Ultrasound-guided synovial biopsy: a safe, well-tolerated and reliable technique for obtaining high-quality synovial tissue from both large and small joints in early arthritis patients.

OBJECTIVE: To determine the tolerability, safety and yield of synovial tissue in an early arthritis cohort using a minimally invasive, ultrasound (US)-guided, synovial biopsy technique in small, medium and large joints. METHODS: 93 sequential biopsy procedures were assessed from a total of 57 patients (baseline and 36 repeat biopsies at 6 months) recruited as part of the 'Pathobiology of Early Arthritis Cohort' study. Patients completed a tolerability questionnaire prior to and following the synovial biopsy procedure. The synovial biopsy was performed under US guidance with US images of the joint recorded prior to each procedure. Synovial tissue was harvested for immunohistochemistry and RNA extraction. RESULTS: Five different joint sites were biopsied (knee, elbow, wrist, metacarpal phalangeal and proximal interphalangeal). No significant complications were reported following the procedure. No difference in pain, swelling and stiffness of the biopsied joint from before and after the procedure was demonstrated. A median of 14 biopsy samples was retrieved from each procedure with 93% of biopsy procedures yielding good quality tissue. RNA yield was good in all joints and in repeat biopsies. Multivariant analysis demonstrated a significantly greater yield of RNA and graded tissue in relation to a high prebiopsy, grey-scale synovitis score (0-3, semiquantitative). CONCLUSIONS: A minimally invasive approach to synovial tissue harvesting, using US guidance, is both safe and well-tolerated by patients. Tissue quality/RNA yield is preserved in subsequent biopsies following therapeutic intervention. A high US grey-scale synovitis score is a predictor of good quality/quantity of tissue and RNA.

Biomarkers and personalised medicine in rheumatoid arthritis: a proposal for interactions between academia, industry and regulatory bodies.

Rheumatoid arthritis (RA) is one of the most appropriate conditions for the application of personalised medicine as a high degree of heterogeneity has been recognised, which remains to be explained. Such heterogeneity is also reflected in the large number of treatment targets and options. A growing number of biologics as well as small molecules are already in use and there are promising new drugs in development. In order to make the best use of treatment options, both targeted and non-targeted biomarkers have to be identified and validated. To this aim, new rules are needed for the interaction between academia and industry under regulatory control. Setting up multi-centre biosample collections with clear definition of access, organising early, possibly non-committing discussions with regulatory authorities, and defining a clear route for the validation, qualification and registration of the biomarker-drug combination are some of the more critical areas where effective collaboration between the drug industry, academia and regulators is needed.

Circulating Reg1α proteins and autoantibodies to Reg1α proteins as biomarkers of ß-cell regeneration and damage in type 1 diabetes.

Type 1 diabetes is an autoimmune disease where ß-cells are in a constant process of death and renewal. Reg genes play a role in ß-cells regeneration. Reg proteins may be target of an autoimmune response in type 1 diabetes with consequent production of autoantibodies and failure of regeneration. The objective of this work was to characterize the role of Reg1α proteins and anti-Reg1α antibodies as biomarkers of ß-cell regeneration and damage. Serum levels of Reg1α protein were investigated in 87 type 1 diabetic subjects (31 newly diagnosed and 56 long standing), 63 type 2 diabetic subjects, 39 subjects with systemic lupus erythematosus (SLE), a nonpancreatic autoimmune disorder, and 64 healthy subjects. The presence of anti-Reg1α antibodies and correlation with metabolic, immune, and genetic parameters were analyzed in diabetic subjects. Increased levels of Reg1α protein were observed in newly diagnosed (p=0.002), and long standing (p=0.001) type 1 diabetes patients and type 2 diabetic subjects (p<0.001). Anti-Reg1α antibodies were found in 47% of patients with type 1 diabetes. No correlation was found with metabolic, immune, and genetic parameters. Patients with SLE showed no increase in Reg1α protein. We report here for the first time raised serum Reg1α protein in type 1 and type 2 diabetes and anti-Reg1α antibodies in type 1 diabetes. Reg1α levels appear not to be influenced by genetic or metabolic control. These findings allow considering future studies on Reg1α protein and autoantibody as new tools in the evaluation and monitoring of ß-cells regeneration and autoimmunity.

The P. gingivalis Autocitrullinome Is Not a Target for ACPA in Early Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a chronic inflammatory disease affecting primarily the joints, is frequently characterized by the presence of autoimmune anticitrullinated protein antibodies (ACPA) during preclinical stages of disease and accumulation of hypercitrullinated proteins in arthritic joints. A strong association has been reported between RA and periodontal disease, and Porphyromonas gingivalis, a known driver of periodontitis, has been proposed as the microbial link underlying this association. We recently demonstrated P. gingivalis-mediated gut barrier breakdown and exacerbation of joint inflammation during inflammatory arthritis. In the present study, we investigated another potential role for P. gingivalis in RA etiopathogenesis, based on the generation of ACPA through the activity of a unique P. gingivalis peptidylarginine deiminase (PPAD) produced by this bacterium, which is capable of protein citrullination. Using a novel P. gingivalis W50 PPAD mutant strain, incapable of protein citrullination, and serum from disease-modifying antirheumatic drug-naïve early arthritis patients, we assessed whether autocitrullinated proteins in the P. gingivalis proteome serve as cross-activation targets in the initiation of ACPA production. We found no evidence for patient antibody activity specific to autocitrullinated P. gingivalis proteins. Moreover, deletion of PPAD did not prevent P. gingivalis-mediated intestinal barrier breakdown and exacerbation of disease during inflammatory arthritis in a murine model. Together, these findings suggest that the enzymatic activity of PPAD is not a major virulence mechanism during early stages of inflammatory arthritis.

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.

A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury.

OBJECTIVES: To generate and validate a murine model of joint surface repair following acute mechanical injury. METHODS: Full thickness defects were generated in the patellar groove of C57BL/6 and DBA/1 mice by microsurgery. Control knees were either sham-operated or non-operated. Outcome was evaluated by histological scoring systems. Apoptosis and proliferation were studied using TUNEL and Phospho-Histone H3 staining, respectively. Type II collagen neo-deposition and degradation were evaluated by immunostaining using antibodies to the CPII telopeptide and C1,2C (Col2-3/4Cshort), respectively. Aggrecanases and matrix metalloproteinases (MMPs) activity were assessed by immunostaining for TEGE(373) and VDIPEN neo-epitopes. RESULTS: Young 8-week-old DBA/1 mice displayed consistent and superior healing of the articular cartilage defect. Age-matched C57BL/6 mice repaired poorly and developed features of osteoarthritis (OA). Compared to C57BL/6, DBA/1 mice displayed a progressive decline of chondrocyte apoptosis, cell proliferation within the repair tissue, persistent type II collagen neo-deposition, less type II collagen degradation, less aggrecanases and more MMP-induced aggrecan degradation. Eight-month-old DBA/1 mice failed to repair, but, in contrast to age-matched C57BL/6 mice, developed no signs of OA. CONCLUSION: We have generated and validated a murine model of cartilage regeneration in which the outcome of joint surface injury is strain and age dependent. This model will allow, for the first time, the dissection of different pathways involved in joint surface regeneration in adult mammals using the powerful technology of mouse genetics.

Cellular immune mechanisms in rheumatoid arthritis and other inflammatory arthritides.

Advances in molecular genetics, cellular immunology and microbiology have offered promise in unravelling the aetiopathogenesis of inflammatory arthritides such as rheumatoid arthritis and reactive arthritis. Such insights are challenging the orthodox view that these diseases are primarily autoimmune in nature, and should lead to exciting and novel therapeutic approaches.

Modulation of cellular annexin I in human leukocytes infiltrating DTH skin reactions.

Based on our previous studies showing endogenous annexin I being depleted from migrated neutrophils (PMN) in vitro, we have tested whether the levels of this glucocorticoid-regulated protein in PMN and mononuclear cells (PBMC) were modified after adhesion to endothelial monolayers in vitro and extravasation into skin blisters in vivo. In vitro, annexin I levels were depleted more significantly (-70%) in post-adherent PMNs than in monocytes (-25%) and lymphocytes (-50%, only in the positive fraction). In vivo, a significant time-dependent increase (approximately threefold, P < 0.05) in cell-associated annexin I was measured in PBMCs recovered from the blisters, whereas no significant changes were detected in extravasated PMNs. This was associated with annexin I release in the blister fluids (approximately 35 ng/mL), whereas no detectable protein was found in matched-paired plasmas. In conclusion, we report for the first time an activation of the annexin I pathway during an ongoing experimental inflammatory response in humans, which is differently regulated between PMNs and PBMCs.

Comparison of the effects of oral versus intravenous methylprednisolone regimens on peripheral blood T lymphocyte adhesion molecule expression, T cell subsets distribution and TNF alpha concentrations in multiple sclerosis.

We investigated in multiple sclerosis the difference between two commonly used oral and intravenous steroid regimens on the level of adhesion molecule expression on blood T lymphocytes, the distribution of circulating T cell subsets, and the concentration of serum tumour necrosis factor alpha. Venous blood samples were collected from a cohort of 22 patients with acute relapses who were participating in a randomised trial comparing intravenous methylprednisolone 1000 mg daily for 3 days with oral methylprednisolone 48 mg daily for one week, 24 mg daily for one week, and finally 12 mg daily for one week. There was a similar significant reduction of T cell LFA-1 surface expression and serum TNF alpha concentrations after 4 days of treatment with each regimen. There was no change in other lymphocyte adhesion molecules expression (ICAM-1, LFA-3 or CD2) at day 4, although LFA-3 and CD2 expression was moderately decreased at day 28 and day 90 respectively; nor was there any change in the distribution of lymphocyte subsets (CD4, CD8, and CD45RA, CD45RO), although a small decrease in CD45RO circulatory T cells was noted at day 28. This study suggests that some of the beneficial effects of glucocorticosteroids may be related to the inhibition of lymphocyte adhesion as well as the modulation of proinflammatory cytokines.

Neuroendocrine, immunologic, and microvascular systems interactions in rheumatoid arthritis: physiopathogenetic and therapeutic perspectives.

OBJECTIVE: To review the "core" systems interactions in rheumatoid arthritis (RA): neuroendocrine, immunologic, and microvascular, and to interpret an integrated physiopathogenesis of the disease, beginning at a preclinical phase of risk factors to the later stages of manifest clinical inflammation. METHODS: Publications on stress reactions, serum hormonal levels, biological mediators of inflammation and vascular alterations in RA during its preclinical phase, course of active disease, including pregnancy, and hormonal therapy of active disease were retrieved. In addition, experimental reports on biological models of the disease were considered. Levels of adrenal and gonadal steroids (ie, glucocorticosteroids [GCS], dehydroepiandrosterone [DHEA], its sulfate [DHEAS], estradiol [E2], and testosterone [T]), as well as prolactin (PRL) and other hormones, biological mediators, vascular endothelial system (VES) interactions with hormones, and immunologic mediators of inflammation in RA, were reviewed and interpreted. RESULTS: Women with premenopausal onset of RA not previously treated with GCS had lower basal serum levels of adrenal androgens, that is, DHEA or DHEAS, both before and after onset of clinical disease, compared with controls. Risk factors, including hormonal, immunologic, and hereditary indicators, were found to be uniformly present many years before clinical onset in such younger women, as compared with a frequency of circa 15% in matched controls. Also, a history of heavy cigarette smoking significantly predicted the onset of RA in perimenopausal women, and in men, suggesting that vascular endothelial alterations predispose to the disease. In the same prospective study, 1 or more of 4 risk factors were present an average of 12 years before clinical onset of disease in 83% of male RA cases versus 26% in matched controls (ie, sensitivity of 83% and specificity of 74%). Early RA patients may have lower serum cortisol levels than normal controls, and less than expected for the degree of ongoing inflammation, as well as having upregulated PRL levels. CONCLUSION: Among persons genetically prone to RA, the "core" systems are hypothesized to become "remodeled" during a long preclinical phase as a result of chronic imbalances in their interactive homeostasis. This hypothesis needs to be critically assessed in further studies of such physiological precursors of disease as well as stressors in the development and course of RA. Optimal hormonal management of biological mediators of RA is also a priority challenge for disease control in the future. RELEVANCE: Evidence indicates that men and women who are susceptible to premenopausal onset of RA can each be identified long before their clinical onsets of disease, and that productive research in primary prevention is an achievable objective. Disease prevention objectives are central in the public health strategy of the National Arthritis Action Plan and of the US Public Health Service "Healthy People 2000" (and 2010 proposed). Success in such prevention goals can be expected to significantly reduce the enormous burden of this common disease, which affects all segments of the population.

Human retroplacental sera inhibit the expression of class II major histocompatibility antigens.

Since factor(s) present in human pregnancy sera may interfere with HLA-DR expression on antigen-presenting cells which could account for fetal immune tolerance, we decided to investigate HLA-DR expression on the human myeloid macrophage cell line U937 using the monoclonal antibody RF DR2 and flow cytometry. Following incubation of U937 cells with recombinant interferon gamma (rIFN gamma) and fetal calf serum, 76% of the cells were HLA-DR positive. In contrast, when U937 cells were incubated with retroplacental serum (RP) only 40% of them were positive for HLA-DR and the mean fluorescent intensity for the cell population was significantly diminished. By performing these experiments at 37 and at 4 degrees C we concluded that a factor or factors present in RP bind onto and mask class II major histocompatibility (MHC) antigen expression.

The vascular endothelial system in the pathogenesis of inflammation and systemic rheumatic diseases: relation to the neuroendocrine system.

There is no doubt that the VES plays a central role in the pathogenesis of immune-mediated and inflammatory conditions. Equally, there is no doubt about the strong influence played by the neuroendocrine-immune system on the pathophysiology and homeostasis of the VES. Nevertheless, much remains to be done to unravel the precise mechanisms by which these systems interact in determining the microvascular dysfunction associated with chronic immune-mediated inflammation.

Pathogenesis of rheumatoid arthritis. The role of T cells and other beasts.

The evidence coming from the different experimental approaches reviewed in this article strongly supports the hypothesis that RA is T-cell driven at all stages of the disease. Although the effector phases responsible for the events that lead to joint destruction involve several different cell types, cytokines, and other mediators, T cells still direct operations behind the scenes. Direct experimental proof of this proposition in patients is still lacking, but the development of nondepleting modulating CD4 monoclonal antibodies may provide new tools to test this hypothesis. In this respect, it is encouraging that using one such reagent, we have recently shown that not only did the activity of the disease improve but, more importantly, the inflammatory indices and production of non-T-cell cytokines were reduced. This is not to dissimilar from the results of experiments described in animals, where by blocking synovial T cells, the production of IL-1 beta and TNF alpha could be decreased by more than 90%. From this perspective, it may be predicted that by modulating T cells in the joint, it is possible to achieve our ultimate goal of permanently switching off the disease.

Adhesion and migration of inflammatory cells.

Adhesion molecules play a major role in many biological functions. From the immunological point of view they are involved in virtually every process involving cell contact from thymic selection to antigen priming, from antigen recognition to cell activation, from cytotoxicity to lymphocyte recirculation. During an inflammatory process leucocyte migration represents one of the most important mechanisms of defense. At the site of the lesion inflammatory cells become activated and differentiate into effector cells. If the insult persists, mononuclear cells are retained in the attempt to clear the noxious stimulus. In this article the mechanisms of cell migration are reviewed together with the role of adhesion molecules in cell activation and local retention. Finally, the new therapeutic modalities against adhesion molecules are briefly considered.

Role of chemokines and chemokine receptors in regulating specific leukocyte trafficking in the immune/inflammatory response.

Antigen recognition, lymphocyte priming and effector responses in inflamed tissues depend on a coordinated and sequential series of events that take place in different anatomical compartments. The integration of these processes is favoured by the dynamic capacity of leukocytes to recirculate between the bloodstream and specific organs and to navigate inside the tissues in a programmed fashion, regulated by a complex interaction of cell adhesion molecules and soluble chemoattractants, in particular chemokines. In this review we discuss the role of chemokines and chemokine receptors in regulating leukocyte trafficking in different anatomical sites in the context of distinct functional phases of the immune/inflammatory response.

The sensitivity and specificity of reduced CD8 lymphocyte levels in the diagnosis of polymyalgia rheumatica/giant cell arteritis.

The aim of this study was to determine whether the sensitivity and specificity of measuring CD8+ T lymphocyte percentages and numbers could be diagnostically useful in the assessment of polymyalgia rheumatica/giant cell arteritis (PMR/GCA). Double immunofluorescence and FACScan analysis were used to identify and quantify the CD3+CD8+ lymphocyte population. The absolute cell numbers were determined by a Coulter Counter. Random disease controls were used for comparison with 30 patients diagnosed as PMR/GCA. Both the percentage of CD3+CD8+ cells (17.4% PMR/GCA group vs 30.5% control) and the absolute numbers of CD8+ lymphocytes (310 cell/ml PMR/GCA vs 723 cells/ml control) were significantly reduced (p < 0.001) in the PMR group. The sensitivity of reduced CD8+ percentages for diagnosis was 73% and the specificity was 85%. Analysis of likelihood ratios showed that CD8+ lymphocyte percentages < 22.15% were five times more likely in patients with PMR/GCA than in controls. Data suggested that reduced CD8+ lymphocyte levels may be useful in the initial clinical assessment of patients with PMR/GCA. Only a larger study can establish whether these parameters should be used as diagnostic criteria.

The role of interleukin-8 and other cytokines in the pathogenesis of Felty's syndrome.

OBJECTIVE: Felty's syndrome (FS) is defined as rheumatoid arthritis (RA) with neutropenia and, in some cases, splenomegaly; the outcome is primarily determined by the risk of infection, which is related to the degree of neutropenia. We analysed whether the clinical manifestations of FS, especially neutropenia, could be explained by abnormalities in cytokine production. METHODS: We examined the production in FS of five cytokines involved in the maturation and activation of polymorphonuclear cells (PMNs): IL-1 beta, TNF alpha, IL-8, G-CSF and GM-CSF. Because of the role of systemic IL-8 in neutrophil migration, serum IL-8 levels were also evaluated. RESULTS: Spontaneous and anti-CD16 stimulated cytokine production was similar in FS, RA and healthy controls (NC). However, anti-CD3 stimulated IL-8 production was significantly increased compared to NC in both RA and FS. FS patients who spontaneously produced G-CSF in culture were protected from bacterial infections. Serum IL-8 levels were elevated in FS and RA compared to NC (p < 0.001 for both groups). In FS, serum IL-8 was higher in patients with a history of bacterial infections compared to those without (p < 0.01) and there was a weak inverse correlation between neutropenia and serum IL-8 levels (Kendal's tau B = -0.31, p = 0.05). CONCLUSION: The neutropenia of FS cannot be explained by changes in peripheral blood cytokine production, although changes in the bone marrow microenvironment cannot be excluded. Our data do suggest a possible role for G-CSF and IL-8 in the development of certain FS complications.

[Glucocorticoids at the threshold of the new millennium: recent findings on the anti-inflammatory and immunomodulator mechanisms of action and future perspectives]. / I glucocorticoidi alle soglie del nuovo millennio: recenti acquisizioni sui meccanismi d'azione antiflogistico-immunomodulanti e prospettive future.

The aim of this work was to provide an updated review of the mechanisms of action of glucocorticoids. We carried out a MEDLINE search (1970 to present) using "glucocorticoids" as the keyword, both on its own (subheadings: "genetics, immunology, metabolism, physiology, therapeutic use") and combined with "inflammation", "glucocorticoid response element", "gene expression regulation", "NF-kappa B", "transcription factor AP-1", "receptors, glucocorticoid", "chemokines", "cytokines", "cytokine receptors", "resistance", "sensitivity", "annexin-I", "apoptosis", "repressors" and "activators", respectively. Original, partially unpublished data from our research group was also reported. Although we reviewed the sources available, we did not adopt any statistic procedures for data extraction, as the review deals only with basic research. The results of this review indicate that glucocorticoids act through different mechanisms: they can regulate the transcription of a number of genes (genomic mechanisms), interfere with cell activation factors (mechanisms of repression of cell activation factors), and inhibit cell activation via a direct interaction with the cell membrane and/or some of its components (non-genomic mechanisms). There is some evidence that most of the anti-inflammatory effects of glucocorticoids are mediated by repression of transcriptor factors, whereas their metabolic effects appear to be predominantly mediated by genomic mechanisms. This observation has prompted the search for new steroid compounds endowed with more selective anti-inflammatory properties than those currently available. We conclude that better understanding of the mechanisms of action of steroids may result in the development of new molecules with a better risk/benefit ratio.

[Role of chemokines in the pathogenesis of chronic synovitis during rheumatoid arthritis]. / Ruolo delle chemochine nella patogenesi della sinovite cronica in corso di artrite reumatoide.

Chemokines play a central role in the pathogenesis of rheumatoid arthritis (RA) synovitis which is characterised by new blood vessel formation, thickening of the lining layer and infiltration of immune cells. The inflammatory infiltrate is generated by a series of events which include the recruitment of leukocytes from the blood stream into the tissue, their local retention and activation to effector cells. All these processes are finely regulated by the interplay of different cell adhesion molecules (CAMs) and chemoattractant factors called chemokines (CK). CK are a superfamily of small proteins that play a crucial role in immune and inflammatory reactions. These chemoattractant cytokines share structural similarities including four conserved cysteine residues which form disulphide bonds in the tertiary structure of the proteins. CK mediate their effects by binding specific receptors (CK-R) characterised by a domain structure which spans the cell membrane seven times and signal through heterotrimeric GPT-binding proteins. Activation of the CK network results in an amplification of the inflammatory cascade and consequently in the progressive destruction of RA joints. The recognition of the central role of CK in inflammation has paved the way to the development of new agents capable of interfering with CK and CK-R. This review will focus in particular on the role of CK in regulating leukocyte trafficking in RA joints.