Automated multi-scale computational pathotyping (AMSCP) of inflamed synovial tissue.

Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states.

Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients.

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-α. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking.

The Role of B Cells in Adult and Paediatric Liver Injury.

B lymphocytes are multitasking cells that direct the immune response by producing pro- or anti-inflammatory cytokines, by presenting processed antigen for T cell activation and co-stimulation, and by turning into antibody-secreting cells. These functions are important to control infection in the liver but can also exacerbate tissue damage and fibrosis as part of persistent inflammation that can lead to end stage disease requiring a transplant. In transplantation, immunosuppression increases the incidence of lymphoma and often this is of B cell origin.

Peripheral blood mononuclear cells preferentially activate 11-oxygenated androgens.

Objective: Androgens are important modulators of immune cell function. The local generation of active androgens from circulating precursors is an important mediator of androgen action in peripheral target cells or tissues. We aimed to characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs).

New Developments in Transcriptomic Analysis of Synovial Tissue.

Transcriptomic technologies are constantly changing and improving, resulting in an ever increasing understanding of gene expression in health and disease. These technologies have been used to investigate the pathological changes occurring in the joints of rheumatoid arthritis patients, leading to discoveries of disease mechanisms, and novel potential therapeutic targets. Microarrays were initially used on both whole tissue and cell subsets to investigate research questions, with bulk RNA sequencing allowing for further elaboration of these findings.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27CD45RB population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues.

Immunoglobulin characteristics and RNAseq data of FcRL4+ B cells sorted from synovial fluid and tissue of patients with rheumatoid arthritis.

This manuscript is a companion paper to Amara et al. [1]. Data shown here include detailed clinical characteristics from anonymized patients, the Ig subclass data generated from B cells sorted from four individual patients, tables detailing variable gene region sequences from sorted cells linked to the patient information and the sequence yields from individual patients.

Autoreactivity to malondialdehyde-modifications in rheumatoid arthritis is linked to disease activity and synovial pathogenesis.

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known.

B cells expressing the IgA receptor FcRL4 participate in the autoimmune response in patients with rheumatoid arthritis.

The clinical efficacy of B cell targeting therapies highlights the pathogenic potential of B cells in inflammatory diseases. Expression of Fc Receptor like 4 (FcRL4) identifies a memory B cell subset, which is enriched in the joints of patients with rheumatoid arthritis (RA) and in mucosa-associated lymphoid tissue. The high level of RANKL production by this B cell subset indicates a unique pathogenic role.

DKK1 expression by synovial fibroblasts in very early rheumatoid arthritis associates with lymphocyte adhesion in an in vitro flow co-culture system.

Background: Synovial fibroblasts play a key role in joint destruction and regulation of the inflammatory infiltrate in established rheumatoid arthritis (RA). The mechanisms by which this occurs in the earliest stages of RA are largely unknown. We investigated the role of Dickkopf-related protein 1 (DKK1) produced by synovial fibroblasts of patients with very early rheumatoid arthritis (VeRA).

Periodontitis prevalence and serum antibody reactivity to periodontal bacteria in primary Sjögren's syndrome: a pilot study.

Aims: The aims of this study were as follows: (i) To assess the prevalence of periodontitis among patients with primary Sjögren's syndrome (pSS) and comparator groups of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). (ii) To perform a pilot study to compare serum antibody responses to 10 oral/periodontal bacteria in these patient groups and a historical comparator group of patients with periodontitis.

Materials And Methods: Standard clinical periodontal assessments were performed on 39 pSS, 36 RA and 23 OA patients and "In-house" antibody ELISAs for serum antibodies against 10 oral/periodontal bacteria were performed in these groups.

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid.

Objective: In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA-protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint.

Differential glucocorticoid metabolism in patients with persistent versus resolving inflammatory arthritis.

Introduction: Impairment in the ability of the inflamed synovium to generate cortisol has been proposed to be a factor in the persistence and severity of inflammatory arthritis. In the inflamed synovium, cortisol is generated from cortisone by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. The objective of this study was to determine the role of endogenous glucocorticoid metabolism in the development of persistent inflammatory arthritis.

Stromal transcriptional profiles reveal hierarchies of anatomical site, serum response and disease and identify disease specific pathways.

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty.

Interferon-α-induced B-lymphocyte stimulator expression and mobilization in healthy and systemic lupus erthymatosus monocytes.

Objective: The aim of this study was to investigate the cellular populations and regulatory factors responsible for B-lymphocyte stimulator (BLyS) overexpression in SLE patients.

Methods: Surface and intracellular BLyS levels were quantified by flow cytometry in healthy and SLE monocytes cultured in the presence of TNF-α, IFN-α, IFN-γ, GM-CSF and SLE immune complexes (SLE-ICs), while soluble BLyS was measured by ELISA. Also, both surface and intracellular BLyS expression by different cell subsets was determined in 23 SLE patients and 16 healthy controls.

Ingenol mebutate: induced cell death patterns in normal and cancer epithelial cells.

We investigated the proposed necrotic mechanism of ingenol mebutate, a natural compound with anti-cancer properties in human keratinocytes, the human squamous cell carcinoma cell line HSC-5, and HeLa cervix carcinoma cells. Topical application of a clinical dose of ingenol mebutate 0.05% (1.

Rituximab abrogates joint destruction in rheumatoid arthritis by inhibiting osteoclastogenesis.

Objectives: To examine how rituximab may result in the inhibition of joint destruction in rheumatoid arthritis (RA) patients.

Methods: Twenty-eight patients with active RA were treated with rituximab. Radiographs of hands and feet before and 1 year after therapy were assessed using the Sharp-van der Heijde score (SHS).

The response of T cells to interleukin-6 is differentially regulated by the microenvironment of the rheumatoid synovial fluid and tissue.

Objective: Interleukin-6 (IL-6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL-6 receptor (IL-6R; CD126) or via trans-signaling, in which soluble IL-6R/IL-6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL-6 in the joints of patients with rheumatoid arthritis (RA).

Enhancing the anti-lymphoma potential of 3,4-methylenedioxymethamphetamine ('ecstasy') through iterative chemical redesign: mechanisms and pathways to cell death.

While 3,4-methylenedioxymethamphetamine (MDMA/'ecstasy') is cytostatic towards lymphoma cells in vitro, the concentrations required militate against its translation directly to a therapeutic in vivo. The possibility of 'redesigning the designer drug', separating desired anti-lymphoma activity from unwanted psychoactivity and neurotoxicity, was therefore mooted. From an initial analysis of MDMA analogues synthesized with a modified α-substituent, it was found that incorporating a phenyl group increased potency against sensitive, Bcl-2-deplete, Burkitt's lymphoma (BL) cells 10-fold relative to MDMA.

Lactoferrin inhibits neutrophil apoptosis via blockade of proximal apoptotic signaling events.

Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined.

Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis.

Objectives: In rheumatoid arthritis (RA), a complex cytokine network drives chronic inflammation and joint destruction. So far, few attempts have been made to identify the cellular sources of individual cytokines systematically. Therefore, the primary objective of this study was systematically to assess the cytokine messenger RNA expression profiles in the five largest cell populations in the synovial fluid and peripheral blood of RA patients.

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression, but little interleukin-22 and interleukin-23R expression.

Introduction: Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.

Methods: Flow cytometry was used to analyse the phenotype and cytokine production of mononuclear cells isolated from peripheral blood (PBMC) (n = 44), synovial fluid (SFMC) (n = 14) and synovium (SVMC) (n = 10) of RA patients and PBMC of healthy controls (n = 13).

Novel antileukemic compound ingenol 3-angelate inhibits T cell apoptosis by activating protein kinase Ctheta.

Members of the protein kinase C (PKC) family of serine-threonine kinases are important regulators of immune cell survival. Ingenol 3-angelate (PEP005) activates a broad range of PKC isoforms and induces apoptosis in acute myeloid leukemia cells by activating the PKC isoform PKCdelta. We show here that, in contrast to its effect on leukemic cells, PEP005 provides a strong survival signal to resting and activated human T cells.

Galectin 3 induces a distinctive pattern of cytokine and chemokine production in rheumatoid synovial fibroblasts via selective signaling pathways.

Objective: High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.