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It is known that the interactions of the graft and recipient microbiome are capable of modulating immune responses, inducing resilience or exacerbation of various inflammatory or fibrotic processes, therefore variations in the lung microbiome are associated with immunological changes in the transplanted lung.
The main objective is to understand the impact of new systems for conditioning and improving suboptimal lung grafts with ex vivo perfusion(EVLP) on the lung microbiome and its association with tissue inflammation.
The hypothesis is that manipulation of lung grafts and perfusion with broad-spectrum antibiotics during EVLP conditioning changes the lung microbiome, conditioning a less pro-inflammatory environment.
The methodology: This is a single-center prospective observational study. 7 consecutive brain-dead donors who do not meet the criteria to be lung donors will be included in the study. They will be carried out:
The following samples will be taken at two times:
Due to the manipulation of the grafts during extraction and use of the technique, which involves extubating the donor and subsequently intubated again the grafts, as well as perfusion for a minimum of 3 hours with antibiotics, the use of EVLP could alter the microbiome of the grafts. This alteration could impact the obtaining of viable organs for transplant, in the immediate postoperative period as well as in the long-term results. There are no studies that analyse the change in the microbiome after conditioning with EVLP or its relationship with inflammatory parameters.
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SAMPLE TAKING AND STORAGE
All samples will be stored in a freezer at -80
Gene expression analysis RNA extraction will be performed using the commercial RNeasy Mini Kit (QIAGEN). Gene expression analysis will be performed by quantitative PCR (qPCR) using the predesigned TransplantRejection panel from SignArray (AnyGenes®, Paris, France) that includes 84 genes that have been described to be related to the immune response in transplant rejection. These are: Genes included in the panel: CX3CR1, ICAM1, ITGA2, ITGAE, ITGAM, PECAM1, THBS1, THBS2, VCAM1, COL1A2, CCR5, CCR7, CD40, CD40LG, CD80, CD86, CTLA4, CXCR3, STAT4, TGFB1, CD44 , CTGF, MMP1, MMP2, MMP7, MMP9, BMP7, CCL11, CCL2, CCL3, CCL4, CCL5, CSF2, CXCL10, IFNG, IL10, IL12A, IL13, IL16, IL1B, IL2, IL2RA, IL3, IL32, IL4, IL5 , IL6, IL8, TNF, TGFB2, TGFB3, TIMP1, VEGFA, MS4A1, CXCL11, CXCL9, CXCR4, ADAM17, C3, CASP1, CASP3, CASP8, CCR2, CCR3, CD14, CD28, CD8A, FAS, FASLG, FCGR1A, GZMA , GZMB, NFKB1, NOS2, PRF1, PSMB9, STAT1, STAT6, TAP1, TLR3, TLR4, TLR9, TNFAIP3, TNFSF10.
Cytokine levels are measured using an immunoassay based on Luminex™ xMAP™ technology that allows for multi parametric analysis of the different cytokines. To this end, a personalized cytokine panel is designed based on the published literature on the effect of statins on the production of cytokines and other proinflammatory chemokines at a systemic level, as well as bibliographic evidence on the cytokines involved in lung transplantation. The cytokines analysed in the panel designed for this purpose are IFN gamma, IL-1 beta, IL-6, IL-8 (CXCL8), IL-18, IP-10 (CXCL10), MCP-1 (CCL2), MIP- 1 alpha (CCL3), TNF alpha, VEGF-D (Custom Procartaplex Multiplex Panel, Invitrogen, ThermoFisher Scientific, MA, USA). The immunological analysis will be carried out using the Invitrogen ProcartaPlex Analyst 1.0 software, supplied with the reagents.
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7 participants in 2 patient groups
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Alberto Sandiumenge Camps, Professor; Irene Bello Rodríguez, Professor
Data sourced from clinicaltrials.gov
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