Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT06250517 |
| Other study ID # |
ExBioma |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
June 1, 2021 |
| Est. completion date |
June 2024 |
Study information
| Verified date |
January 2024 |
| Source |
Vall d'Hebron Institute Research |
| Contact |
Irene Bello Rodríguez, Professor |
| Phone |
+34620664172 |
| Email |
irene.bello.rodriguez[@]gmail.com |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
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.
Description:
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.
- Cytokine analysis The determination of cytokines in the perfusion fluid will be carried out
using immunoassays based on Luminex™ xMAP™ technology (multi-analyte profiling) that allow
the simultaneous quantification and detection of different secreted proteins (cytokines,
chemokines, growth factors, etc.) We will use panels designed specifically for the gene
products of interest.
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.
- Bioinformatic analysis of the sequences To obtain the microbial composition of each sample,
we will use the QIIME software. QIIME is a software pipeline that uses phylogenetic
information and multivariate statistical techniques to compare microbial communities and
determine, for example, whether they are statistically different. The program also identifies
the species that contribute the most to these differences and discovers patterns of various
types that characterize specific groups of samples.
