RAGE gene expression of RAW 264.7 macrophages and BMDM.

<p>A) Agarose gel electrophoresis of RT-PCR products indicates RAGE band at expected band size of 350 bp and β-actin as reference gene with band size 500 bp. Mouse lung sample used as positive control (+ve), with negative control (-ve) containing water in place of cDNA. B) Western blot for RAGE showing predicted 50 KDa band for RAW264.7 macrophages and BMDM and for positive control (+ve) mouse lung.</p

Treatment of macrophages with S100B does not reduce cell viability as indicated by cell morphology and acid phosphatase assay.

<p>Images to show BMDM untreated (A) and treated with 2 μM S100B for 24 h (B) and RAW 264.7 untreated (C) and treated with 2 μM S100B for 24 h (D). Scale bars = 50 μM. Acid phosphatase cell viability assay on RAW 264.7 untreated or treated with 2μM or 5 μM S100B for 24 h (E) and on BMDM (F). No significant difference in viability following treatment with 2μM S100B for 24 h. Triton (0.1%) and DMSO (10%) as positive controls for the assay. Error bars indicate SEM n = 6.</p

S100B treatment of macrophages results in increased pro-IL1β expression.

<p>Representative dot plots showing flow cytometric analysis of RAW 264.7 cells using anti-CD11b (PerCP-Cy 5.5) to gate macrophages and anti-pro-IL1β (PE) to detect intracellular staining in untreated cells (<b>A</b>) and in cells treated with 2μM S100B (<b>B</b>). Significant increase in the percentage of CD11b positive cells expressing pro-IL-1β in response to S100B is shown graphically in both RAW 264.7 macrophage cells (*P<0.05) (<b>C</b>) and BMDM (***P<0.001) (<b>D</b>). Error bars indicate SEM n = 4.</p

CCL22 production by macrophages in response to S100B treatment for 24 h as determined by ELISA on supernatants.

<p>CCL22 production by RAW 264.7 cells (A) and BMDM cells (B). There was a significant increase in CCL22 with 1 μM S100B and 2 μM S100B in RAW 264.7 macrophage cells and with 2 μM in BMDM cells compared to untreated control cultures. Representative of 3 individual experiments. *P<0.05, **P<0.001 (ANOVA). Error bars indicate SEM n = 3.</p

Increase in S100B was detected in retinal sections from WT mice with EAU compared to untreated mice using immunohistochemistry.

<p><b>(A)</b> Positive staining was observed in the naïve retina, specifically in the outer plexiform layer (a), inner plexiform layer (b) and retinal ganglion layer (c). Increased staining was observed in EAU diseased sections <b>(B)</b> in the retinal ganglion layer (d) and in the rod outer segments where positive staining was located around infiltrating cells (e). No positive staining was observed in control sections, where S100B antibody was replaced with PBS in naïve retina <b>(C)</b> or in EAU diseased retina <b>(D).</b> Serial sections from naïve and diseased mice taken at spaced intervals throughout the eye.</p

EAU TEFI grading in C57BL/6 and S100B KO mice at day 15 and day 21 pi.

<p><b>A</b>, vascular cuffing (a) indicating inflammation in C57BL/6 day 15 pi. Severe inflammation in C57BL/6 at day 21 pi, with swollen, barely visible optic disk (b), cell infiltrates (c), and progression of vascular cuffing. In comparison, S100B KO images showing reduced disease but evidence of inflammation. Optic disk appears swollen day 21 pi (d) with vascular cuffing occurring on both sides of vessels (e). Approximate diameter of eye 3.3 mm. <b>B</b>, TEFI clinical grading scores for EAU at day 15 pi and <b>C,</b> day 21 pi indicating significantly reduced disease in S100B KO mice (*<i>P</i><0.05). TEFI scores for individual eyes from the same mouse were averaged and each point represents one animal. Results from two independent experiments were combined.(C57BL/6 n = 18 mice S100B KO n = 22 mice). Error bars indicate SEM.</p

Confirmation of increased CCL22 and IL1β expression in RAW 264.7 macrophages in response to increasing dose of S100B.

<p>Relative fold increase in mRNA expression for CCL22 (A) and IL-1β (B) determined by real-time PCR analysis. Error bars indicate SEM. n = 6. * <i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i>≤0.001 (ANOVA).</p

PCR array for inflammatory cytokines, chemokines and receptors on RAW 264.7 macrophages treated with or without S100B (2 μM for 6 h).

<p>Graph shows relative fold change in response to S100B for those genes with >2 fold change after S100B treatment (2^-ΔΔCT). Fold regulation normalised to GAPDH. Mean of two individual experiments each with 3 pooled samples.</p

Image_5_The impact of pre-transplant donor specific antibodies on the outcome of kidney transplantation – Data from the Swiss transplant cohort study.jpeg

BackgroundPre-transplant donor specific antibodies (DSA), directed at non-self human leukocyte antigen (HLA) protein variants present in the donor organ, have been associated with worse outcomes in kidney transplantation. The impact of the mean fluorescence intensity (MFI) and the target HLA antigen of the detected DSA has, however, not been conclusively studied in a large cohort with a complete virtual cross-match (vXM).MethodsWe investigated the effect of pre-transplant DSA on the risk of antibody-mediated rejection (ABMR), graft loss, and the rate of eGFR decline in 411 DSA positive transplants and 1804 DSA negative controls.ResultsPre-transplant DSA were associated with a significantly increased risk of ABMR, graft loss, and accelerated eGFR decline. DSA directed at Class I and Class II HLA antigens were strongly associated with increased risk of ABMR, but only DSA directed at Class II associated with graft loss. DSA MFI markedly affected outcome, and Class II DSA were associated with ABMR already at 500-1000 MFI, whereas Class I DSA did not affect outcome at similar low MFI values. Furthermore, isolated DSA against HLA-DP carried comparable risks for ABMR, accelerated eGFR decline, and graft loss as DSA against HLA-DR.ConclusionOur results have important implications for the construction and optimization of vXM algorithms used within organ allocation systems. Our data suggest that both the HLA antigen target of the detected DSA as well as the cumulative MFI should be considered and that different MFI cut-offs could be considered for Class I and Class II directed DSA.</p

Table_3_The impact of pre-transplant donor specific antibodies on the outcome of kidney transplantation – Data from the Swiss transplant cohort study.docx

BackgroundPre-transplant donor specific antibodies (DSA), directed at non-self human leukocyte antigen (HLA) protein variants present in the donor organ, have been associated with worse outcomes in kidney transplantation. The impact of the mean fluorescence intensity (MFI) and the target HLA antigen of the detected DSA has, however, not been conclusively studied in a large cohort with a complete virtual cross-match (vXM).MethodsWe investigated the effect of pre-transplant DSA on the risk of antibody-mediated rejection (ABMR), graft loss, and the rate of eGFR decline in 411 DSA positive transplants and 1804 DSA negative controls.ResultsPre-transplant DSA were associated with a significantly increased risk of ABMR, graft loss, and accelerated eGFR decline. DSA directed at Class I and Class II HLA antigens were strongly associated with increased risk of ABMR, but only DSA directed at Class II associated with graft loss. DSA MFI markedly affected outcome, and Class II DSA were associated with ABMR already at 500-1000 MFI, whereas Class I DSA did not affect outcome at similar low MFI values. Furthermore, isolated DSA against HLA-DP carried comparable risks for ABMR, accelerated eGFR decline, and graft loss as DSA against HLA-DR.ConclusionOur results have important implications for the construction and optimization of vXM algorithms used within organ allocation systems. Our data suggest that both the HLA antigen target of the detected DSA as well as the cumulative MFI should be considered and that different MFI cut-offs could be considered for Class I and Class II directed DSA.</p