Impact of the Ex-vivo Pulmonary Perfusion System on the Microbiome of Lung Grafts and Their Inflammatory Reaction.
试验速览
- 阶段
- 不适用
- 状态
- 招募中
- 入组人数
- 7
- 试验地点
- 2
- 主要终点
- Analysing with quantitative PCR of the genetic expression of 84 genes related to immune response in lung transplantation after cold storage
研究概览
简要总结
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:
- P1. Detection: The donor without criteria to be a lung donor or rejected by all the transplant teams.
- P2. Extraction.
- P3. Cold preservation: The left lung will be cold-preserved
- P4. EVLP Conservation: The right lung will be prepared and conditioned for 3 hours using EVLP
The following samples will be taken at two times:
- T0: At the end of the extraction
- Bronchoalveolar lavage (BAL): Before tracheal clamping, BAL will be taken from the left main bronchus using bronchoscopy. The BAL will be performed on the right lung just before starting P4.
- Lung biopsy: Lung biopsy of the lower lobe of both grafts will be performed
- Preservation liquid or Perfusion liquid: 20 mL of preservation liquid that is in contact with the left graft before storage, as a sterility control (P3) and 20 mL of perfusion liquid before conditioning, as a sterility control (P4).
- T1: At the end of the conservation protocols (P3 or P4).
- B.A.L.
- Lung biopsy: left lower lobe.
- Preservation liquid or Infusion liquid: 20 mL of preservation liquid that is in contact with the left graft or 20 mL of perfusion fluid.
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.
详细描述
SAMPLE TAKING AND STORAGE
- BAL:
- Technique: The bronchoscope will be inserted in segment 6, aliquots of sterile saline will be instilled. The procedure will be repeated in segments 3 and 5, in the case of the right lung, and in segment 6 and lingula in the left lung. 20-30cc of BAL will be completed for each graft.
- Storage: Samples will be frozen immediately after collection and transferred to the laboratory with a cold accumulator to ensure that they do not thaw.
- Analysis: The lung microbiome will be analysed.
- Lung biopsy:
- Technique: Lung biopsy of a minimum of 2cm3 will be taken using non-absorbable automatic suture.
- Storage: Samples will be preserved in RNA Stabilization solution Reagent (QIAGEN), which allows specimens to be kept at room temperature until frozen, avoiding RNA degradation.
- Analysis: RNA sequencing of transcription of inflammatory signals.
- Ex vivo perfusion liquid solution:
- Technique: Taking samples sterile with a 20cc syringe.
- Storage: Samples will be frozen immediately after collection and transferred to the laboratory with a cold accumulator to ensure that they do not thaw.
- Analysis: Microbiome analysis
- Preservation liquid solution:
- Technique: Taking samples sterile with a 20cc syringe.
- Storage: Samples will be frozen immediately after collection and transferred to the laboratory with a cold accumulator to ensure that they do not thaw.
- Analysis: Microbiome and cytokine analysis
All samples will be stored in a freezer at -80
- Microbiome analysis For the analysis of microbial diversity, the V4 variable region of the 16S gene will be amplified from bacterial DNA by PCR. Amplicons will be sequenced using Illumina (MiSeq) technology. Samples will be processed and more than 10,000 300bp sequences per sequence per sample will be generated.
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.
研究设计
- 研究类型
- Interventional
- 分配方式
- Non Randomized
- 干预模型
- Parallel
- 主要目的
- Treatment
- 盲法
- None
入排标准
- 年龄范围
- 18 Years 至 100 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •brain-death donors and donors after cardio circulatory death rejected for lung transplantation
排除标准
- •Unilateral pneumonia
- •Lack of consent for the donor's family.
结局指标
主要结局
Analysing with quantitative PCR of the genetic expression of 84 genes related to immune response in lung transplantation after cold storage
时间窗: After 3 hours of cold storage
Analyse with quantitative PCR of the genetic expression of 84 genes related with immune response in lung transplantation: 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 in lung samples before and after the cold storage, comparing them
Analyse the concentration of inflammatory cytokines after exvivo lung perfusion
时间窗: After 3 hours of perfusion in ex vivo lung perfusion
Analyse the concentration of inflammatory cytokines: 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, with Luminex xMAP technique, in perfusion solution before and after the use of exvivo lung perfusion and comparing them
Change in individual operational taxonomic units (OTUs) in lung parenchyma after cold storage
时间窗: After 3 hours of cold storage
Analyse the lung microbiome and alpha diversity before and after the cold storage and compare the micrological samples.
Change in individual operational taxonomic units (OTUs) in lung parenchyma after the exvivo lung perfusion during 3 hours
时间窗: After 3 hours of perfusion in ex vivo lung perfusion
Analyse the lung microbiome and alpha diversity before the use of exvivo lung perfusion and after compared the micrological samples
Analysing with quantitative PCR of the genetic expression of 84 genes related to immune response in lung transplantation after the lung perfusion during 3h in exvivo lung perfusion system.
时间窗: After 3 hours of perfusion in ex vivo lung perfusion
Analyse with quantitative PCR of the genetic expression of 84 genes related with immune response in lung transplantation: 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 in lung samples before the use of exvivo lung perfusion and after, comparing them
Analysing the concentration of inflammatory citokines after cold storage
时间窗: After 3 hours of cold storage
Analyse the concentration of inflammatory cytokines: 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, with Luminex xMAP technique, in perfusion solution before and after the cold storage and comparing them
次要结局
未报告次要终点
研究者
Irene Bello
Professor
Vall d'Hebron Institute Research