B Cell Synovitis and Clinical Phenotypes in Rheumatoid Arthritis: Relationship to Disease Stages and Drug Exposure.

OBJECTIVE: To define the relationship of synovial B cells to clinical phenotypes at different stages of disease evolution and drug exposure in rheumatoid arthritis (RA). METHODS: Synovial biopsy specimens and demographic and clinical data were collected from 2 RA cohorts (n = 329), one of patients with untreated early RA (n = 165) and one of patients with established RA with an inadequate response to tumor necrosis factor inhibitors (TNFi-IR; n = 164). Synovial tissue was subjected to hematoxylin and eosin and immunohistochemical staining and semiquantitative assessment for the degree of synovitis (on a scale of 0-9) and of CD20+ B cell infiltrate (on a scale of 0-4). B cell scores were validated by digital image analysis and B cell lineage-specific transcript analysis (RNA-Seq) in the early RA (n = 91) and TNFi-IR (n = 127) cohorts. Semiquantitative CD20 scores were used to classify patients as B cell rich (≥2) or B cell poor (<2). RESULTS: Semiquantitative B cell scores correlated with digital image analysis quantitative measurements and B cell lineage-specific transcripts. B cell-rich synovitis was present in 35% of patients in the early RA cohort and 47.7% of patients in the TNFi-IR cohort (P = 0.025). B cell-rich patients showed higher levels of disease activity and seropositivity for rheumatoid factor and anti-citrullinated protein antibody in early RA but not in established RA, while significantly higher histologic synovitis scores in B cell-rich patients were demonstrated in both cohorts. CONCLUSION: We describe a robust semiquantitative histologic B cell score that closely replicates the quantification of B cells by digital or molecular analyses. Our findings indicate an ongoing B cell-rich synovitis, which does not seem to be captured by standard clinimetric assessment, in a larger proportion of patients with established RA than early RA

Measurement and clinical significance of biomarkers of oxidative stress in humans

Oxidative stress is the result of the imbalance between reactive oxygen species (ROS) formation and enzymatic and nonenzymatic antioxidants. Biomarkers of oxidative stress are relevant in the evaluation of the disease status and of the health-enhancing effects of antioxidants. We aim to discuss the major methodological bias of methods used for the evaluation of oxidative stress in humans. There is a lack of consensus concerning the validation, standardization, and reproducibility of methods for the measurement of the following: (1) ROS in leukocytes and platelets by flow cytometry, (2) markers based on ROS-induced modifications of lipids, DNA, and proteins, (3) enzymatic players of redox status, and (4) total antioxidant capacity of human body fluids. It has been suggested that the bias of each method could be overcome by using indexes of oxidative stress that include more than one marker. However, the choice of the markers considered in the global index should be dictated by the aim of the study and its design, as well as by the clinical relevance in the selected subjects. In conclusion, the clinical significance of biomarkers of oxidative stress in humans must come from a critical analysis of the markers that should give an overall index of redox status in particular conditions

Effect of Turmeric and Carrot Seed Extracts on Serum Liver Biomarkers and Hepatic Lipid Peroxidation, Antioxidant Enzymes and Total Antioxidant Status in Rats

Introduction: Pathogenic role of free radicals are well known in various metabolic diseases. They originate from internal and external sources of body. Essential roles of antioxidant defense system for cellular redox regulation and free radical scavenging activity were described in this study. Many in vitro investigations have shown that turmeric (TE) and carrot seed extract (CSE) exhibits to possess antioxidant activities. In this study, we evaluated the antioxidant potentials of ethanolic TE and CSE based on in vivo experiment in the rats. Methods: Animals were assigned to six groups: the 1st and 2nd groups were control groups and 2nd group received 0.2 ml dimethyl sulphoxide as vehicle treated group; other four experimental groups received different doses of TE (100, 200 mg/kg b.w.) and CSE (200, 400 mg/kg b.w.) by gavages, respectively for a period of one month. The indicators of oxidative stress, lipids peroxidation, markers of hepatocyte injury and biliary function markers were measured. Results: The levels of superoxide dismutase, catalase, and glutathione peroxidase were significantly stimulated in the hepatic tissue of treatment groups. The malondialdehyde contents of liver tissue were significantly reduced in the groups fed with TE and CSE. Serum levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, in treated groups were found to be significantly decreased, whereas albumin and total protein increased as compared to the control groups (P<0.05). Conclusion: this study showed that the regular intake of TE and CSE through the diet can improve antioxidant status and inhibit peroxidation activity in the liver tissue so that using these extracts may protect tissue oxidative stress

Pharmacological Modulation of Inflammation, Bone Loss and Pain in a Murine Model of Inflammatory Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune condition, affecting approximately 1% of the population. RA is characterised by a chronic inflammatory response resulting in destruction of soft and hard tissues within the synovial joints. Disease progression in RA is complex, with multiple signalling pathways identified as crucial to T-cell mediated inflammation and increased osteoclastogenesis in the progression of joint destruction. Cell-adhesion molecules and alterations in apoptotic and autophagic pathways in cells located in the synovial joints have recently emerged as key components of the progression of inflammation and bone destruction in RA. Despite the growing knowledge of these mechanisms, control of bone destruction is still challenging and the prognosis of joint pain is often poor despite optimal control of inflammation. For this reason, it is imperative to utilise appropriate cell culture and in vivo models to identify key signalling pathways and develop targeted therapeutics that may inhibit inflammation, bone destruction and pain concomitantly upon diagnosis or prior to the onset of visible symptoms. The aim of this research was to use in vitro human osteoclast assays in conjunction with a modified collagen-antibody induced arthritis (CAIA) murine model of inflammatory arthritis to determine the effects of emerging novel compounds on inflammation, bone loss and pain-like behaviour. These studies also explored the pathology and progression of pain in a mild and moderate form of the CAIA model whilst extending micro-computed tomography analysis to include assessment of local inflammation and bone volume in the hind paws. The results of these studies support that novel compounds targeting cell adhesion molecules and NF-kB intracellular signalling have the potential to treat inflammatory induced bone loss. The findings presented also highlight the complex mechanisms associated with progression of joint destruction and pain-like behaviour in inflammatory arthritis. Further studies are necessary to elucidate the specific roles of each novel compound and further test the effectiveness of these compounds as potential therapies for RA.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Established and Emerging Cardiovascular Magnetic Resonance Imaging Techniques in the Evaluation of Subclinical Cardiovascular Disease

Introduction: Cardiovascular disease (CVD) remains the number one cause of mortality in the world for both men and women, thus improving its diagnosis and treatment is a priority. Rheumatoid Arthritis (RA) is a common auto-immune disease associated with high rates of CVD. Cardiovascular magnetic resonance (CMR) offers a multi-parametric, quantitative approach to the assessment of the heart and cardiovascular system with a host of techniques allowing assessment of anatomy, ventricular function, myocardial composition, myocardial perfusion, vascular performance and myocardial metabolism during a single scan. Its quantitative nature and lack of ionising radiation lend themselves ideally to the longitudinal study of subclinical CVD. Aims: To assess 1) whether blood longitudinal relaxation (T1) can be used to estimate blood haematocrit value to allow calculation of extracellular myocardial volume fraction (ECV), 2) whether CMR feature tracking (CMR-FT) is a feasible means of assessing aortic stiffness, 3) whether global longitudinal strain (GLS) is reduced in patients with prior MI with preserved left ventricular ejection fraction versus healthy controls, 4) whether aortic stiffness is present at all time points in the disease course of RA and 5) whether subclinical CV abnormalities in newly diagnosed RA improve with treatment and if the anti-tumour necrosis α Inhibitor Etanercept offers additional benefit over standard treatment. Methods: Patients were recruited between and February 2011 and February 2017. All patients underwent a comprehensive, multi-parametric CMR study including cine and late Gadolinium enhancement imaging at either 1.5 or 3.0T. Results: 1) estimation of blood haematocrit from blood T1 value provides accurate estimation of ‘synthetic’ ECV, 2) CMR-FT assessment of aortic stiffness is feasible and provides reproducible values for descending and ascending aortic strain values, 3) GLS is reduced in prior MI patients versus healthy controls (-17.3 ± 3.7% versus -19.3 ± 1.9% respectively, p=0.012). A GLS cut-off value of 18% correctly identifies prior MI with a sensitivity of 60% and specificity of 72.5%, 4) Aortic stiffness is evident in at risk RA individuals, newly diagnosed RA as well as established RA and 5) Aortic distensibility and left ventricular mass improve significantly in newly diagnosed RA patients following treatment. Etanercept appears to offer additional benefit over standard treatment evidenced by numerical improvements in aortic distensibility

Non-contact measures to monitor hand movement of people with rheumatoid arthritis using a monocular RGB camera

Hand movements play an essential role in a person’s ability to interact with the environment. In hand biomechanics, the range of joint motion is a crucial metric to quantify changes due to degenerative pathologies, such as rheumatoid arthritis (RA). RA is a chronic condition where the immune system mistakenly attacks the joints, particularly those in the hands. Optoelectronic motion capture systems are gold-standard tools to quantify changes but are challenging to adopt outside laboratory settings. Deep learning executed on standard video data can capture RA participants in their natural environments, potentially supporting objectivity in remote consultation. The three main research aims in this thesis were 1) to assess the extent to which current deep learning architectures, which have been validated for quantifying motion of other body segments, can be applied to hand kinematics using monocular RGB cameras, 2) to localise where in videos the hand motions of interest are to be found, 3) to assess the validity of 1) and 2) to determine disease status in RA. First, hand kinematics for twelve healthy participants, captured with OpenPose were benchmarked against those captured using an optoelectronic system, showing acceptable instrument errors below 10°. Then, a gesture classifier was tested to segment video recordings of twenty-two healthy participants, achieving an accuracy of 93.5%. Finally, OpenPose and the classifier were applied to videos of RA participants performing hand exercises to determine disease status. The inferred disease activity exhibited agreement with the in-person ground truth in nine out of ten instances, outperforming virtual consultations, which agreed only six times out of ten. These results demonstrate that this approach is more effective than estimated disease activity performed by human experts during video consultations. The end goal sets the foundation for a tool that RA participants can use to observe their disease activity from their home.Open Acces

PU.1 controls fibroblast polarization and tissue fibrosis

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs

Meta-analysis of genome-wide DNA methylation identifies shared associations across neurodegenerative disorders

Background: People with neurodegenerative disorders show diverse clinical syndromes, genetic heterogeneity, and distinct brain pathological changes, but studies report overlap between these features. DNA methylation (DNAm) provides a way to explore this overlap and heterogeneity as it is determined by the combined effects of genetic variation and the environment. In this study, we aim to identify shared blood DNAm differences between controls and people with Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Results: We use a mixed-linear model method (MOMENT) that accounts for the effect of (un)known confounders, to test for the association of each DNAm site with each disorder. While only three probes are found to be genome-wide significant in each MOMENT association analysis of amyotrophic lateral sclerosis and Parkinson’s disease (and none with Alzheimer’s disease), a fixed-effects meta-analysis of the three disorders results in 12 genome-wide significant differentially methylated positions. Predicted immune cell-type proportions are disrupted across all neurodegenerative disorders. Protein inflammatory markers are correlated with profile sum-scores derived from disease-associated immune cell-type proportions in a healthy aging cohort. In contrast, they are not correlated with MOMENT DNAm-derived profile sum-scores, calculated using effect sizes of the 12 differentially methylated positions as weights. Conclusions: We identify shared differentially methylated positions in whole blood between neurodegenerative disorders that point to shared pathogenic mechanisms. These shared differentially methylated positions may reflect causes or consequences of disease, but they are unlikely to reflect cell-type proportion differences.</p