc: Acadèmia de Ciències Mèdiques (Major de Can Caralleu 1-7, Barcelona). Data i hora: 3 de juliol de 2014, a les 18.30h. "Regulation of macrophage functions by LXR Nuclear Receptors". Conferència a càrrec del Dr. Antonio Castrillo Viguera (IIB-UAM, Madrid); Lloc: Parc de Recerca Biomèdica de Barcelona (PRBB), sala Xipre 1ª planta (c/Doctor Aiguader 88, Barcelona).

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List of participants

Abril-Gil, Mar ........................................ 38, 39 Almansa, Raquel........................................ 16 Alonso, Pedro L. .................................. 22, 44 Alsina, Laia .......................................... 29, 43 Altisent, C. ................................................. 36 Antón, Mireia ........................................ 29, 43 Antonijoan, Rosa M. ................................... 22 Aponte, John J. .......................................... 44 Arevalo-Herrera, Myriam ............................ 25 Arostegui, Juan I. ................................. 29, 43 Arpa, B. ...................................................... 30 Auer, Herbert ............................................. 13 Autonell, I. .................................................. 30 Azucena Salas ........................................... 35 Badell, Isabel ............................................. 11 Ballester, Maria Rosa ................................. 22 Bardají, Azucena........................................ 25 Barquinero, Jordi........................................ 47 Barrios, Diana ...................................... 22, 25 Bartra, Joan ............................................... 31 Bas, Jordi ................................................... 28 Benítez-Ribas, Daniel ................................ 24 Bermejo-Martin, Jesús ............................... 16 Cabezón, Raquel ....................................... 19 Calderón, Elisabeth .............................. 19, 35 Cambras, Trinitat ....................................... 39 Campo, Joseph J. ................................ 25, 44 Camps-Bossacoma, Mariona ..................... 38 Cantó, Elisabet .................................... 20, 46 Carbó, Jose Maria ...................................... 13 Carrascal, J. ............................................... 30 Casacuberta, Sílvia .................................... 47 Casanovas, A. ........................................... 30 Castell, M. ...................................... 38, 39, 40 Castellote, C. ............................................. 40 Celada, Antonio ............................. 13, 14, 15 Celhar, Teja ............................................... 14 Chitnis, Chetan .......................................... 25 Cisteró, Pau ............................................... 22 Climent, Joan ............................................. 28 Colobran, Roger .................. 8, 12, 26, 32, 33 Comalada, Mònica ..................................... 38 Córdoba, Lorena ........................................ 16 Corral, J. .................................................... 36 Corral-San Miguel, Rubén .......................... 48 Costa, Carme ............................................. 47 Costa, Manuela .......................................... 21 Crompton, Peter D. .................................... 44 De la Calle-Martín, Oscar ........................... 11

De la Cruz, Xavier ...................................... 12 De la Haba, Carlos ..................................... 23 De la Morena-Barrio, M.E. ......................... 36 De Lazzari, Elisa ........................................ 25 De Moner, Noemí ................................. 42, 43 Diaz-Lopez, Cesar ..................................... 20 Diaz-Torné, Cesar ...................................... 20 Digón, Montserrat ...................................... 37 Dobaño, Carlota ............................. 22, 25, 44 Dotti, Isabella ............................................. 35 Eixarch, Herena ......................................... 47 Ercilla, Guadalupe ...................................... 37 Escolà-Gil, Joan Carles.............................. 13 España, Carolina ....................................... 24 Espejo, Carmen ......................................... 47 Esteve-Sole, Ana ....................................... 29 Fairhurst, Anna-Marie ................................ 14 Farrera, Consol .......................................... 15 Felgner, Philip L. ........................................ 44 Fernández Sanmartín, Marco A. ................ 41 Fernández, Marco A. ........................... 27, 41 Flórez-Grau, Georgina ............................... 24 Franch, A. ...................................... 38, 39, 40 Franco-Jarava, Clara ................................... 8 García, Milagros......................................... 34 Garcia-Just, Alba ....................................... 39 García-López, R......................................... 36 Garcia-Martín, Carme ................................ 21 García-Peñarrubia, Pilar ............................ 48 Giese, Thomas .......................................... 41 Gifre, Laia .................................................. 32 Giménez-Barcons, Mireia ........................... 26 Gómez Pau, Ana........................................ 26 Gómez, Alba .............................................. 47 Gómez-Pérez, Patricia ............................... 22 González, Azucena ........................ 29, 34, 43 González, Europa A. .................................. 37 González, Eva ..................................... 29, 43 Guarner, Carlos ......................................... 45 Guilarte, Mar .............................................. 12 Guillén, E. .................................................. 36 Heredia, Gemma........................................ 17 Hernández-Caselles, T. ....................... 36, 48 Hernández-González, Manuel.................... 12 Hoffman, Stephen ...................................... 22 Jaeken, J. .................................................. 36 Jaraquemada, Dolores ............................... 21 Jimenez Bernal, Isabel ............................... 11 Jimenez, Alfons ......................................... 22

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Juan, Manel ............................. 29, 31, 42, 43 Juárez, Cándido ............................. 20, 45, 46 Julià Panés ................................................ 42 Julià, Germán ............................................ 46 Julve, Josep ............................................... 13 Knipping, K. ............................................... 40 Kristensen, S.R. ......................................... 36 Legarda, Almudena .................................... 22 León, Theresa ............................................ 13 Liang, Li ..................................................... 44 Lloberas, Jorge .................................... 14, 15 Llobet Agulló, M.Pilar ................................. 10 Llobet, Josep M. ........................................ 20 Lois, Sergio ................................................ 12 López, María .............................................. 17 López, Sergio ............................................. 47 López-Botet, Miguel ................................... 17 Lozano, M.L. .............................................. 36 Lozano-Ramos S.I. ...................................... 9 Mansilla, Mª José ....................................... 47 Martí, Mercè ............................................... 21 Martín, Andrea ................................. 8, 32, 33 Martínez Orellana, Pamela ........................ 16 Martínez, Jorge .......................................... 16 Martínez, Paz ............................................. 23 Martínez-Cáceres, Eva M. ............... 9, 27, 41 Martínez-Gallo, Mónica ........................ 32, 33 Martínez-Martínez, I. .................................. 36 Martínez-Martinez, Laura ........................... 11 Martínez-Torró, Carlos ............................... 21 Martín-Nalda, Andrea ............................. 8, 33 Martorell, Jaume ........................................ 37 Martorell, Lluís ........................................... 47 Masó, Montserrat ................................. 29, 43 Massot-Cladera, Malen ........................ 38, 39 Matalonga, Jonathan ................................. 13 Mayor, Alfredo ........................................... 25 Menegon, Michela ...................................... 25 Menéndez, Clara........................................ 25 Mensa, Anna ........................................ 31, 43 Mestre, Mariona ......................................... 28 Milà, Jordi .................................................. 31 Millán, Lucía ............................................... 31 Minguell, Laura ............................................ 8 Miñano, A................................................... 36 Molina, Douglas M. .................................... 44 Moncunill, Gemma ..................................... 22 Montalban, Xavier ...................................... 47 Montoya, María .......................................... 16 Mora, C. ..................................................... 30 Mora, Rut ............................................. 19, 35 Morandeira, Francisco ............................... 28 Moraru, Manuela ........................................ 17 Morros, Antoni ........................................... 23 Mueller, Ivo ................................................ 25

Muntasell, Aura .......................................... 17 Muñoz, José .............................................. 22 Nakajima-Sasaki, Rie ................................. 44 Nieto, Juan C. ...................................... 20, 45 Ome, Maria ................................................ 25 Ortiz, M. Angels ......................................... 20 Palacio, José R. ......................................... 23 Palkovics, Tamas ....................................... 23 Panés, Julià ......................................... 19, 24 Parra, David ............................................... 18 Pascal, Mariona ................................... 31, 43 Pascual-García, Monica ............................. 13 Peñafiel, Judith .......................................... 17 Pereira-Lopes, Selma ................................ 14 Pérez, Nuria ............................................... 34 Pérez-Cano, Francisco J. ............... 38, 39, 40 Pérez-Dueñas, B........................................ 36 Pérez-Navarro, Esther ............................... 13 Pintos-Morell G. ........................................... 9 Piquer, Mónica ........................................... 29 Pizarro, Eduardo ........................................ 21 Planell, Núria ............................................. 35 Plaza, Ana Mª ............................................ 29 Puig-Domingo, Manel ................................ 27 Pujol-Borrell, Ricardo ..................... 12, 26, 41 Quintana, Luis ............................................ 34 Quirant, Bibiana ................................... 27, 41 Raquel Cabezón ........................................ 24 Requena, Pilar ........................................... 25 Rigo-Adrover, M. ........................................ 40 Rigol-Monzó, Elisabet ................................ 37 Rodrigo C..................................................... 9 Rodríguez, Rosa ........................................ 42 Rogerson, Stephen .................................... 25 Román, Eva ............................................... 45 Romera, Maria Antonia .............................. 42 Romero, Cristina ........................................ 45 Romo, Neus ............................................... 17 Rosanas, Anna .......................................... 25 Rosell, E. ................................................... 30 Roura-Mir, Carme ...................................... 21 Rubiales, Maria Victoria ............................. 11 Rué, Laura ................................................. 13 Ruiz, Estibaliz ............................................ 43 Ruiz-Alcaraz, Antonio José ........................ 48 Rus-Merchán, Amanda .......................... 9, 41 Sábado, J................................................... 30 Sacarlal, Jahit ............................................ 44 Salas, Azucena .......................................... 19 Sánchez, Elisabet ...................................... 45 Sanchis, Ana .............................................. 32 Sanmartí, Raimon ...................................... 42 Sans-Fons, Gloria ...................................... 14 Sanz, Sergi ................................................ 25 Sentís, Alexis ............................................. 42

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Serra, Maria ............................................... 14 Serra-Pagès, Carles .................................. 37 Severini, Carlo ........................................... 25 Sevivas, T. ................................................. 36 Siba, Peter M. ............................................ 25 Sim, Kim Lee ............................................. 22 Skinner, Jeff ............................................... 44 Soldevila, Berta .......................................... 27 Soler, Pere ....................................... 8, 32, 33 Soriano, Germán........................................ 45 Steffensen, Knutt R. ................................... 13 Suárez, Belén ............................................ 42 Sunyer, Oriol .............................................. 18 Szekeres-Barthó, Júlia ............................... 23 Teniente Serra, Aina ........................ 9, 27, 41 Umbers, Alex ............................................. 25 Usero, Lorena ............................................ 21 Valledor, Annabel....................................... 13

Valverde-Estrella, Lorena ........................... 15 Vera, Andrea .............................................. 17 Verdaguer, J. ............................................. 30 Vicente, Filipa ............................................ 38 Vicente, V. ................................................. 36 Vidal, Sílvia .................................... 20, 45, 46 Vidaña, Beatriz .......................................... 16 Vila, Joan ................................................... 17 Vilardell, N. ................................................ 40 Vilches, Carlos ........................................... 17 Vilella, Ramon ............................................ 31 Villarroel, Patricia ........................... 34, 37, 42 Viñas, Odette ............................................. 34 Vives-Pi, M. ................................................ 30 Wangnapi, Regina ..................................... 25 Xufré, Cristina ............................................ 21 Yagüe, Jordi ............................................... 42 Zamora, Carlos .......................................... 20

Premi a la millor comunicació oral i al millor pòster en el VIIè Congrés SCI: Per quart any consecutiu i gràcies al patrocini de Miltenyi Biotec, aquest any s’atorgarà també el premi a la millor comunicació oral (500 €) i al millor pòster (250 €) del present congrés. El comité cientific i la Junta de la SCI seleccionaran entre les comunicacions presentades en cada categoria aquella que destaca tant pels seus valors cientícs com per aspectes relacionats amb la pròpia presentació. En les deliberacions estarà present amb veu però sense vot i un membre de l’empresa Miltenyi. Les resolucions es faran públiques a la fi del congrés.

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Other useful information and notes

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VIIè CONGRÉS Societat Catalana d’Immunologia (SCI)

XIè SIMPOSI adjunt Grupo Español de Inmunodeficiencias Primarias (GEIDP) y Registro Español de Inmunodeficiencias Primarias (REDIP)

Barcelona, 14 i 15 de novembre 2013

This Congress has received the support of the SEI (Sociedad Española de Inmunología)

Sociedad Española de Inmunología

The contents of this congress will be accessible in few months in our website www.congressci.com by using the Virtual Congress tool

immunitat innata immunodeficiènciesgrupo español de inmunodeficiencias primarias (geidp) y...

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