Marion E. Brunck

Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients.

Citrullinated peptide-exposed DCs induced immune regulatory effects in HLA risk genotype–positive RA patients. Immunotherapy out of joint Autoantibodies to anti–citrullinated peptides (ACPA) are found in most patients with rheumatoid arthritis (RA), especially those with HLA-DRB1 risk alleles. Benham et al. report a first-in-human phase 1 trial of a single injection of autologous dendritic cells modified with an NF-κB inhibitor that have been exposed to four citrullinated peptide antigens. They find that HLA risk genotype–positive RA patients had reduced numbers of effector T cells and decreased production of proinflammatory cytokines compared with untreated RA patient controls. The therapy was safe and did not induce disease flares. These data support larger studies of antigen-specific immunotherapy for RA. In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritis in an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti–citrullinated peptide autoantibodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated with HLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCs modified with a nuclear factor κB (NF-κB) inhibitor exposed to four citrullinated peptide antigens, designated “Rheumavax,” in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered once intradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype–positive RA patients with citrullinated peptide–specific autoimmunity. Sixteen RA patients served as controls. Rheumavax was well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a reduction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15 (IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin447–455–Cit450 relative to controls. Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreased within 1 month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety and biological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides, and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen immunomodulatory therapy in RA.

Targeted sequencing for gene discovery and quantification using RNA CaptureSeq

RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼7 d.

Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing

We compared quantitative RT-PCR (qRT-PCR), RNA-seq and capture sequencing (CaptureSeq) in terms of their ability to assemble and quantify long noncoding RNAs and novel coding exons across 20 human tissues. CaptureSeq was superior for the detection and quantification of genes with low expression, showed little technical variation and accurately measured differential expression. This approach expands and refines previous annotations and simultaneously generates an expression atlas.

Universal Alternative Splicing of Noncoding Exons

The human transcriptome is so large, diverse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.

Absolute counting of neutrophils in whole blood using flow cytometry

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked‐in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual‐platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra‐assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time‐course study of chemotherapy induced neutropenia using four drug regimens.

Fms-like tyrosine kinase 3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse

The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans, and mice. We recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. In the study described here, we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3-L caused whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However, unlike G-CSF, Flt3-L had an indirect effect on EI macrophages, as it was not detected at the surface of EI macrophages or erythroid progenitors.

Concise Review: Next-Generation Cell Therapies to Prevent Infections in Neutropenic Patients

High‐dose chemotherapy is accompanied by an obligate period of neutropenia. Resulting bacterial and fungal infections are the leading cause of morbidity and mortality in neutropenic patients despite prophylactic antimicrobials and hematopoietic growth factor supplements. Replacing neutrophils in the patient through transfusion of donor cells is a logical solution to prevent fulminant infections. In the past, this strategy has been hampered by poor yield, inability to store collected cells, and possible donor morbidity caused by granulocyte colony‐stimulating factor injections and apheresis. Today, neutrophil‐like cells can be manufactured in the laboratory at the clinical scale from hematopoietic stem and progenitor cells enriched from umbilical cord blood. This article reviews the rationale for focusing research efforts toward ex vivo neutrophil production and explores clinical settings for future trials.

Filling the void: allogeneic myeloid cells for transplantation.

Purpose of reviewThe success of allogeneic haematopoietic stem and progenitor cell (HSPC) transplantations remains inconsistent. Umbilical cord blood (UCB) is a promising source of HSPCs for transplantation, but low cell yield hampers its widespread use. Multiple strategies are being developed to manipulate UCB to either increase HSPC content or enhance bone marrow homing upon transfusion. Recent findingsSeveral ex-vivo manipulation protocols have increased engraftment success in recent phase I/II clinical trials. Additionally, by exploiting knowledge of the transcriptome, mature cells were dedifferentiated into induced haematopoietic stem cells capable of self-renewal and reconstitution of haematopoiesis in vivo. SummaryUCB is a more readily available source of allogeneic transplant material compared with bone marrow and mobilized peripheral blood. However, the number of HSPCs in a graft is correlated to the rate and success of engraftment and UCB grafts typically contain 10 times less cells compared with bone marrow or mobilized peripheral blood grafts that contain around 1 × 108 CD34+ cells. Recently, research efforts have focused on increasing UCB engrafting cells in addition to the methods to accelerate engraftment or to provide transient protection and support until engraftment succeeds.

Outcome of a phase I trial of rheumavax in patients with rheumatoid arthritis

Aim: The aim of this study was to identify miRNA that target infl ammatory pathways during arthritis. Specifi cally we are interested in production of the key infl ammatory cytokine IL-1β, which is generated by a complex of proteins known as the infl ammasome. Methods: Synovial fl uid monocytes were isolated from patients with rheumatoid or psoriatic arthritis and analysis of miRNA expression was performed. NLRP3, a key component of the infl ammasome, was identifi ed as a potential target of miR-223 and this was validated by several approaches. Results: We have identifi ed the fi rst miRNA that targets an infl ammasome complex. This is miR-223, which has a single, highly conserved binding site in the NLRP3 3′UTR. miR-223 expression decreases as monocytes differentiate into macrophages, and NLRP3 protein increases during this time. However overexpression of miR-223 prevents accumulation of endogenous NLRP3 protein levels, as for monocytic Thp-1 cells differentiated into macrophages with PMA. NLRP3 function was also impacted by miR-223, with decreased IL-1β production after stimulation with nigericin or uric acid crystals, but not poly dAdT (AIM2) or salmonella (NLRC4). Consistent with this, mice lacking miR-223 are reported to have spontaneous infl ammatory disease associated with increased NLRP3 protein expression. miR-223 is known to be decreased in type 2 diabetes, Crohn’s disease, and now we show a specifi c decrease in synovial monocytes from rheumatoid and psoriatic arthritis patients. All of these diseases are associated with increased IL-1β and the effect of miR-223 on NLRP3 could be a mechanism to account for this. Conclusions: In summary we have identifi ed an endogenous miRNA that limits NLRP3 infl ammatory capacity during myeloid differentiation, and is decreased in synovial fl uid monocytes from patients with rheumatoid or psoriatic arthritis.