Caracterización de los antagonistas del receptor de muerte CD95/FAS/APO-1, FAIM y Lifeguard en el sistema nervioso

La apoptosis es un mecanismo fisiológico que contribuye a regular el número de células de un organismo de tal forma que aquéllas que realizan funciones transitorias, que están lesionadas, o en exceso, serán eliminadas. Éste es un proceso estrictamente regulado durante el desarrollo embrionario e íntimamente ligado con el inicio o progresión de determinadas patologías. Así pues, el exceso de apoptosis contribuye al desarrollo de enfermedades neurodegenerativas mientras que su defecto sería el origen de neoplasias. El principal regulador del proceso apoptótico es la activación de las caspasas, cisteína-proteasas con especificidad para residuos de aspartato. Los principales mecanismos que activan las caspasas son la salida de citocromo C de la mitocondria por una alteración de la función mitocondrial, y la activación de proteínas de membrana denominadas receptores de muerte (DRs, Death Receptors). Estos últimos han sido ampliamente caracterizados en el sistema inmune, mientras que en tejidos como el sistema nervioso sus funciones están en las fases iniciales de caracterización.El objetivo del presente trabajo es contribuir a esclarecer los mecanismos moleculares que regulan la actividad de estos receptores en el sistema nervioso, a través de la caracterización funcional de dos nuevas proteínas, FAIM y Lifeguard, propuestas inicialmente como antagonistas del receptor de muerte CD95/Fas/APO-1. Para ello se han usado las líneas celulares PC12 y SH-SY5Y, ampliamente utilizadas en modelos de diferenciación y muerte celular, junto con cultivos primarios de neuronas corticales y neuronas granulares de cerebelo.En la primera parte de este trabajo se describe la clonación del ortólogo de ratón de Lifeguard, y su caracterización como antagonista funcional de CD95 en el sistema nervioso. Hemos demostrado que su sobreexpresión es capaz de bloquear la muerte inducida por CD95 en el modelo del neuroblastoma humano SH-SY5Y así como en neuronas corticales murinas. También hemos comprobado que la disminución de sus niveles endógenos sensibiliza a las neuronas granulares y las corticales de ratón. Además, se ha constatado que su mecanismo molecular de acción depende de su localización exclusiva en microdominios de membrana llamados Lipid Rafts, donde puede interaccionar con CD95 e inhibir la activación de caspasas iniciadoras.De los resultados obtenidos en la segunda parte del trabajo se deduce que los niveles de la isoforma larga del antagonista FAIM, FAIML, específica del sistema nervioso, aumentan durante el desarrollo embrionario. Su máxima expresión es en los períodos del desarrollo en los que se modelan y ajustan las estructuras neurales que darán lugar al cerebro adulto. Funcionalmente, hemos demostrado que no participa en procesos de neuritogénesis (a diferencia de la isoforma corta de FAIM, FAIMS) y que no bloquea la muerte apoptótica inducida a través de estímulos mitocondriales. Sin embargo, FAIML es capaz de antagonizar la apoptosis inducida a través de los DRs CD95 y TNFR1. Los ensayos >con RNA de interferencia nos han permitido elucidar que FAIML es, al menos en parte, responsable del bloqueo de la actividad de caspasas iniciadoras activadas por DRs.Por tanto, este trabajo aporta claves sobre las bases moleculares que regulan la actividad de los DRs en el sistema nervioso y que pueden constituir una base para el desarrollo de estrategias terapéuticas en neuropatologías en las que los DRs participan de forma relevante.Apoptosis is a physiological process by which the number of cells in metazoan organisms is regulated. Thus, cells with transient function, supranumerary cells or damaged ones are selectively eliminated by this process. Apoptosis is strictly regulated and recently it has been suggested that it could be involved in the pathogenesis of some nervous system diseases. In that sense, an excess of cell death could contribute to neurodegenerative disorders and, on the other hand, a defect could be one of the reasons for neoplasia development. The main regulator of apoptosis is the activation of caspases. These are cysteine-proteases which have cleavage specificity for aspartic residues. Caspases are activated by two main mechanisms: (1) release of citocrome C from altered mitochondria to the cytoplasm and (2) activation of membrane receptors called death receptors (DRs). These proteins have been widely characterized in the immune system, whereas in the nervous system their functions are at the initial stages of characterization.The present project is focussed on the characterization of two novel antagonists of the death receptor CD95/Fas/APO-1 which are specifically expressed in the nervous system, Lifeguard and FAIM. For this purpose PC12 and SH-SY5Y cell lines, widely used in models of differentiation and cell death, have been used along with primary cultures of cortical neurons and cerebellar granule neurons.Results obtained in the first part of this study allowed us to clone mouse Lifeguard, and describe its function as an antagonist of CD95 in the nervous system. We have demonstrated that its overexpression is able to block CD95 induced cell death in the human neuroblastoma cell line SH-SY5Y, and in mouse embryonic cortical neurons. Furthermore, reduction of Lifeguard endogenous levels sensitizes both, mouse granular and cortical neurons to CD95 induced apoptosis. In addition, it has been stated that the molecular mechanism of action of Lifeguard depends on its exclusive location in plasma membrane microdomains called Lipid Rafts, where it can interact with CD95 and inhibit the activation of initiator caspases.In the second part of this study, our results demonstrate that the expression of the long form of the CD95 antagonist FAIM, FAIML, specific of the nervous system, increases during the embryonic development. It reaches maximum levels in the periods of the development in which the neural structures are being defined, thus creating adult brain structures.Functionally, we have demonstrated that FAIML, in contrast to the short form of FAIM (FAIMS), does not participate in neurotrophic factor induced neurite outgrowth and it is not able to block apoptotic induced cell death through mitochondrial stimuli. Nevertheless, FAIML is able to antagonize the apoptosis induced through DRs CD95 and TNFR1. FAIML RNA interference efficiently reduced its endogenous levels and allowed us to conclude that FAIML is one of the molecules responsible for maintaining initiator caspases inactive upon receptor engagement.Therefore, this work will contribute to the understanding of the molecular basis for DRs activity regulation in the nervous system, and it could constitute a starting point for the development of new therapeutic strategies for DRs associated neuropathologies

Interplay Between ncRNAs and Cellular Communication: A Proposal for Understanding Cell-Specific Signaling Pathways

Cancer; Cell signaling; EpigeneticsCàncer; Comunicació cel·lular; EpigenèticaCáncer; Comunicación celular; EpigenéticaIntercellular communication is essential for the development of specialized cells, tissues, and organs and is critical in a variety of diseases including cancer. Current knowledge states that different cell types communicate by ligand-receptor interactions: hormones, growth factors, and cytokines are released into the extracellular space and act on receptors, which are often expressed in a cell-type-specific manner. Non-coding RNAs (ncRNAs) are emerging as newly identified communicating factors in both physiological and pathological states. This class of RNA encompasses microRNAs (miRNAs, well-studied post-transcriptional regulators of gene expression), long non-coding RNAs (lncRNAs) and other ncRNAs. lncRNAs are diverse in length, sequence, and structure (linear or circular), and their functions are described as transcriptional regulation, induction of epigenetic changes and even direct regulation of protein activity. They have also been reported to act as miRNA sponges, interacting with miRNA and modulating its availability to endogenous mRNA targets. Importantly, lncRNAs may have a cell-type-specific expression pattern. In this paper, we propose that lncRNA-miRNA interactions, analogous to receptor-ligand interactions, are responsible for cell-type-specific outcomes. Specific binding of miRNAs to lncRNAs may drive cell-type-specific signaling cascades and modulate biochemical feedback loops that ultimately determine cell identity and response to stress factors

A high-throughput screening identifies microRNA inhibitors that influence neuronal maintenance and/or response to oxidative stress

Oxidative stress; Small RNA sequencing; NeurodegenerationEstrés oxidativo; Secuenciación de ARN pequeño; NeurodegeneraciónEstrès oxidatiu; Seqüenciació d'ARN petit; NeurodegeneracióSmall non-coding RNAs (sncRNAs), including microRNAs (miRNAs) are important post-transcriptional gene expression regulators relevant in physiological and pathological processes. Here, we combined a high-throughput functional screening (HTFS) platform with a library of antisense oligonucleotides (ASOs) to systematically identify sncRNAs that affect neuronal cell survival in basal conditions and in response to oxidative stress (OS), a major hallmark in neurodegenerative diseases. We considered hits commonly detected by two statistical methods in three biological replicates. Forty-seven ASOs targeting miRNAs (miRNA-ASOs) consistently decreased cell viability under basal conditions. A total of 60 miRNA-ASOs worsened cell viability impairment mediated by OS, with 36.6% commonly affecting cell viability under basal conditions. In addition, 40 miRNA-ASOs significantly protected neuronal cells from OS. In agreement with cell viability impairment, damaging miRNA-ASOs specifically induced increased free radical biogenesis. miRNAs targeted by the detrimental ASOs are enriched in the fraction of miRNAs downregulated by OS, suggesting that the miRNA expression pattern after OS contributes to neuronal damage. The present HTFS highlighted potentially druggable sncRNAs. However, future studies are needed to define the pathways by which the identified ASOs regulate cell survival and OS response and to explore the potential of translating the current findings into clinical applications.This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER funds (SAF2014-60551-R and SAF2017-88452-R). We acknowledge the support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and the Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208). We acknowledge the support of the Spanish Ministry of Science Innovation and Universities, Maria Maeztu Unit of Excellence Programme. We thank the staff of the Genomics Unit for the preparation of sRNA libraries and sequencing and the staff of the Biomolecular Screening and Protein Technologies Unit for their help in the setting up the high-throughput screening

Engineering DNA-Grafted Quatsomes as Stable Nucleic Acid-Responsive Fluorescent Nanovesicles

Fluorescence; Nanovesicles; Responsive nanomaterialsFluorescencia; Nanovesículas; Nanomateriales sensiblesFluorescència; Nanovesícules; Nanomaterials sensiblesThe development of artificial vesicles into responsive architectures capable of sensing the biological environment and simultaneously signaling the presence of a specific target molecule is a key challenge in a range of biomedical applications from drug delivery to diagnostic tools. Herein, the rational design of biomimetic DNA-grafted quatsome (QS) nanovesicles capable of translating the binding of a target molecule to amphiphilic DNA probes into an optical output is presented. QSs are synthetic lipid-based nanovesicles able to confine multiple organic dyes at the nanoscale, resulting in ultra-bright soft materials with attractiveness for sensing applications. Dye-loaded QS nanovesicles of different composition and surface charge are grafted with fluorescent amphiphilic nucleic acid-based probes to produce programmable FRET-active nanovesicles that operate as highly sensitive signal transducers. The photophysical properties of the DNA-grafted nanovesicles are characterized and the highly selective, ratiometric detection of clinically relevant microRNAs with sensitivity in the low nanomolar range are demonstrated. The potential applications of responsive QS nanovesicles for biosensing applications but also as functional nanodevices for targeted biomedical applications is envisaged.This work was financially supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement “Nano-Oligo Med” (No 778133), Ministry of Science and Innovation (MINECO), Spain, through the “MOL4BIO” project (PID2019-105622RB-I00) and by Instituto de Salud Carlos III (DTS20/00018), Italian Ministry of University and Research (Project of National Interest, PRIN, 2017Y2PAB8_004 through the project “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”. M.R. was supported from a Fondazione Umberto Veronesi postdoctoral fellowship. Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centers of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Quatsome production and their physicochemical characterization has been performed by the Biomaterial Processing and Nanostructuring Unit (U6) of the ICTS “NANBIOSIS”, a unit of the CIBER network in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC)

Multi-Smart and Scalable Bioligands-Free Nanomedical Platform for Intratumorally Targeted Tambjamine Delivery, a Difficult to Administrate Highly Cytotoxic Drug

Nanocàpsules anfòteres; Tractament del càncer de pulmó; Sistemes de lliurament de medicaments dirigitsNanocápsulas anfóteras; Tratamiento del cáncer de pulmón; Sistemas de administración de fármacos dirigidosAmphoteric nanocapsules; Lung cancer treatment; Targeted drug delivery systemsCancer is one of the leading causes of mortality worldwide due, in part, to limited success of some current therapeutic approaches. The clinical potential of many promising drugs is restricted by their systemic toxicity and lack of selectivity towards cancer cells, leading to insufficient drug concentration at the tumor site. To overcome these hurdles, we developed a novel drug delivery system based on polyurea/polyurethane nanocapsules (NCs) showing pH-synchronized amphoteric properties that facilitate their accumulation and selectivity into acidic tissues, such as tumor microenvironment. We have demonstrated that the anticancer drug used in this study, a hydrophobic anionophore named T21, increases its cytotoxic activity in acidic conditions when nanoencapsulated, which correlates with a more efficient cellular internalization. A biodistribution assay performed in mice has shown that the NCs are able to reach the tumor and the observed systemic toxicity of the free drug is significantly reduced in vivo when nanoencapsulated. Additionally, T21 antitumor activity is preserved, accompanied by tumor mass reduction compared to control mice. Altogether, this work shows these NCs as a potential drug delivery system able to reach the tumor microenvironment, reducing the undesired systemic toxic effects. Moreover, these nanosystems are prepared under scalable methodologies and straightforward process, and provide tumor selectivity through a smart mechanism independent of targeting ligands.This research was funded by Consejería de Educación de la Junta de Castilla y León (BU092U16 and BU067P20), Instituto de Salud Carlos III (grants PI18/00441 and DTS20/00018), ACCIÓ (Agency for business competitiveness; Generalitat de Catalunya) (Nuclis d’R+D EMC/2755/2017); co-funded by the European Regional Development Fund (ERDF); Asociación Española Contra el Cáncer (LABAE18009SEGU), and supported by the “Pla de Doctorats Industrials de la Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya (grant number 2013 DI 028)

Krüppel-like factor 4 (KLF4) regulates the miR-183~96~182 cluster under physiologic and pathologic conditions

Cèl·lules mare embrionàries; Melanoma; MicroARNCélulas madre embrionarias; Melanoma; MicroARNEmbryonic stem cells; Melanoma; MicroRNAMicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that post-transcriptionally control the translation and stability of target mRNAs in a sequence-dependent manner. MiRNAs are essential for key cellular processes including proliferation, differentiation, cell death and metabolism, among others. Consequently, alterations of miRNA expression contribute to developmental defects and a myriad of diseases. The expression of miRNAs can be altered by several mechanisms including gene copy number alterations, aberrant DNA methylation, defects of the miRNA processing machinery or unscheduled expression of transcription factors. In this work, we sought to analyze the regulation of the miR-182 cluster, located at the 7q32 locus, which encodes three different miRNAs that are abundantly expressed in human embryonic stem cells and de-regulated in cancer. We have found that the Krüppel-like factor 4 (KLF4) directly regulates miR-182 cluster expression in human embryonic stem cells (hESCs) and in melanoma tumors, in which the miR-182 cluster is highly expressed and has a pro-metastatic role. Furthermore, higher KLF4 expression was found to be associated with metastatic progression and poor patient outcome. Loss of function experiments revealed that KLF4 is required for melanoma cell maintenance. These findings provide new insights into the regulation of the miR-182 cluster expression and new opportunities for therapeutic intervention in tumors in which the KLF4-miR-182 cluster axis is deregulated.This work was supported by NCI/NIH Grant (5R01CA155234), Instituto de Salud Carlos III (CP11/00052 and RD12/0036/0016) co-financed by the European Regional Development Fund (ERDF), and European Commission’s Framework Programme 7 through the Marie Curie Career Integration Grants

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics

Nanovesicles; Neuroblastoma; Pediatric cancerNanovesículas; Neuroblastoma; Cáncer pediátricoNanovesícules; Neuroblastoma; Càncer pediàtricMicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.The funding was received by Ministerio de Educación, Cultura y Deporte (Grant no. FPU16/01099), Ministerio de Economía, Industria y Competividad (Grants MAT2016-80820-R, MAT2016-80826-R and SAF2016-75241-R), the Ministry of Science and Innovation (MINECO) of Spain through grant PID2019-105622RB-I00, from Instituto de Salud Carlos III (Grant no. CP16/00006, PI17/00564, PI20/00530, DTS20/00018) (Co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”), from the EuroNanoMed II platform through the NanoVax project, from CIBER-BBN through grant TAG-SMARTLY, Joan Petit Foundation, Asociación Matem Lo Bitxo and Asociación Española Contra el Cáncer (Grant no. LABAE18009SEGU), as well as, Generalitat de Catalunya through the Centres de Recerca de Catalunya (CERCA) programme and grant no. 2017-SGR-918, and from Agency for Management of University and Research Grants (AGAUR) (Grant no 2018LLAV0064 and SIFECAT IU68-010017). Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S)

FAIM-L Is an IAP-Binding Protein That Inhibits XIAP Ubiquitinylation and Protects from Fas-Induced Apoptosis

The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.This work was funded by the Spanish Government Ministerio de Sanidad y Consumo (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, CB06/05/1104 to J.X.C.), Ministerio de Economía y Competitividad (SAF2010–19953 to J.X.C.; SAF2012–31485 to V.J.Y.), Instituto de Salud Carlos III (CP11/00052 to M.F.S.), and the Generalitat de Catalunya (Suport als Grups de Recerca Consolidats 2009SGR346). F.M.-F. and L.P.-F. are supported by postgraduate fellowships from the Spanish Government Ministerio de Educación y Ciencia. J.U. is supported by a postgraduate fellowship from the Generalitat de Catalunya. R.S.M. and V.J.Y. were under the Juan de la Cierva and the Ramon y Cajal programs, respectively, from the Ministerio de Educación y Ciencia (Spain), cofinanced by the European Social Fund. M.F.S. is under the Miguel Servet program from the Instituto de Salud Carlos III and cofinanced by the European Regional Development Fund

Loss of microRNA-135b Enhances Bone Metastasis in Prostate Cancer and Predicts Aggressiveness in Human Prostate Samples

Bone metastasis; MiRNAs; Prostate cancerMetàstasi òssia; MiRNAs; Càncer de pròstataMetástasis ósea; MiRNAs; Cáncer de próstataAbout 70% of advanced-stage prostate cancer (PCa) patients will experience bone metastasis, which severely affects patients’ quality of life and progresses to lethal PCa in most cases. Hence, understanding the molecular heterogeneity of PCa cell populations and the signaling pathways associated with bone tropism is crucial. For this purpose, we generated an animal model with high penetrance to metastasize to bone using an intracardiac percutaneous injection of PC3 cells to identify PCa metastasis-promoting factors. Using genomic high-throughput analysis we identified a miRNA signature involved in bone metastasis that also presents potential as a biomarker of PCa progression in human samples. In particular, the downregulation of miR-135b favored the incidence of bone metastases by significantly increasing PCa cells’ migratory capacity. Moreover, the PLAG1, JAKMIP2, PDGFA, and VTI1b target genes were identified as potential mediators of miR-135b’s role in the dissemination to bone. In this study, we provide a genomic signature involved in PCa bone growth, contributing to a better understanding of the mechanisms responsible for this process. In the future, our results could ultimately translate into promising new therapeutic targets for the treatment of lethal PCa.The study was supported by grants CPII18/00027 and PI18/01017 to A.S.; PI17/02248 to J.M.; grants PI09/00496, PI13/00173 and postdoctoral fellowship CD12/00475 of Instituto de Salud Carlos III (ISCIII), pre-doctoral fellowship of Vall d’Hebron Research Institute (VHIR), postdoctoral fellowship PERIS of Departament de Salut Govern de Catalunya to M.O.; BBVA and PID2019-104948RB-100 to M.G. (Marc Guiu) and R.R.G.; RD12/0036/0035 of Red Temática de investigación cooperativa en cancer (RTICC), and 2014SGR1330 from “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya”

Targeting the Hedgehog Pathway in Rhabdomyosarcoma

Embryonic pathways; Paediatric cancer; Soft tissue sarcomasVies embrionàries; Càncer pediàtric; Sarcomes de teixits tousVías embrionarias; Cáncer pediátrico; Sarcomas de tejidos blandosAberrant activation of the Hedgehog (Hh) signalling pathway is known to play an oncogenic role in a wide range of cancers; in the particular case of rhabdomyosarcoma, this pathway has been demonstrated to be an important player for both oncogenesis and cancer progression. In this review, after a brief description of the pathway and the characteristics of its molecular components, we describe, in detail, the main activation mechanisms that have been found in cancer, including ligand-dependent, ligand-independent and non-canonical activation. In this context, the most studied inhibitors, i.e., SMO inhibitors, have shown encouraging results for the treatment of basal cell carcinoma and medulloblastoma, both tumour types often associated with mutations that lead to the activation of the pathway. Conversely, SMO inhibitors have not fulfilled expectations in tumours—among them sarcomas—mostly associated with ligand-dependent Hh pathway activation. Despite the controversy existing regarding the results obtained with SMO inhibitors in these types of tumours, several compounds have been (or are currently being) evaluated in sarcoma patients. Finally, we discuss some of the reasons that could explain why, in some cases, encouraging preclinical data turned into disappointing results in the clinical setting.This article was funded by grants from: Institut Català d’Oncologia (ICO); Instituto de Salud Carlos III (PI18/00398 and FI18/00178); ACCIÓ (COMRDI15-1-0014); Fundació la Marató de TV3; Fundació Albert Bosch; Rotary Clubs Barcelona Eixample, Barcelona Diagonal, Santa Coloma de Gramanet, München-Blutenburg, Deutschland Gemeindienst e.V. and others from Barcelona and province; Fundation Amics Joan Petit; Del Hospital a la cathedral Initiative by Xavi Vallès; and Mi compañero de viaje Association