Incident heart failure in chronic kidney disease: proteomics informs biology and risk stratification.

BACKGROUND AND AIMS: Incident heart failure (HF) among individuals with chronic kidney disease (CKD) incurs hospitalizations that burden patients and health care systems. There are few preventative therapies, and the Pooled Cohort equations to Prevent Heart Failure (PCP-HF) perform poorly in the setting of CKD. New drug targets and better risk stratification are urgently needed. METHODS: In this analysis of incident HF, SomaScan V4.0 (4638 proteins) was analysed in 2906 participants of the Chronic Renal Insufficiency Cohort (CRIC) with validation in the Atherosclerosis Risk in Communities (ARIC) study. The primary outcome was 14-year incident HF (390 events); secondary outcomes included 4-year HF (183 events), HF with reduced ejection fraction (137 events), and HF with preserved ejection fraction (165 events). Mendelian randomization and Gene Ontology were applied to examine causality and pathways. The performance of novel multi-protein risk models was compared to the PCP-HF risk score. RESULTS: Over 200 proteins were associated with incident HF after adjustment for estimated glomerular filtration rate at P < 1 × 10-5. After adjustment for covariates including N-terminal pro-B-type natriuretic peptide, 17 proteins remained associated at P < 1 × 10-5. Mendelian randomization associations were found for six proteins, of which four are druggable targets: FCG2B, IGFBP3, CAH6, and ASGR1. For the primary outcome, the C-statistic (95% confidence interval [CI]) for the 48-protein model in CRIC was 0.790 (0.735, 0.844) vs. 0.703 (0.644, 0.762) for the PCP-HF model (P = .001). C-statistic (95% CI) for the protein model in ARIC was 0.747 (0.707, 0.787). CONCLUSIONS: Large-scale proteomics reveal novel circulating protein biomarkers and potential mediators of HF in CKD. Proteomic risk models improve upon the PCP-HF risk score in this population.

Highly Multiplexed Image Analysis of Intestinal Tissue Sections in Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Significant progress has been made since the first report of inflammatory bowel disease (IBD) in 1859, after decades of research that have contributed to the understanding of the genetic and environmental factors involved in IBD pathogenesis. Today, a range of treatments is available for directed therapy, mostly targeting the overactive immune response. However, the mechanisms by which the immune system contributes to disease pathogenesis and progression are not fully understood. One challenge hindering IBD research is the heterogeneous nature of the disease and the lack of understanding of how immune cells interact with one another in the gut mucosa. Introduction of a technology that enables expansive characterization of the inflammatory environment of human IBD tissues may address this gap in knowledge. METHODS: We used the imaging mass cytometry platform to perform highly multiplex image analysis of IBD and healthy deidentified intestine sections (6 Crohn's disease compared to 6 control ileum; 6 ulcerative colitis compared to 6 control colon). The acquired images were graded for inflammation severity by analysis of adjacent H&E tissue sections. We assigned more than 300,000 cells to unique cell types and performed analyses of tissue integrity, epithelial activity, and immune cell composition. RESULTS: The intestinal epithelia of patients with IBD exhibited increased proliferation rates and expression of HLA-DR compared to control tissues, and both features were positively correlated with the severity of inflammation. The neighborhood analysis determined enrichment of regulatory T cell interactions with CD68+ macrophages, CD4+ T cells, and plasma cells in both forms of IBD, whereas activated lysozyme C+ macrophages were preferred regulatory T cell neighbors in Crohn's disease but not ulcerative colitis. CONCLUSIONS: Altogether, our study shows the power of imaging mass cytometry and its ability to both quantify immune cell types and characterize their spatial interactions within the inflammatory environment by a single analysis platform.

Proteins Associated with Risk of Kidney Function Decline in the General Population.

BACKGROUND: Proteomic profiling may allow identification of plasma proteins that associate with subsequent changesin kidney function, elucidating biologic processes underlying the development and progression of CKD. METHODS: We quantified the association between 4877 plasma proteins and a composite outcome of ESKD or decline in eGFR by ≥50% among 9406 participants in the Atherosclerosis Risk in Communities (ARIC) Study (visit 3; mean age, 60 years) who were followed for a median of 14.4 years. We performed separate analyses for these proteins in a subset of 4378 participants (visit 5), who were followed at a later time point, for a median of 4.4 years. For validation, we evaluated proteins with significant associations (false discovery rate <5%) in both time periods in 3249 participants in the Chronic Renal Insufficiency Cohort (CRIC) and 703 participants in the African American Study of Kidney Disease and Hypertension (AASK). We also compared the genetic determinants of protein levels with those from a meta-analysis genome-wide association study of eGFR. RESULTS: In models adjusted for multiple covariates, including baseline eGFR and albuminuria, we identified 13 distinct proteins that were significantly associated with the composite end point in both time periods, including TNF receptor superfamily members 1A and 1B, trefoil factor 3, and ß-trace protein. Of these proteins, 12 were also significantly associated in CRIC, and nine were significantly associated in AASK. Higher levels of each protein associated with higher risk of 50% eGFR decline or ESKD. We found genetic evidence for a causal role for one protein, lectin mannose-binding 2 protein (LMAN2). CONCLUSIONS: Large-scale proteomic analysis identified both known and novel proteomic risk factors for eGFR decline.

Patient-Reported Sinonasal Symptoms and Risk of Relapse in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: Relapses are frequent and difficult to predict in antineutrophil cytoplasmic antibody-associated vasculitis (AAV), resulting in long-term use of immunosuppression. Although sinonasal disease is associated with relapse of AAV, detailed characterization of sinonasal symptoms is lacking. Using a patient-reported outcome, the 22-item SinoNasal Outcome Test (SNOT-22), we investigated the relationship between sinonasal symptoms and disease activity in AAV. METHODS: This was a prospective, longitudinal study of individual with AAV and healthy individuals. Relapse was defined as a Birmingham Vasculitis Activity Score for Wegner's Granulomatosis score >0. Higher SNOT-22 scores indicate worse symptoms. Generalized estimating equation and Cox proportional hazard models evaluated the association between SNOT-22 and relapse. RESULTS: There were 773 visits (106 active disease visits) from 168 patients with AAV and 51 controls. Median SNOT-22 at remission was higher in AAV versus controls (20 vs 5; P < 0.001) and higher during active disease versus remission (P < 0.001). In all AAV, and particularly within granulomatosis with polyangiitis, higher SNOT-22 scores were observed months to years before relapse and were associated with increased risk of relapse (hazard ratio 2.7, 95% confidence interval 1.2-6.2; P = 0.02). Similar findings were seen when examining patients with versus without sinonasal disease and after removing relapses limited to the ear, nose, and throat. CONCLUSION: A patient-reported outcome measure of sinonasal disease, the SNOT-22, not only changes with disease activity in AAV, but also is associated with a higher risk of relapse within two years. These findings support the possibility that the SNOT-22 score may enhance prediction of relapse and that persistent sinonasal disease may be important in the pathophysiology of relapse.

Dynamic Tracking of Native Polyclonal Hematopoiesis in Adult Mice.

Hematopoietic dysfunction has been associated with a reduction in the number of active precursors. However, precursor quantification at homeostasis and under diseased conditions is constrained by the scarcity of available methods. To address this issue, we optimized a method for quantifying a wide range of hematopoietic precursors. Assuming the random induction of a stable label in precursors following a binomial distribution, the estimation depends on the inverse correlation between precursor numbers and the variance of precursor labeling among independent samples. Experimentally validated to cover the full dynamic range of hematopoietic precursors in mice (1 to 105), we utilized this approach to demonstrate that thousands of precursors, which emerge after modest expansion during fetal-to-adult transition, contribute to native and perturbed hematopoiesis. We further estimated the number of precursors in a mouse model of Fanconi Anemia, showcasing how repopulation deficits can be segregated into autologous (cell proliferation) and non-autologous causes (lack of precursor). Our results support an accessible and reliable approach for precursor quantification, emphasizing the contemporary perspective that native hematopoiesis is highly polyclonal.

Changes in fecal lipidome after treatment with ivacaftor without changes in microbiome or bile acids.

BACKGROUND: Alterations in gastrointestinal health are prominent manifestations of cystic fibrosis (CF) and can independently impact pulmonary function. Ivacaftor has been associated with robust improvements in pulmonary function and weight gain, but less is known about the impact of ivacaftor on the fecal microbiome, lipidome, and bile acids. METHODS: Stool samples from 18 patients with CF and gating mutations (ages 6-61 years, 13 pancreatic insufficient) were analyzed for fecal microbiome and lipidome composition as well as bile acid concentrations at baseline and after 3 months of treatment with ivacaftor. Microbiome composition was also assessed in a healthy reference cohort. RESULTS: Alpha and beta diversity of the microbiome were different between CF and reference cohort at baseline, but no treatment effect was seen in the CF cohort between baseline and 3 months. Seven lipids increased with treatment. No differences were seen in bile acid concentrations after treatment in CF. At baseline, 403 lipids and unconjugated bile acids were different between pancreatic insufficient (PI-CF) and sufficient (PS-CF) groups and 107 lipids were different between PI-CF and PS-CF after 3 months of treatment. CONCLUSIONS: The composition and diversity of the fecal microbiome were different in CF as compared to a healthy reference, and did not change after 3 months of ivacaftor. We detected modest differences in the fecal lipidome with treatment. Differences in lipid and bile acid profiles between PS-CF and PI-CF were attenuated after 3 months of treatment.

Analytical and Biological Variability of a Commercial Modified Aptamer Assay in Plasma Samples of Patients with Chronic Kidney Disease.

BACKGROUND: We carried out a study of the aptamer proteomic assay, SomaScan V4, to evaluate the analytical and biological variability of the assay in plasma samples of patients with moderate to severe chronic kidney disease (CKD). METHODS: Plasma samples were selected from 2 sources: (a) 24 participants from the Chronic Renal Insufficiency Cohort (CRIC) and (b) 49 patients from the Brigham and Women's Hospital-Kidney/Renal Clinic. We calculated intra-assay variability from both sources and examined short-term biological variability in samples from the Brigham clinic. We also measured correlations of aptamer measurements with traditional biomarker assays. RESULTS: A total of 4656 unique proteins (4849 total aptamer measures) were analyzed in all samples. Median (interquartile range [IQR] intra-assay CV) was 3.7% (2.8-5.3) in CRIC and 5.0% (3.8-7.0) in Brigham samples. Median (IQR) biological CV among Brigham samples drawn from one individual on 2 occasions separated by median (IQR) 7 (4-14) days was 8.7% (6.2-14). CVs were independent of CKD stage, diabetes, or albuminuria but were higher in patients with systemic lupus erythematosus. Rho correlations between aptamer and traditional assays for biomarkers of interest were cystatin C = 0.942, kidney injury model-1 = 0.905, fibroblast growth factor-23 = 0.541, tumor necrosis factor receptors 1 = 0.781 and 2 = 0.843, P < 10-100 for all. CONCLUSIONS: Intra-assay and within-subject variability for SomaScan in the CKD setting was low and similar to assay variability reported from individuals without CKD. Intra-assay precision was excellent whether samples were collected in an optimal research protocol, as were CRIC samples, or in the clinical setting, as were the Brigham samples.

Dynamic Changes in the Nasal Microbiome Associated With Disease Activity in Patients With Granulomatosis With Polyangiitis.

OBJECTIVE: Little is known about temporal changes in nasal bacteria in granulomatosis with polyangiitis (GPA). This study was undertaken to examine longitudinal changes in the nasal microbiome in association with relapse in GPA patients. METHODS: Bacterial 16S ribosomal RNA gene sequencing was performed on nasal swabs from 19 patients with GPA who were followed up longitudinally for a total of 78 visits, including 9 patients who experienced a relapse and 10 patients who remained in remission. Relative abundance of bacteria and ratios between bacteria were examined. Generalized estimating equation models were used to evaluate the association between bacterial composition and 1) disease activity and 2) levels of antineutrophil cytoplasmic antibody (ANCA) with specificity for proteinase 3 (PR3), adjusted for medication. RESULTS: Corynebacterium and Staphylococcus were the most abundant bacterial genera across all nasal samples. Patients with quiescent disease maintained a stable ratio of Corynebacterium to Staphylococcus across visits. In contrast, in patients who experienced a relapse, a significantly lower ratio was observed at the visit prior to relapse, followed by a higher ratio at the time of relapse (adjusted P < 0.01). Species-level analysis identified an association between a higher abundance of nasal Corynebacterium tuberculostearicum and 1) relapse (adjusted P = 0.04) and 2) higher PR3-ANCA levels (adjusted P = 0.02). CONCLUSION: In GPA, significant changes occur in the nasal microbiome over time and are associated with disease activity. The occurrence of these changes months prior to the onset of relapse supports a pathogenic role of nasal bacteria in GPA. Our results uphold existing hypotheses implicating Staphylococcus as an instigator of disease and have generated a novel finding involving Corynebacterium as a potential mediator of disease in GPA.

Immunogenicity and Immune Silence in Human Cancer.

Despite recent advances in cancer immunotherapy, the process of immunoediting early in tumorigenesis remains obscure. Here, we employ a mathematical model that utilizes the Cancer Genome Atlas (TCGA) data to elucidate the contribution of individual mutations and HLA alleles to the immunoediting process. We find that common cancer mutations including BRAF-V600E and KRAS-G12D are predicted to bind none of the common HLA alleles, and are thus "immunogenically silent" in the human population. We identify regions of proteins that are not presented by HLA at a population scale, coinciding with frequently mutated hotspots in cancer, and other protein regions broadly presented across the population in which few mutations occur. We also find that 9/29 common HLA alleles contribute disproportionately to the immunoediting of early oncogenic mutations. These data provide insights into immune evasion of common driver mutations and a molecular basis for the association of particular HLA genotypes with cancer susceptibility.