Single-cell transcriptomics reveals targeted modulation of inflammatory repertoire by SOCE blockers.

Store-operated calcium entry (SOCE) plays a critical role in regulating intracellular calcium signaling and is essential for immune cell functions. SOCE blockade with pyrazole derivative BTP2 has been explored as an anti-inflammatory strategy in preclinical models and Zegocractin (CM4620) is being investigated in Phase 2 clinical trials as an immunoregulatory agent. However, the mode of action and differential effects of SOCE blockade on diverse immune cell types remain largely unknown, limiting the precision of current therapeutic applications.

Impact of Ultrasound-guided Percutaneous Core Needle Biopsy on Biomarkers of Human Kidney Allograft Status.

Background: Deciphering the impact of invasive percutaneous core needle biopsy of the kidney allograft on diagnostic biomarkers may help guide their clinical usage.

Methods: We prospectively enrolled 39 adult kidney allograft recipients (patients) who underwent 41 clinically indicated, ultrasound-guided, percutaneous core needle biopsies. Pre- and post-biopsy urines were analyzed for urinary cell 3-gene signature score (UroMap), and the bloods for peripheral blood gene expression score (AlloMap Kidney) and plasma donor-derived cell-free DNA percentage (dd-cfDNA).

Antibiotic subclasses differentially perturb the gut microbiota in kidney transplant recipients.

Introduction: The impact of antibiotics on the gut microbiota in kidney transplant recipients is not well characterized. In this study, we determine the impact of different subclasses of antibiotics on the gut microbiota in a cohort of 168 kidney transplant recipients.

Methods: Gut microbiome profiling was performed on 510 fecal specimens using 16S rRNA gene sequencing of the V4-V5 hypervariable region.

Blockade of store-operated calcium entry by BTP2 preserves anti-inflammatory gene expression in human peripheral blood mononuclear cells.

Store-operated calcium entry (SOCE) is essential for cellular signaling. Earlier studies of the pyrazole derivative BTP2, an efficient inhibitor SOCE, identified that SOCE blockade suppresses proinflammatory gene expression. The impact of SOCE blockade on gene expression at the whole transcriptome level, however, is unknown.

A quantitative comparison of urine centrifugation and filtration for the isolation and analysis of urinary nucleic acid biomarkers.

Urine is a rich source of nucleic acid biomarkers including cell-free DNA (cfDNA) and RNA for monitoring the health of kidney allografts. In this study, we aimed to evaluate whether urine filtration can serve as an alternative to the commonly used method of centrifugation to collect urinary fluid and cell pellets for isolating cfDNA and cellular messenger RNA (mRNA). We collected urine specimens from kidney allograft recipients and obtained the urine supernatant and cell pellet from each specimen using both filtration and centrifugation for paired analyses.

RNA-sequencing of Human Kidney Allografts and Delineation of T-Cell Genes, Gene Sets, and Pathways Associated With Acute T Cell-mediated Rejection.

Background: Delineation of T-cell genes, gene sets, pathways, and T-cell subtypes associated with acute T cell-mediated rejection (TCMR) may improve its management.

Methods: We performed bulk RNA-sequencing of 34 kidney allograft biopsies (16 Banff TCMR and 18 no rejection [NR] biopsies) from 34 adult recipients of human kidneys. Computational analysis was performed to determine the differential intragraft expression of T-cell genes at the level of single-gene, gene set, and pathways.

Urinary Cell Gene Signature of Acute Rejection in Kidney Allografts.

Introduction: A kidney allograft biopsy may display acute T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (MR). Development of noninvasive biomarkers diagnostic of all three types of acute rejection is a useful addition to the diagnostic armamentarium.

Methods: We developed customized RT-qPCR assays and measured urinary cell mRNA copy number in 145 biopsy-matched urine samples from 126 kidney allograft recipients and calculated urinary cell three-gene signature score from log -transformed values for the 18S-normalized CD3E mRNA, 18S-normalized CXCL10 mRNA and 18S rRNA.

Transcriptomic signatures of chronic active antibody-mediated rejection deciphered by RNA sequencing of human kidney allografts.

Natural killer (NK) cells mediate spontaneous cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. This dual functionality could enable their participation in chronic active antibody-mediated rejection (CA-ABMR). Earlier microarray profiling studies have not subcategorized antibody-mediated rejection into CA-ABMR and active-ABMR, and the gene expression pattern of CA-ABMR has not been compared with that of T cell-mediated rejection (TCMR).

Evaluation of Immunocompetence and Biomarkers of Tolerance in Chimeric and Immunosuppression-free Kidney Allograft Recipients.

Background: Thirty-seven patients have received a living-donor kidney transplant in a phase 2 study designed to induce tolerance with facilitated allogeneic hematopoietic stem cell transplant. The study protocol is based on tolerogenic CD8 + /T-cell receptor - facilitating cells (FCR001; also including hematopoietic stem cells and αβ-T-cell receptor + T cells) and low-dose, nonmyeloablative conditioning. Persistent chimerism allowing full immunosuppression (IS) withdrawal was achieved in 26 patients (time off IS 36-123 mo).

Selective modulation of gene expression in activated normal human peripheral blood mononuclear cells by store-operated calcium entry blocker BTP2.

Calcium is a critical signaling molecule in many cell types including immune cells. The calcium-release activated calcium channels (CRAC) responsible for store-operated calcium entry (SOCE) in immune cells are gated by STIM family members functioning as sensors of Ca store content in the endoplasmic reticulum. We investigated the effect of SOCE blocker BTP2 on human peripheral blood mononuclear cells (PBMC) stimulated with the mitogen phytohemagglutinin (PHA).

Urine as liquid gold: the transcriptional landscape of acute rejection defined by urinary cell mRNA profiling of kidney allograft recipients.

Purpose Of Review: Because all functioning nephrons contribute to urine formation, we reasoned that urine would be a suitable substitute to kidney allograft biopsy to discern human kidney allograft status. In view of compelling data that ribonucleic acid (RNA) sequencing outperforms microarray-based profiling, we performed RNA sequencing of urinary cells and kidney allograft biopsies to define the transcriptional landscape of allograft rejection.

Recent Findings: Whole genome transcriptome profiling identified unique and shared gene signatures of acute T cell mediated rejection (TCMR) and antibody mediated rejection (AMR).

Urinary cell mRNA profiling of kidney allograft recipients: Development of a portable protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy.

Background: We developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 80C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 80C, and shipment in cold containers for mRNA profiling.

Chronic reduction of store operated Ca entry is viable therapeutically but is associated with cardiovascular complications.

Loss of function mutations in store-operated Ca entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition.

Development of a Bak gene based standard curve for absolute quantification of BK virus in real time quantitative PCR assay and noninvasive diagnosis of BK virus nephropathy in kidney allograft recipients.

Background: BK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay.

Methods: We determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays.

A metagenomic DNA sequencing assay that is robust against environmental DNA contamination.

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present Sample-Intrinsic microbial DNA Found by Tagging and sequencing (SIFT-seq) a metagenomic sequencing assay that is robust against environmental DNA contamination introduced during sample preparation. The core idea of SIFT-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing.

Detection of infiltrating fibroblasts by single-cell transcriptomics in human kidney allografts.

We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune injury. We selected 3 biopsies of kidney cortex from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy, tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We did not study T-cell-mediated rejections.

T-Cell-Positive, B-Cell-Negative Flow Cytometry Crossmatch: Frequency, HLA Locus Specificity, and Mechanisms Among 3073 Clinical Flow Cytometry Crossmatch Tests.

Objectives: A T-cell-positive and B-cell-negative flow cytometry crossmatch result remains a conundrum since HLA class I antigens are expressed on both T and B cells. We investigated the frequency, donor HLA specificity of the antibodies, and mechanisms for these crossmatch results.

Materials And Methods: We analyzed 3073 clinical flow cytometry crossmatch tests performed in an American Society of Histocompatibility and Immunogeneticsaccredited histocompatibility laboratory.

A metagenomic DNA sequencing assay that is robust against environmental DNA contamination.

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing.

Design and Methods of the Validating Injury to the Renal Transplant Using Urinary Signatures (VIRTUUS) Study in Children.

Unlabelled: Lack of noninvasive diagnostic and prognostic biomarkers to reliably detect early allograft injury poses a major hindrance to long-term allograft survival in pediatric kidney transplant recipients.

Methods: Validating Injury to the Renal Transplant Using Urinary Signatures Children's Study, a North American multicenter prospective cohort study of pediatric kidney transplant recipients, aims to validate urinary cell mRNA and metabolite profiles that were diagnostic and prognostic of acute cellular rejection (ACR) and BK virus nephropathy (BKVN) in adult kidney transplant recipients in Clinical Trials in Organ Transplantation-4. Specifically, we are investigating: (1) whether a urinary cell mRNA 3-gene signature (-normalized mRNA, and ribosomal RNA) discriminates biopsies with versus without ACR, (2) whether a combined metabolite profile with the 3-gene signature increases sensitivity and specificity of diagnosis and prognostication of ACR, and (3) whether mRNA levels in urinary cells are diagnostic of BKVN and prognostic for allograft failure.

Measurement Biases Distort Cell-Free DNA Fragmentation Profiles and Define the Sensitivity of Metagenomic Cell-Free DNA Sequencing Assays.

Background: Metagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing.

Urinary cell mRNA profiling of kidney allograft recipients: A systematic investigation of a filtration based protocol for the simplification of urine processing.

Background: Kidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality.

Kidney Allograft Function Is a Confounder of Urine Metabolite Profiles in Kidney Allograft Recipients.

Noninvasive biomarkers of kidney allograft status can help minimize the need for standard of care kidney allograft biopsies. Metabolites that are measured in the urine may inform about kidney function and health status, and potentially identify rejection events. To test these hypotheses, we conducted a metabolomics study of biopsy-matched urine cell-free supernatants from kidney allograft recipients who were diagnosed with two major types of acute rejections and no-rejection controls.