Search

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. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former. Results We analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the Affymetrix® HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules (HSP-70, HSP-110, Calreticulin, gp96), pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators (Granulysin, Granzyme A). Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001]. Conclusions The upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response.

Background: Kinases are intracellular signalling mediators and key to sustaining the inflammatory process in rheumatoid arthritis (RA). Oral inhibitors of Janus Kinase family (JAKs) are widely used in RA, while inhibitors of other kinase families e.g. phosphoinositide 3-kinase (PI3K) are under development. Most current biomarker platforms quantify mRNA/protein levels, but give no direct information on whether proteins are active/inactive. Phosphoproteome analysis has the potential to measure specific enzyme activation status at tissue level., Methods: We validated the feasibility of phosphoproteome and total proteome analysis on 8 pre-treatment synovial biopsies from treatment-naive RA patients using label-free mass spectrometry, to identify active cell signalling pathways in synovial tissue which might explain failure to respond to RA therapeutics., Results: Differential expression analysis and functional enrichment revealed clear separation of phosphoproteome and proteome profiles between lymphoid and myeloid RA pathotypes. Abundance of specific phosphosites was associated with the degree of inflammatory state. The lymphoid pathotype was enriched with lymphoproliferative signalling phosphosites, including Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted novel kinases not previously linked to RA, such as Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotype. Several phosphosites correlated with clinical features, such as Disease-Activity-Score (DAS)-28, suggesting that phosphosite analysis has potential for identifying novel biomarkers at tissue-level of disease severity and prognosis., Conclusions: Specific phosphoproteome/proteome signatures delineate RA pathotypes and may have clinical utility for stratifying patients for personalised medicine in RA., (© 2024. The Author(s).)

Follicular dendritic cells (FDCs) fundamentally contribute to the formation of synovial ectopic lymphoid-like structures in rheumatoid arthritis (RA) which is associated with poor clinical prognosis. Despite this critical role, regulation of FDC development in the RA synovium and its correlation with synovial pathotype differentiation remained largely unknown. Here, we demonstrate that CNA.42 + FDCs distinctively express the pericyte/fibroblast-associated markers PDGFR-β, NG2, and Thy-1 in the synovial perivascular space but not in established follicles. In addition, synovial RNA-Seq analysis revealed that expression of the perivascular FDC markers was strongly correlated with PDGF-BB and fibroid synovitis, whereas TNF-α/LT-β was significantly associated with lymphoid synovitis and expression of CR1, CR2, and FcγRIIB characteristic of mature FDCs in lymphoid follicles. Moreover, PDGF-BB induced CNA.42 + FDC differentiation and CXCL13 secretion from NG2 + synovial pericytes, and together with TNF-α/LT-β conversely regulated early and late FDC differentiation genes in unsorted RA synovial fibroblasts (RASF) and this was confirmed in flow sorted stromal cell subsets. Furthermore, RASF TNF-αR expression was upregulated by TNF-α/LT-β and PDGF-BB; and TNF-α/LT-β-activated RASF retained ICs and induced B cell activation in in vitro germinal center reactions typical of FDCs. Additionally, FDCs trapped peptidyl citrulline, and strongly correlated with IL-6 expression, and plasma cell, B cell, and T cell infiltration of the RA synovium. Moreover, synovial FDCs were significantly associated with RA disease activity and radiographic features of tissue damage. To the best of our knowledge, this is the first report describing the reciprocal interaction between PDGF-BB and TNF-α/LT-β in synovial FDC development and evolution of RA histological pathotypes. Selective targeting of this interplay could inhibit FDC differentiation and potentially ameliorate RA in clinically severe and drug-resistant patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 El Shikh, El Sayed, Aly, Prediletto, Hands, Fossati-Jimack, Bombardieri, Lewis and Pitzalis.)

Aims: To determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment., Methods: PTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium., Results: Circulating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders., Conclusion: This study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required., Competing Interests: AM and BB are inventors of patents on pentraxin-3 and obtain royalties on related reagents. The authors declare that there are no other commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Boutet, Nerviani, Lliso-Ribera, Leone, Sironi, Hands, Rivellese, Del Prete, Goldmann, Lewis, Mantovani, Bottazzi and Pitzalis.)

Objectives: To determine the relationship between synovial versus skin transcriptional/histological profiles in patients with active psoriatic arthritis (PsA) and explore mechanistic links between diseased tissue pathology and clinical outcomes., Methods: Twenty-seven active PsA patients were enrolled in an observational/open-label study and underwent biopsies of synovium and paired lesional/non-lesional skin before starting anti-tumour necrosis factor (TNF) (if biologic-naïve) or ustekinumab (if anti-TNF inadequate responders). Molecular analysis of 80-inflammation-related genes and protein levels for interleukin (IL)-23p40/IL-23p19/IL-23R were assessed by real-time-PCR and immunohistochemistry, respectively., Results: At baseline, all patients had persistent active disease as per inclusion criteria. At primary end-point (16-weeks post-treatment), skin responses favoured ustekinumab, while joint responses favoured anti-TNF therapies. Principal component analysis revealed distinct clustering of synovial tissue gene expression away from the matched skin. While IL12B, IL23A and IL23R were homogeneously expressed in lesional skin, their expression was extremely heterogeneous in paired synovial tissues. Here, IL-23 transcriptomic/protein expression was strongly linked to patients with high-grade synovitis who, however, were not distinguishable by conventional clinimetric measures., Conclusions: PsA synovial tissue shows a heterogeneous IL-23 axis profile when compared with matched skin. Synovial molecular pathology may help to identify among clinically indistinguishable patients those with a greater probability of responding to IL-23 inhibitors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Biomarkers are needed for predicting the effectiveness of disease modifying antirheumatic drugs (DMARDs). Here, using functional lipid mediator profiling and deeply phenotyped patients with early rheumatoid arthritis (RA), we observe that peripheral blood  specialized pro-resolving mediator (SPM) concentrations are linked with both DMARD responsiveness and disease pathotype. Machine learning analysis demonstrates that baseline plasma concentrations of resolvin D4, 10S, 17S-dihydroxy-docosapentaenoic acid, 15R-Lipoxin (LX)A 4 and n-3 docosapentaenoic-derived Maresin 1 are predictive of DMARD responsiveness at 6 months. Assessment of circulating SPM concentrations 6-months after treatment initiation establishes that differences between responders and non-responders are maintained, with a decrease in SPM concentrations in patients resistant to DMARD therapy. These findings elucidate the potential utility of  plasma SPM concentrations as biomarkers of DMARD responsiveness in RA.

Background: Clinical outcomes in elderly-onset rheumatoid arthritis (EORA), starting after the age of 60, are conflicting. Thus, we aimed to investigate in a unique biopsy-driven, treatment-naïve early arthritis cohort, the relationship between synovial pathobiology of elderly- (EORA) and younger-onset rheumatoid arthritis (YORA) patients through clinical, imaging and treatment response outcome-measures., Methods: Patients (n = 140) with early RA (<12months) starting before (YORA, n = 99) or after (EORA, n = 41) age 60 had an ultrasound-guided synovial biopsy prior to conventional immunosuppressive therapy and after 6 months. Clinical, ultrasound and radiographic data were collected prospectively and compared between groups and against immunohistological features. Using multivariate logistic regression, we determined predictors of clinical response (disease activity score-28-erythrocyte sedimentation rate [DAS28-ESR]<3.2) at 6 months and radiographic progression (≥1-unit-increase in Sharp van der Heijde [SvdH] score) at 12 months., Results: EORA patients were more frequently male and presented most commonly with an abrupt, polymyalgia rheumatica-like onset and extra-articular features. Both before and after treatment, DAS28-ESR was similar but ultrasound synovial-thickening (p<0.05) and power-Doppler (p<0.01) synovitis and SvdH (p<0.001) scores were higher in EORA patients. EORA was independently associated with poor treatment response at 6 months (OR=0.28, p = 0.047) and radiographic progression at 12 months (OR=4.08, p = 0.029). Synovial pathotype, synovitis scores and cellular infiltration were similar before treatment, but a pauci-immune-fibroid pathotype tended to be more common in YORA at 6 months (p = 0.093). Moreover, YORA patients had a marked improvement of all synovitis parameters (p<0.001), whereas EORA presented only mild decreases in synovitis (p<0.05), sublining macrophage (p<0.05) and T cell scores (p<0.05), with no significant changes in lining macrophages, B cells or plasma cells., Conclusion: Early EORA presents differently and has a worse overall prognosis than YORA, with poorer clinical, histological, ultrasonographic and radiographic outcomes., Competing Interests: Declaration of Competing Interest VCR reports personal fees and non-financial support from Pfizer and Janssen; non-financial support from Merck Sharp and Dohme, Lilly and Roche, outside the submitted work. SK reports personal fees from UCB Pharma, Janssen and Pfizer, outside the submitted work. VRR reports personal fees from AstraZeneca, outside the submitted work. DvdH reports personal fees from AbbVie, Amgen, Astellas, AstraZeneca, BMS, Boehringer Ingelheim, Celgene, Daiichi, Eli-Lilly, Galapagos, Gilead, Glaxo-Smith-Kline, Janssen, Merck, Novartis, Pfizer, Regeneron, Roche, Sanofi, Takeda and UCB, outside the submitted work; and is Director of Imaging Rheumatology bv. JEF reports grants and personal fees from AbbVie, Merck Sharp and Dohme, Pfizer, Roche, UCB Pharma, Janssen, Novartis and Sanofi, outside the submitted work. CP reports grants and personal fees from Abbott / AbbVie, Astellas, AstraZeneca / MedImmune, Bristol-Myers Squibb, Janssen / Johnson & Johnson, Merck Sharp and Dohme, Pfizer, Roche / Genentech / Chugai and UCB Pharma, outside the submitted work. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Objectives: To assess whether the histopathological features of the synovium before starting treatment with the TNFi certolizumab-pegol could predict clinical outcome and examine the modulation of histopathology by treatment. Methods: Thirty-seven RA patients fulfilling UK NICE guidelines for biologic therapy were enrolled at Barts Health NHS trust and underwent synovial sampling of an actively inflamed joint using ultrasound-guided needle biopsy before commencing certolizumab-pegol and after 12-weeks. At 12-weeks, patients were categorized as responders if they had a DAS28 fall >1.2. A minimum of 6 samples was collected for histological analysis. Based on H&E and immunohistochemistry (IHC) staining for CD3 (T cells), CD20 (B cells), CD138 (plasma cells), and CD68 (macrophages) patients were categorized into three distinct synovial pathotypes (lympho-myeloid, diffuse-myeloid, and pauci-immune). Results: At baseline, as per inclusion criteria, DAS28 mean was 6.4 ± 0.9. 94.6% of the synovial tissue was retrieved from the wrist or a metacarpophalangeal joint. Histological pathotypes were distributed as follows: 58% lympho-myeloid, 19.4% diffuse-myeloid, and 22.6% pauci-immune. Patients with a pauci-immune pathotype had lower levels of CRP but higher VAS fatigue compared to lympho- and diffuse-myeloid. Based on DAS28 fall >1.2, 67.6% of patients were deemed as responders and 32.4% as non-responders. However, by categorizing patients according to the baseline synovial pathotype, we demonstrated that a significantly higher number of patients with a lympho-myeloid and diffuse-myeloid pathotype in comparison with pauci-immune pathotype [83.3% (15/18), 83.3 % (5/6) vs. 28.6% (2/7), p = 0.022) achieved clinical response to certolizumab-pegol. Furthermore, we observed a significantly higher level of post-treatment tender joint count and VAS scores for pain, fatigue and global health in pauci-immune in comparison with lympho- and diffuse-myeloid patients but no differences in the number of swollen joints, ESR and CRP. Finally, we confirmed a significant fall in the number of CD68+ sublining macrophages post-treatment in responders and a correlation between the reduction in the CD20+ B-cells score and the improvement in the DAS28 at 12-weeks. Conclusions: The analysis of the synovial histopathology may be a helpful tool to identify among clinically indistinguishable patients those with lower probability of response to TNFα-blockade., (Copyright © 2020 Nerviani, Di Cicco, Mahto, Lliso-Ribera, Rivellese, Thorborn, Hands, Bellan, Mauro, Boutet, Giorli, Lewis, Kelly, Bombardieri, Humby and Pitzalis.)

Objective: To establish whether synovial pathobiology improves current clinical classification and prognostic algorithms in early inflammatory arthritis and identify predictors of subsequent biological therapy requirement., Methods: 200 treatment-naïve patients with early arthritis were classified as fulfilling RA1987 American College of Rheumatology (ACR) criteria (RA1987) or as undifferentiated arthritis (UA) and patients with UA further classified into those fulfilling RA2010 ACR/European League Against Rheumatism (EULAR) criteria. Treatment requirements at 12 months (Conventional Synthetic Disease Modifying Antirheumatic Drugs (csDMARDs) vs biologics vs no-csDMARDs treatment) were determined. Synovial tissue was retrieved by minimally invasive, ultrasound-guided biopsy and underwent processing for immunohistochemical (IHC) and molecular characterisation. Samples were analysed for macrophage, plasma-cell and B-cells and T-cells markers, pathotype classification (lympho-myeloid, diffuse-myeloid or pauci-immune) by IHC and gene expression profiling by Nanostring., Results: 128/200 patients were classified as RA1987, 25 as RA2010 and 47 as UA. Patients classified as RA1987 criteria had significantly higher levels of disease activity, histological synovitis, degree of immune cell infiltration and differential upregulation of genes involved in B and T cell activation/function compared with RA2010 or UA, which shared similar clinical and pathobiological features. At 12-month follow-up, a significantly higher proportion of patients classified as lympho-myeloid pathotype required biological therapy. Performance of a clinical prediction model for biological therapy requirement was improved by the integration of synovial pathobiological markers from 78.8% to 89%-90%., Conclusion: The capacity to refine early clinical classification criteria through synovial pathobiological markers offers the potential to predict disease outcome and stratify therapeutic intervention to patients most in need., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders., (Copyright © 2019. Published by Elsevier Ltd.)

There is a current imperative to unravel the hierarchy of molecular pathways that drive the transition of early to established disease in rheumatoid arthritis (RA). Herein, we report a comprehensive RNA sequencing analysis of the molecular pathways that drive early RA progression in the disease tissue (synovium), comparing matched peripheral blood RNA-seq in a large cohort of early treatment-naive patients, namely, the Pathobiology of Early Arthritis Cohort (PEAC). We developed a data exploration website (https://peac.hpc.qmul.ac.uk/) to dissect gene signatures across synovial and blood compartments, integrated with deep phenotypic profiling. We identified transcriptional subgroups in synovium linked to three distinct pathotypes: fibroblastic pauci-immune pathotype, macrophage-rich diffuse-myeloid pathotype, and a lympho-myeloid pathotype characterized by infiltration of lymphocytes and myeloid cells. This is suggestive of divergent pathogenic pathways or activation disease states. Pro-myeloid inflammatory synovial gene signatures correlated with clinical response to initial drug therapy, whereas plasma cell genes identified a poor prognosis subgroup with progressive structural damage., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: To unravel the hierarchy of cellular/molecular pathways in the disease tissue of early, treatment-naïve rheumatoid arthritis (RA) patients and determine their relationship with clinical phenotypes and treatment response/outcomes longitudinally., Methods: 144 consecutive treatment-naïve early RA patients (<12 months symptoms duration) underwent ultrasound-guided synovial biopsy before and 6 months after disease-modifying antirheumatic drug (DMARD) initiation. Synovial biopsies were analysed for cellular (immunohistology) and molecular (NanoString) characteristics and results compared with clinical and imaging outcomes. Differential gene expression analysis and logistic regression were applied to define variables correlating with treatment response and predicting radiographic progression., Results: Cellular and molecular analyses of synovial tissue demonstrated for the first time in early RA the presence of three pathology groups: (1) lympho-myeloid dominated by the presence of B cells in addition to myeloid cells; (2) d iffuse-myeloid with myeloid lineage predominance but poor in B cells nd (3) pauci-immune characterised by scanty immune cells and prevalent stromal cells. Longitudinal correlation of molecular signatures demonstrated that elevation of myeloid- and lymphoid-associated gene expression strongly correlated with disease activity, acute phase reactants and DMARD response at 6 months. Furthermore, elevation of synovial lymphoid-associated genes correlated with autoantibody positivity and elevation of osteoclast-targeting genes predicting radiographic joint damage progression at 12 months. Patients with predominant pauci-immune pathology showed less severe disease activity and radiographic progression., Conclusions: We demonstrate at disease presentation, prior to pathology modulation by therapy, the presence of specific cellular/molecular synovial signatures that delineate disease severity/progression and therapeutic response and may pave the way to more precise definition of RA taxonomy, therapeutic targeting and improved outcomes., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Objective: To evaluate whether the choice of synovial biopsy technique (arthroscopy, blind needle [BN] biopsy, ultrasound [US]-guided portal and forceps [P&F], or US-guided needle biopsy [NB]) translates to significant variation in synovial tissue quality and quantity, with the aim of informing recommendations for the choice of synovial sampling technique within clinical trials., Methods: In total, 159 procedures from 5 academic rheumatology centers were evaluated. Hematoxylin and eosin-stained, paraffin-embedded synovial tissue sections from patients with inflammatory arthritis were assessed in order to determine the proportion of graded synovial fragments, total area of graded synovial tissue, and synovitis score per procedure. RNA quantity (μg of RNA) and quality (RNA integrity number) per procedure were also assessed in the synovial samples., Results: In this study, 84 of the 159 procedures performed on large joints at baseline (25 arthroscopic, 35 US-P&F, 11 US-NB, and 13 BN biopsies), 41 of the 159 procedures performed on small joints at baseline (11 US-P&F, 20 US-NB, and 10 BN biopsies), and 34 sequential biopsy procedures were evaluated. Compared to all other techniques evaluated in the small and large joints, fewer small joint BN biopsies and a significantly lower proportion of large joint BN biopsies yielded graded synovial tissue. No significant difference in either the proportion of graded tissue samples or total graded synovial tissue area between the US-NB and arthroscopic large joint procedures was demonstrated. Among the sequential biopsy procedures evaluated (small joint US-NB, large joint arthroscopy, US-P&F biopsy, and BN biopsy), no significant difference in the proportion of graded synovial tissue or total graded synovial tissue area was demonstrated. All procedures yielded RNA of significant quality and quantity for subsequent transcriptomic analysis., Conclusion: These data support the integration of US-guided methods along with arthroscopic biopsy for clinical trial protocols in which sequential sampling of synovium from the large and small joints is needed for both histologic and molecular analysis. BN biopsy may be considered if graded synovial tissue is not required for subsequent analyses., (© 2018, American College of Rheumatology.)

Objective: To examine in a cohort of rheumatoid arthritis (RA) patients undergoing serial ultrasound (US)-guided biopsies of small joints in the context of clinical trials whether sufficient synovial tissue could be obtained at both baseline and second biopsy to: 1) accurately evaluate the synovial immune phenotype, 2) permit adequate RNA extraction to determine molecular signatures, and 3) sensitively detect change in the number of synovial sublining macrophages (CD68+) following effective therapy., Methods: Synovial samples from RA patients undergoing US-guided biopsy of small joints as part of 2 clinical trials (Barts Early Arthritis Cohort [n = 18] and the Clinical and Pathological Response to Certolizumab Pegol (CLIP-Cert) study [n = 17]) were examined, and the quality and quantity of histologic samples and RNA extracted per joint were determined and compared to synovial thickness and power Doppler scores determined by US before biopsy. Modulation of the number of CD68+ sublining macrophages was correlated with clinical response to treatment., Results: Good quality synovial tissue that accurately reflected the synovial immune phenotype of the total joint was obtained in 80% of US-guided procedures when synovial thickness (higher than grade 2) was documented before biopsy. In 100% of the procedures, sufficient RNA was extracted to permit molecular analysis. There was a significant correlation between change in CD68+ sublining macrophage number and clinical response to treatment., Conclusion: This study provides minimum standards for sample retrieval for small joint biopsy. Furthermore, our findings confirm the clinical utility of the procedure in the largest reported cohort of US-guided small joint biopsies. The demonstration that small joint synovial tissue can be readily accessed by a technically simple, minimally invasive procedure is likely to facilitate critical advancements in the knowledge of RA pathobiology and personalized health care., (© 2015, American College of Rheumatology.)

Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal-vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.

IGF-binding protein-3 (IGFBP-3) has been reported to exert a protective influence on the pathogenesis of colorectal cancer. This may reflect its modulation of IGF-I bioactivity as well as IGF-I-independent effects on cell proliferation and apoptosis. Although local expression of IGF-I in the colon is increasingly recognised as having important regulatory consequences, the role of locally expressed IGFBP-3 remains unknown. The aims of the present study were: (i) to quantify and localise the expression of IGFBP-3 in human normal and malignant colon; (ii) to relate this expression to that of other components of the IGF-I axis; and (iii) to investigate the effects of IGFBP-3 on colonic epithelial cell proliferation and apoptosis. RNA was extracted from 46 paired samples of normal and malignant colonic tissue. IGFBP-3, IGF-I, IGF-I receptor and GH receptor mRNA levels were quantified using real-time RT-PCR. Laser-capture microdissection of the same samples was used to isolate mRNA from epithelium and stromal components and localise mRNA expression. Expression was confirmed at a protein level by immunohistochemistry. Human colorectal cancer HT-29 and CaCo-2 cells were cultured with IGFBP-3 (200 ng/ml), +/- IGF-I (20 ng/ml), +/- sodium butyrate (5 mM). Cell number was assessed by an MTS assay (a modification of the MTT assay), and apoptosis assessed by cell morphology and FACS analysis using both annexin and propidium iodide staining. UO146, a MAP kinase inhibitor, and wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI-3K) pathway, were used to determine the contribution of these signalling pathways on the effects of IGFBP-3. IGFBP-3 mRNA was detected in all samples (mean copy number/mug total RNA in normal colon, 2.6 x 10(6) compared with 1.3 x 10(7) in the cancers; P < 0.0001). Immunohistochemistry confirmed the expression and showed it to be equally distributed between epithelial and stromal components in normal tissue, but to be mainly restricted to the stromal component of malignant tissue. This differential expression was confirmed by RT-PCR of RNA from laser-capture microdissected samples. IGF-I mRNA was detected in 31 samples of normal colon; mean IGFBP-3 copy number was higher in the IGF-I-positive samples compared with IGF-I-negative samples. IGFBP-3 on its own induced apoptosis in HT-29 cells (P < 0.001). Co-incubation of 200 ng/ml IGFBP-3 with butyrate (5 mM) resulted in the potentiation of its apoptosis (P < 0.0001), which was not rescued by co-incubation with IGF-I (P < 0.0001). The addition of UO126 caused a decrease in cell number and increased the effects of IGFBP-3. IGFBP-3 is differentially expressed between stromal and epithelial components of normal and malignant colon, which may reflect its pro-apoptotic, IGF-I-independent effect on colonic epithelial cells. These effects are mediated in part by the PI-3K pathway in contrast to the MAP kinase pathway used by IGF-I.

Conventional histopathologic staging of primary colorectal cancers does not allow accurate prognostic stratification within a given tumour stage. Therefore, PCR-based assays are increasingly used to try to predict more accurately the likelihood of disease progression for the individual patient. Real-time reverse transcription PCR (RT-PCR) assays were used to detect and quantitate cytokeratin 20 (ck20), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC) mRNA in 149 lymph nodes (LN) from 17 patients with benign disease and 302 LN from 42 patients with colorectal cancer who had curative (R0) resections. None of the markers were specific, with ck20, CEA and GCC mRNA detected in 47%, 89% and 13% of 149 LN, respectively, from patients with benign disease. The sensitivity of all 3 markers was very high, with mRNA detected in 93%, 100% and 97% of 30 histologically involved LN, respectively. There was significant overlap in the mRNA levels of all 3 markers between histologically involved and uninvolved LN. There was no association between mRNA levels and distant recurrence (median follow-up: 3.94 years, range 3.35-5.12). We conclude that the use of molecular techniques to detect occult disease in LN may suffer from the same limitations as conventional methods. Instead, accurate prognostic stratification requires careful assessment of the likely metastatic potential of the primary cancer., (Copyright 2003 Wiley-Liss, Inc.)

Tumour development and metastasis are associated with altered gene expression profiles. The aim of this study was to identify the transcriptional differences in normal, tumour and metastatic tissue. We used oligonucleotide arrays to identify differential expression patterns of insulin-like growth factor 2 (IGF 2) between 139 primary colorectal tumour specimens and 42 tumour-adjacent mucosa specimens from colorectal cancer (CRC) patients. The expression levels of the IGF 2 gene were significantly increased in primary tumours compared with adjacent mucosae. This was concordant with our real-time RT-PCR quantification of 48 matched tumour mucosa samples. IGF 2 expression levels were also measured by RT-PCR quantitative analysis in 18 liver metastases and 10 normal tissues from patients without cancer. The mRNA levels were significantly under-expressed in liver metastases compared with either colorectal tumours or adjacent normal mucosae. The non- malignant normal tissue expressed significantly lower IGF 2 levels than adjacent normal tissue, and this was not due to a field effect originating from the tumour. In addition, our microarray data demonstrated that IGF 2 expression was down-regulated in sporadic microsatellite instability (MSI-H) CRC and parallels under-expression of hMLH1 and IGF 2 receptor genes in these patients. We conclude that IGF 2 plays an important role in CRC development. Also, individuals with loss of genomic imprinting (LOI) causing over-expression of IGF 2 may be at greater risk of developing CRC. However, this LOI may be reversed in MSI-H patients., (Copyright 2004 S. Karger AG, Basel)

An outpatient diagnostic index was used to analyse data relating to a sample of new patients attending the Southampton Eye Hospital in 1979 and 1986. Our main findings are an increased demand for cataract surgery by an increasingly aged population and a trend towards operating on cataracts at an earlier stage in their development. These and other results are discussed.

Although it had been known for several years that central venous injections of hypertonic salt solutions with added dextran could effectively resuscitate animals from hemorrhagic shock, it was not known whether peripheral injections could result in the same beneficial effects. Chronically instrumented, unrestrained, and unanesthetized sheep were subjected to a moderate degree of hemorrhagic shock and then resuscitated with a 2-minute infusion of 7.5% NaCl/6% dextran 70 in a volume of 5 ml/kg body weight. Infusions were made into the cephalic vein, the femoral artery, or, centrally, the superior vena cava. All three routes of injection promptly reestablished arterial pressure and cardiac output. All gave equivalently good restoration of plasma volume. None of the injections damaged the vessels, as determined either by gross inspection or by histologic examination. Thus the solution was safe and effective when given peripherally. It might be useful in the field resuscitation of hypovolemic patients.

We resuscitated unanesthetized bled sheep (bled volume = 1.2-1.7 liters) with 200 ml of hypertonic saline/dextran 70 infused either through a peripheral vein (n = 6) or directly into the red marrow of the sternum (n = 6). Intraosseous infusion of the viscous 7.5% NaCl/6% dextran solution required 2-4 min. Plasma sodium was rapidly increased to the same level in both groups demonstrating equally rapid entry into the vascular space. Both regimens provide rapid and sustained normalization of arterial pressure and cardiac output. No significant differences between the two groups were apparent for any measured variable. Intraosseous infusion of hypertonic resuscitation fluids merits further research to evaluate the safety and efficacy for prehospital treatment of hypovolemia and trauma.