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, the nephrologist (A.S.) who reviewed the biopsies specimens were blinded to clinical and immunological information
, Renal allograft lesions were graded according to the Banff classification, 2011.
, anti-CD68 (macrophages) and anti-66b (granulocytes) were performed by immunochemistry on paraffin embedded sections using an anti-human CD34 (clone QBEnd10, Double stainings with anti-CD34 (endothelial cells) and respectively one antibody among anti-CD56 (NK cells), anti-CD3 (T cells)
The algorithm allows for precise quantification of inflammatory cells in the renal allograft. The quantification of inflammatory cells in mvi+DSA-patients' biopsies was compared to the one of mvi+DSA+C3d-patients' biopsies ,
, Detection of anti-HLA antibodies
, Donor-recipient HLA typing were performed by PCR-SSO reverse (One Lambda
, Serum samples banked at the time of biopsy from patients with significant microvascular inflammation were tested for the presence of donor specific anti-HLA antibodies using Screening Flow Beads
, the Etablissement Français du Sang
Two primary cell lines of coronary endothelial cells (#407 and 408) (Promocell, fact mvi+DSA-graft survival was similar to the one of patients diagnosed with conventional antibody-mediated rejection due to non-complement binding DSA, 2012. ,
, Mvi+DSA-patients and mvi-DSA-patients displayed similar characteristics (Table 2), especially regarding the number of HLA mismatches, except for microvascular inflammation, vol.0001, p.0
, Mvi+DSA-patients and mvi+DSA+C3d-had also similar baseline characteristics, including regarding the severity of histological lesions
, PBMCs were cultured overnight at 37°C in 5% CO2 in complete culture medium (RPMI 1640 containing glutamine and supplemented with 10 % FBS, hepes and penicillin-streptomycin) supplemented with 500
, UI/ml recombinant human IL-2 (R&Dsystems) or were maintained at 4°C in complete culture medium
, Simultaneously, endothelial cells were seeded (100 000 in each well) in wells of a flat bottom 96-well plate coated with gelatin 1% (Sigma)
, Then, purified NK cells were resuspended at 0.5 millions/ml in complete RPMI. One hundred thousand NK cells were added in each well containing endothelial cells after removing endothelial cell culture medium. Five microliter of anti-human CD 107a FITC (eBIOH4A3, ebioscience) was added in each well at the beginning of the co-culture. One hour after the beginning of the co-culture, golgi stop (DB biosciences) was added in each well. Then cells were co-cultured for 3 hours. After the co-culture, cells were detached with trypsin and recovered in V bottom-96-well plates, Then cells were stained 20 min at room temperature in 50 µl of the following antibodies diluted in PBS1x: CD3 APC-H7 (SK7) BD biosciences, vol.1, p.25
, CD56 PE CF594 (NCAM16.2) BD biosciences, vol.1, p.25
, KIR2DL3 APC (180701) R&Dsystems 1/10e
, KIR3DL1 BV 711 (DX9) BD bisociences 1/25e
, CD 107a FITC (eBIOH4A3) ebiosciences 1/50e
, Fixable viability dye eFluor 506 ebiosciences 1/1000e
, Without washing, the following antibodies were added in 50 µl in PBS1x for an incubation of 15 minutes at room temperature: KIR2DL1/S1 PE CY7 (EB6B) Beckman Coulter, vol.1, p.25
, KIR2DL2-3/S2 PE CY5.5 (GL183) Beckman Coulter, vol.1, p.25
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