We studied the clinical, histologic, and molecular features distinguishing DSA-negative from DSA-positive molecularly defined antibody-mediated rejection (mABMR). We analyzed mABMR biopsies with available DSA assessments from the INTERCOMEX study: 148 DSA-negative versus 248 DSA-positive, compared with 864 no rejection (excluding TCMR and Mixed). DSA-positivity varied with mABMR stage: early-stage (EABMR) 56%; fully developed (FABMR) 70%; and late-stage (LABMR) 58%. DSA-negative patients with mABMR were usually sensitized, 60% being HLA antibody-positive. Compared with DSA-positive mABMR, DSA-negative mABMR was more often C4d-negative; earlier by 1.5 years (average 2.4 vs. 3.9 years); and had lower ABMR activity and earlier stage in molecular and histology features. However, the top ABMR-associated transcripts were identical in DSA-negative versus DSA-positive mABMR, for example, NK-associated (e.g., KLRD1 and GZMB) and IFNG-inducible (e.g., PLA1A). Genome-wide class comparison between DSA-negative and DSA-positive mABMR showed no significant differences in transcript expression except those related to lower intensity and earlier time of DSA-negative ABMR. Three-year graft loss in DSA-negative mABMR was the same as DSA-positive mABMR, even after adjusting for ABMR stage. Thus, compared with DSA-positive mABMR, DSA-negative mABMR is on average earlier, less active, and more often C4d-negative but has similar graft loss, and genome-wide analysis suggests that it involves the same mechanisms. SUMMARY SENTENCE: In 398 kidney transplant biopsies with molecular antibody-mediated rejection, the 150 DSA-negative cases are earlier, less intense, and mostly C4d-negative, but use identical molecular mechanisms and have the same risk of graft loss as the 248 DSA-positive cases.

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory condition affecting the colonic epithelium, with potential roles for the inflammasome, complement activation, T cells, and the microbiome in pathogenesis. We applied an established method of microarray-based gene expression analysis to a set of 128 UC biopsies (from 113 patients), to elucidate the molecular changes associated with active UC. Aims Our aim was to describe the molecules most associated with UC disease activity (the endoscopic Mayo score) and to annotate these molecules into biological processes. Methods 128 UC colonic biopsies were collected at the University of Alberta Hospital (Edmonton, Alberta) and Cedars-Sinai Hospital (Los Angeles, California) during standard of care colonoscopy. Biopsies were processed using Affymetrix microarrays. Gene expression data from the population was visualized using volcano plots (showing fold change and association between genes and endoscopic Mayo score), and heatmaps (showing expression of the top 30 genes in a previously established cell panel). Overexpression of top genes was analyzed using Gene Ontology and KEGG pathways. Results The volcano plot (Figure 1A) showed strong associations between the endoscopic Mayo score and components of innate immunity, e.g. complement factor B (CFB), C1-inhibitor (also known as SERPING1), chitinase 3-like 1 (CHI3L1), and inflammasome genes (ZBP1 and PIM2). Moderate associations with calprotectin (S100A8 and S100A9), other inflammasome components (CASP1 and NLRP3), and T cell transcripts (i.e. CTLA4, PDL1) were observed. Targets of biologic therapy (TNFA, ITGA4/B7, IL12B) were weakly associated with the endoscopic Mayo score. Expression of the top genes in a cell panel (Figure 1B) showed primary expression in monocytes, macrophages, dendritic cells, and polymorphonucleocytes, with some expression in colon epithelial and endothelial cells. Minimal expression was found in CD4/CD8 T cells or NK cells. Pathway analysis represented extracellular matrix remodeling, complement regulation, and TNFA signaling, but revealed no pathways associated with adaptive immunity (Table 1). Conclusions UC disease activity, as assessed by the endoscopic Mayo score, was strongly associated with tissue remodeling and molecules of innate immunity that were largely found in myeloid cells, colon epithelium, and endothelium. Cognate T cells were not dominant features of UC disease activity. These data suggest that the driver of ongoing UC activity is independent of the cognate T cell response. Funding Agencies None

Transplanted lungs suffer worse outcomes than other organ transplants with many developing chronic lung allograft dysfunction (CLAD), diagnosed by physiologic changes. Histology of transbronchial biopsies (TBB) yields little insight, and the molecular basis of CLAD is not defined. We hypothesized that gene expression in TBBs would reveal the nature of CLAD and distinguish CLAD from changes due simply to time posttransplant. Whole-genome mRNA profiling was performed with microarrays in 498 prospectively collected TBBs from the INTERLUNG study, 90 diagnosed as CLAD. Time was associated with increased expression of inflammation genes, for example, CD1E and immunoglobulins. After correcting for time, CLAD manifested not as inflammation but as parenchymal response-to-wounding, with increased expression of genes such as HIF1A, SERPINE2, and IGF1 that are increased in many injury and disease states and cancers, associated with development, angiogenesis, and epithelial response-to-wounding in pathway analysis. Fibrillar collagen genes were increased in CLAD, indicating matrix changes, and normal transcripts were decreased-dedifferentiation. Gene-based classifiers predicted CLAD with AUC 0.70 (no time-correction) and 0.87 (time-corrected). CLAD related gene sets and classifiers were strongly prognostic for graft failure and correlated with CLAD stage. Thus, in TBBs, molecular changes indicate that CLAD primarily reflects severe parenchymal injury-induced changes and dedifferentiation.

Introduction: Ulcerative colitis (UC) is a chronic inflammatory condition affecting the colonic epithelium. We used an established microarray-based system to analyze a set of 128 UC biopsies (115 patients), assessing gene expression associated with the colon’s response to injury in UC. Methods: 128 UC colon biopsies were collected at the University of Alberta Hospital (Edmonton, AB) and Cedars-Sinai Hospital (Los Angeles, CA) during standard of care colonoscopy and processed using Affymetrix microarrays. Principal component analysis (PCA) and archetypal analysis (AA) visualized relationships between biopsies and previously annotated injury-associated transcript sets. AA assigned each biopsy to one of three groups, and scores to each biopsy relating it to all three groups. Results: Spearman correlations (Table 1) were highest between the endoscopic Mayo score and the injury-repair-associated transcripts (IRRAT, 0.64, P=4.7x10-16), immunoglobulin transcripts highly associated with chronic injury and fibrosis (IGT, 0.63, P=3.0x10-15), endothelial transcripts (ENDAT, 0.61, P=1.8x10-14), and parenchymal dedifferentiation i.e. epithelial solute carrier loss (CT2, -0.60, P=6.5x10-14). PCA separated injury from no injury in PC1 (Figure 1A). T cell transcripts (QCATs), interferon-gamma inducible transcripts (GRITs) and targets of biologics (IL12, TNFA, ITGA4/B7) separated from injury transcripts in PC2. We assigned three AA groups and visualized biopsies in PCA (Figure 1B, colored by AA membership). Group 1 (grey, N=44) biopsies had little parenchymal dedifferentiation and low expression of injury-associated transcripts. Groups 2 (red, N=44) and 3 (blue, N=40) had increased expression of injury-associated transcript sets and dedifferentiation compared to Group 1 (Table 2). Although Group 3 was endoscopically similar to Group 1 (P >0.05), Group 3 showed elevated injury-associated transcript set expression (e.g. IRRAT) and increased parenchymal dedifferentiation (CT2). Conclusion: Assessment of UC biopsies using AA and previously annotated injury-associated gene sets reveals two groups of biopsies that are endoscopically similar though one group has increased molecular abnormalities, thus revealing heterogeneity unrelated to the Mayo score. A molecular system based around PCA and AA could enhance and refine UC disease assessment by allowing for quantitation and qualification of injury in biopsies obtained at endoscopy i.e. a level of resolution beyond conventional endoscopic scoring.Table 1.: Transcript set correlation with Endoscopic Mayo Score (0-3) in ulcerative colitis biopsies (N=128).Table 2.: Mean transcript set scores (expression) and mean clinical scores/counts in each of the three Archetype Groups.Figure 1.: (A) PCA factor analysis of injury and inflammation transcripts in UC biopsies, (B) Three AA groups of 128 UC biopsies visualized in PCA space.