Materials and methods
This case-control study was conducted according to the Declaration of Helsinki and the Good Clinical Practice guidelines. The study was approved by the local ethical committee (trial number: NL72704.078.20).
Five patients that developed SRF, HRS, and diffuse RPE atrophy after uncomplicated RPE-choroid transplantation were included at the REH (Rotterdam, the Netherlands), or OSC (Negrar (Verona), Italy). Considering that presence of auto-antibodies has been described previously in AMD patients and even after laser photocoagulation [8, 9] four control groups were included each consisting of five patients: 1) patients that underwent uncomplicated RPE-choroid transplantation, 2) patients that underwent uncomplicated FMT, 3) AMD patients treated with anti-VEGF injections, and 4) healthy age-matched controls. Exclusion criteria were a history of uveitis, diabetic retinopathy, and a systemic autoimmune disease.
Retrospectively, the following data were collected from medical files of the included patients with the inflammatory reaction: demographics, ophthalmic history, general history, indication for surgery, intra- and postoperative complications, and best-corrected visual acuity (BCVA) before surgery, after surgery and at last visit. Spectral Domain – optical coherence tomography (SD-OCT) images, fundus photography, fluoresceine angiography images and fundus autofluorescence (FAF) were collected. From the subjects of the control groups, demographics, ocular history and general history were reported. After informed consent was obtained, peripheral blood was collected providing both serum, and mononuclear cell samples for laboratory tests. The samples were stored at a temperature of − 80 °C until analyses.
In one patient the RPE-choroid graft was explanted because of a recurrent submacular hemorrhage and histopathologic examination was performed. The choroidal explant tissue was fixated in neutral buffered-formalin 10% and after processing, the formalin fixed paraffin embedded block was cut at 4 μm sections which were mounted on slides for histochemical and immunohistochemical staining. Routine staining protocols were used for standard haematoxylin and eosin staining using an automated staining system (HE600, Ventana Medical Systems, Tucsen, AZ, USA). Immunohistochemistry was performed with an automated, validated and accredited staining system (Ventana Benchmark ULTRA, Ventana Medical Systems, Tucsen, AZ, USA) using ultraview universal DAB detection kit. In brief, following deparaffinisation and heat-induced antigen retrieval, the tissue samples were incubated according to their optimized time with the antibody of interest. Incubation was followed by haematoxylin II counter stain for 8 minutes and then a blue coloring reagent for 8 minutes according to the manufacturer’s instructions (Ventana). Tonsil tissue was used as a positive control for all antibodies.
Serum anti-RPE-choroid antibodies and anti-retinal antibodies were determined as described previously, with slight optimization of the protocol . In short, biochip primate retinal slides (Euroimmun, Lübeck, Germany) were incubated with patients’ serum for 30 minutes at room temperature (RT) (dilution 1:100). After incubation, the slides were washed with phosphate-buffered saline (PBS) for 15 minutes and incubated with goat anti-human IgG Cy5 (ab97172; Abcam, Cambridge, UK) for 30 minutes at RT. Tissue slides were washed again with PBS for 15 minutes, embedded in glycerol and covered with a coverslip. Slides were analysed and photographed using an Axioplan2 fluorescence microscope with AxioCam MRm at 160x magnification and an exposure time of 6 seconds (Zeiss, Thornwood, NY, USA). In case of a weak result the procedure was repeated with a serum dilution 1:50 and 1:25. PBS and serum negative for anti-nuclear antibodies (ANA) were used as negative references. Recombinant rabbit anti-RPE65 antibody (ab231782; dilution 1:250; Abcam, Cambridge, UK), recombinant anti-collagen type 1 (ab138492; dilution 1:500; Abcam, Cambridge, UK), and serum of an anti-retinal antibody positive, and ANA-positive subject (dilution 1:100) were used as positive references.
Western blot protein extracts
Human RPE and choroid tissue extracts for Western blot were prepared as described previously, implementing slight changes in the protocol . RPE and choroid were obtained from a healthy donor eye post-mortem. After careful removal of the retina, RPE and choroid were dissected and subsequently homogenized in RIPA buffer with Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, USA) for 30 minutes. After homogenization the samples were centrifuged at 17000G at 4 °C for 15 minutes, the supernatants were separated, and protein yield was assessed by colorimetric Bradford assay. In addition to the human donor RPE-choroid protein extract, an RPE cell line was used to provide an RPE protein extract . ARPE-19 cells were cultured in culture flasks in DME/F (HyClone, Logan, UT) containing 10% fetal calf serum, and 1% penicillin/streptomycin. Two times (72 h and 24 h before the harvest) 50 μg/ml ascorbic acid and 200 μM L-Proline were added to the cell culture, and thrombin (5 U/ml) was added 48 h before the harvest. Confluent grown ARPE19 cells were harvested and subsequently homogenized in cold RIPA buffer with Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, USA). After homogenization, the samples were centrifuged at 17000G at 4 °C for 15 minutes, the supernatants were separated, and protein yield was assessed by colorimetric Bradford assay. Extracts were stored at a temperature of − 80 °C until further use.
Western blot procedure
Protein extract were diluted in RIPA buffer, with 4x Laemmli sample buffer (Bio-rad, Hercules, California, USA), and incubated at 95 °C for 5 minutes. The lysate was loaded on 4–12% mini-PROTEAN TGX Stain-Free Precast Gels (Bio-rad, Hercules, California, USA), and transferred to nitrocellulose membranes. Membranes were blocked with trix-buffered saline (TBS) containing 3% bovine serum albumine (BSA), and subsequently incubated with diluted samples (serum 1:100 TBS-Tween 0,1% containing 3% BSA, other antibodies as mentioned) for 1 hour at RT. Membranes were washed multiple times using an automated laboratory workstation (auto-LIA 48; Fujirebio, Japan), and incubated with goat anti-rabbit IgG (IRDye 680; dilution 1:4000; Licor, Lincoln, NE, USA), or goat anti-Human IgG (IRDye 680; dilution 1:15000; Licor, Lincoln, NE, USA) for 30 minutes. Visualization was performed with the imaging system Odyssey CLx (Thermo Fisher Scientific, Waltham, USA). Recombinant rabbit anti-RPE65 antibody, anti-recoverin antibody, and recombinant anti-collagen type I antibody were used as references.
Human leukocyte antigen typing
DNA was collected from whole blood using a QIAamp DNA Blood Mini kit, or using PAXgene blood tubes and a PAXgene Blood DNA Kit (Qiagen, Hilden, Germany), following manufacturer protocol. HLA class I and II typing was performed on all samples except for the five healthy participants. The procedure was performed as described by van Sonderen et al.  The HLA type of the study subjects was compared to the frequency in 5604 Dutch healthy blood donors .
Because of the small number of patients, and the explorative nature of the study, no statistical analysis was performed. The data have been evaluated descriptively.
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