Phenotype-specific therapeutic efficacy of ilofotase alfa in patients with sepsis-associated acute kidney injury.

BACKGROUND: There is no effective treatment for sepsis-associated acute kidney injury (SA-AKI). Ilofotase alfa (human recombinant alkaline phosphatase) has been shown to exert reno-protective properties, although it remains unclear which patients might be most likely to benefit. We aimed to identify a clinical phenotype associated with ilofotase alfa's therapeutic efficacy. METHODS: Data from 570 out of 650 patients enrolled in the REVIVAL trial were used in a stepwise machine learning approach. First, clinical variables with increasing or decreasing risk ratios for ilofotase alfa treatment across quartiles for the main secondary endpoint, Major Adverse Kidney Events up to day 90 (MAKE90), were selected. Second, linear regression analysis was used to determine the therapeutic effect size. Finally, the top-15 variables were used in different clustering analyses with consensus assessment. RESULTS: The optimal clustering model comprised two phenotypes. Phenotype 1 displayed relatively lower disease severity scores, and less pronounced renal and pulmonary dysfunction. Phenotype 2 exhibited higher severity scores and creatinine, with lower eGFR and bicarbonate levels. Compared with placebo treatment, ilofotase alfa significantly reduced MAKE90 events for phenotype 2 patients (54% vs. 68%, p = 0.013), but not for phenotype 1 patients (49% vs. 46%, p = 0.54). CONCLUSION: We identified a clinical phenotype comprising severely ill patients with underlying kidney disease who benefitted most from ilofotase alfa treatment. This yields insight into the therapeutic potential of this novel treatment in more homogeneous patient groups and could guide patient selection in future trials, showing promise for personalized medicine in SA-AKI and other complex conditions.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells.

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-ß. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

Polymorphisms in the multidrug-resistance 1 gene related to glucocorticoid response in rheumatoid arthritis treatment.

A substantial proportion of rheumatoid arthritis (RA)-patients experience an insufficient response to glucocorticoids, an important therapeutic agent in RA. The multidrug-resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is an efflux pump that actively transports substrates, such as glucocorticoids, out of the cell. We investigated if the variation in response might be explained by single-nucleotide polymorphisms (SNPs) in the MDR1 gene. RA-patients treated with intravenous methylprednisolone pulses (n = 18) or oral prednisone/prednisolone (n = 22) were included in a prospective cohort, and clinical response was measured after 5 and 30 days, respectively. The C1236T, G2677A/T, and C3435T SNPs were determined, and the functionality of P-gp was assessed by flow cytometry (Rhodamine efflux assay). Carriage of the G2677A/T SNP was significantly associated with response (OR = 6.18, p = 0.035), the other SNPs showed trends. Stratified for received treatment, the effect was only present in methylprednisolone treated patients. Mutant allele carriage significantly decreased functionality of P-gp in B cells, though had a smaller impact in other PBMC subtypes. Carriage of a MDR1 SNP was related to a response to methylprednisolone in this study, which his suggests that RA-patients carrying wild-type alleles might benefit from P-gp inhibition or administration of glucocorticoid analogues that are non-P-gp substrates.

Tolerability in man following inhalation dosing of the selective TLR7 agonist, AZD8848.

BACKGROUND: Many patients with asthma have a T-helper type 2 (Th2) driven inflammation of the lung, whereas toll-like receptor 7 (TLR7) agonists, by inducing type I interferons, inhibit Th2 responses. In man, oral or parenteral TLR7 agonists can induce influenza-like symptoms through systemic induction of type I interferons. Design of a TLR7 agonist that is only active in the lung could reduce the risk of side effects and offer a new means for treating asthma. We developed a TLR7 agonist antedrug, AZD8848, to determine its local and systemic effects in preclinical models and man. METHODS: In vitro cellular potencies for the TLR7 antedrug agonist, AZD8848, were determined along with pharmacokinetics and efficacy in a rat allergy model. Sputum and blood biomarkers were measured in single ascending and multiple ascending dose clinical studies following inhalation delivery of AZD8848 and tolerability assessed. RESULTS: AZD8848 was potent in cellular assays and pharmacokinetics confirmed lack of systemic exposure to AZD8848. Weekly lung dosing in an animal model showed efficacy 26 days beyond the final dose. In healthy volunteers, AZD8848 was initially well tolerated with target engagement being demonstrated by induction of CXCL10 in sputum. A second inhaled dose, given 1 week later, amplified the systemic interferon signal in more than half the participants and resulted in significant influenza-like symptoms. CONCLUSIONS: The antedrug design restricted the direct actions of AZD8848 to the lung. However, the type I interferon induced locally by TLR7 spilled over systemically, limiting the utility of this inhaled antedrug approach. TRIAL REGISTRATION NUMBER: NCT01560234, NCT01818869.

Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment.

INTRODUCTION: Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis. Alefacept (a lymphocyte function-associated antigen (LFA)-3 Ig fusion protein that binds to CD2 and functions as an antagonist to T-cell activation) has been shown to result in improvement in psoriasis but has limited effectiveness in PsA. Interleukin-20 (IL-20) is a key proinflammatory cytokine involved in the pathogenesis of psoriasis. The effects of alefacept treatment on IL-20 expression in the synovium of patients with psoriasis and PsA are currently unknown. METHODS: Eleven patients with active PsA and chronic plaque psoriasis were treated with alefacept (7.5 mg per week for 12 weeks) in an open-label study. Skin biopsies were taken before and after 1 and 6 weeks, whereas synovial biopsies were obtained before and 4 and 12 weeks after treatment. Synovial biopsies from patients with rheumatoid arthritis (RA) (n = 10) were used as disease controls. Immunohistochemical analysis was performed to detect IL-20 expression, and stained synovial tissue sections were evaluated with digital image analysis. Double staining was performed with IL-20 and CD68 (macrophages), and conversely with CD55 (fibroblast-like synoviocytes, FLSs) to determine the phenotype of IL-20-positive cells in PsA synovium. IL-20 expression in skin sections (n = 6) was analyzed semiquantitatively. RESULTS: IL-20 was abundantly expressed in both PsA and RA synovial tissues. In inflamed PsA synovium, CD68+ macrophages and CD55+ FLSs coexpressed IL-20, and its expression correlated with the numbers of FLSs. IL-20 expression in lesional skin of PsA patients decreased significantly (P = 0.04) 6 weeks after treatment and correlated positively with the Psoriasis Area and Severity Index (PASI). IL-20 expression in PsA synovium was not affected by alefacept. CONCLUSIONS: Conceivably, the relatively limited effectiveness of alefacept in PsA patients (compared with anti-tumor necrosis factor (TNF) therapy) might be explained in part by persistent FLS-derived IL-20 expression.

CCR5 blockade in rheumatoid arthritis: a randomised, double-blind, placebo-controlled clinical trial.

OBJECTIVE: C-C chemokine receptor type 5 (CCR5), a chemokine receptor expressed on T cells and macrophages, and its ligands are found in inflamed synovial tissue (ST) of patients with rheumatoid arthritis (RA). The rationale for testing CCR5 blockade in patients with RA was supported by the effects of a CCR5 antagonist in collagen-induced arthritis in rhesus monkeys. The effects of CCR5 blockade in patients with active RA were explored. METHODS: In this phase Ib randomised, placebo-controlled trial, treatment with an oral CCR5 inhibitor (SCH351125) in patients with active RA was evaluated. Clinical efficacy was assessed using European League Against Rheumatism and American College of Rheumatology response criteria. ST biopsies were taken before and after 28 days of treatment, and analysed for CCR5+ cells. In a subset of patients, MRIs of an inflamed joint were obtained before and after treatment. RESULTS: In all, 32 patients were included; 20 received SCH351125 and 12 placebo. Three patients who received SCH351125 did not complete the study due to adverse events; none of these were serious. No improvement was observed in the active treatment group compared to placebo. Results were consistent for clinical evaluation, ST analysis and MRI. CONCLUSION: This proof of concept study does not support the use of CCR5 blockade as a therapeutic strategy in patients with active RA.

Expression of chemokine receptor CXCR3 by lymphocytes and plasmacytoid dendritic cells in human psoriatic lesions.

In psoriasis, leukocytes that infiltrate skin lesions have been shown to be involved in the pathogenesis of this disease. Previous investigations reporting the presence of CXCR3(+) T lymphocytes in psoriatic lesional skin have suggested a role of this receptor in the recruitment of T cells into the lesion. The purpose of this study was to quantify the mRNA levels of CXCR3 and to perform a systematic analysis of the cell populations that express CXCR3 in human lesional and non-lesional psoriatic biopsies. We showed by real-time reverse transcriptase-polymerase chain reaction that the mRNA levels of CXCR3 and its ligands, CXCL9-11, were significantly elevated in psoriatic lesions, as compared to non-lesional samples. Serial cryostat sections of psoriasis skin biopsies were evaluated by immunohistochemistry. The number of CXCR3(+) cells was low in non-lesional tissues. Quantitative image analysis demonstrated significant increases in the number of both epidermal and dermal CXCR3(+) cells in lesional compared with non-lesional biopsies. The majority of CXCR3(+) cells were located in the dermis of the lesional skin and 74% were demonstrated to be CD3(+) T lymphocytes. A small number of CXCR3(+) cells were CD68(+) myeloid cells. In addition, we found that nearly all BDCA-2(+) plasmacytoid dendritic cells in the psoriatic biopsies were CXCR3(+). These findings support and extend prior reports suggesting the potential role for CXCR3 in the pathophysiology of plaque psoriasis, by mediating the recruitment of plasmacytoid dendritic cells and T cells into the developing lesions.

Involvement of breast cancer resistance protein expression on rheumatoid arthritis synovial tissue macrophages in resistance to methotrexate and leflunomide.

OBJECTIVE: To determine whether multidrug-resistance efflux transporters are expressed on immune effector cells in synovial tissue from patients with rheumatoid arthritis (RA) and compromise the efficacy of methotrexate (MTX) and leflunomide (LEF). METHODS: Synovial tissue biopsy samples obtained from RA patients before treatment and 4 months after starting treatment with MTX (n = 17) or LEF (n = 13) were examined by immunohistochemical staining and digital image analysis for the expression of the drug efflux transporters P-glycoprotein, multidrug resistance-associated protein 1 (MRP-1) through MRP-5, MRP-8, MRP-9, and breast cancer resistance protein (BCRP), and the relationship to clinical efficacy of MTX and LEF was assessed. RESULTS: BCRP expression was observed in all RA synovial biopsy samples, both pretreatment and posttreatment, but not in control noninflammatory synovial tissue samples from orthopedic patients. BCRP expression was found both in the intimal lining layer and on macrophages and endothelial cells in the synovial sublining. Total numbers of macrophages in RA patients decreased upon treatment; in biopsy samples with persistently high macrophage counts, 2-fold higher BCRP expression was observed. Furthermore, median BCRP expression was significantly increased (3-fold) in nonresponders to disease-modifying antirheumatic drugs (DMARDs) compared with responders to DMARDs (P = 0.048). Low expression of MRP-1 was found on synovial macrophages, along with moderate expression in T cell areas of synovial biopsy specimens from one-third of the RA patients. CONCLUSION: These findings show that the drug resistance-related proteins BCRP and MRP-1 are expressed on inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP-1, and LEF is a high-affinity substrate for BCRP, these transporters may contribute to reduced therapeutic efficacy of these DMARDs.

Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients.

OBJECTIVE: To determine the expression of folate receptor beta (FRbeta) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FRbeta than methotrexate (MTX). METHODS: Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FRbeta protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FRbeta messenger RNA (mRNA) levels were determined by reverse transcription-polymerase chain reaction analysis. Binding affinities of FRbeta for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FRbeta-transfected cells. Efficacy of FRbeta-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FRbeta-transfected cells. RESULTS: Immunohistochemical staining of RA synovial tissue showed high expression of FRbeta on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FRbeta mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FRbeta had a high binding affinity (20-77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FRbeta-transfected cells. CONCLUSION: Abundant FRbeta expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity-binding folate antagonists, of which BCG 945 may be a prototypical representative.

Quantitative image analysis of synovial tissue.

Quantitative image analysis is a form of imaging that includes microscopic histological quantification, video microscopy, image analysis, and image processing. Hallmarks are the generation of reliable, reproducible, and efficient measurements via strict calibration and step-by-step control of the acquisition, storage and evaluation of images with dedicated hardware and software. Major advantages of quantitative image analysis over traditional techniques include sophisticated calibration systems, interaction, speed, and control of inter- and intraobserver variation. This results in a well controlled environment, which is essential for quality control and reproducibility, and helps to optimize sensitivity and specificity. To achieve this, an optimal quantitative image analysis system combines solid software engineering with easy interactivity with the operator. Moreover, the system also needs to be as transparent as possible in generating the data because a "black box design" will deliver uncontrollable results. In addition to these more general aspects, specifically for the analysis of synovial tissue the necessity of interactivity is highlighted by the added value of identification and quantification of information as present in areas such as the intimal lining layer, blood vessels, and lymphocyte aggregates. Speed is another important aspect of digital cytometry. Currently, rapidly increasing numbers of samples, together with accumulation of a variety of markers and detection techniques has made the use of traditional analysis techniques such as manual quantification and semi-quantitative analysis unpractical. It can be anticipated that the development of even more powerful computer systems with sophisticated software will further facilitate reliable analysis at high speed.

Expression of the chemokine receptor CCR5 in psoriasis and results of a randomized placebo controlled trial with a CCR5 inhibitor.

Several reports have indicated that the chemokine receptor CCR5 and its ligands, especially CCL5 (formerly known as RANTES), may play a role in the pathogenesis of psoriasis. The purpose of this investigation was to examine the expression of CCR5 and its ligands in chronic plaque psoriasis and to evaluate the clinical and immunohistochemical effect of a CCR5 receptor inhibitor. Immunohistochemical analysis showed low but significant increased total numbers of CCR5 positive cells in epidermis and dermis of lesional skin in comparison to non-lesional skin. However, relative expression of CCR5 proportional to the cells observed revealed that the difference between lesional and non-lesional skin was only statistically significant in the epidermis for CD3 positive cells and in the dermis for CD68 positive cells. Quantification of mRNA by reverse transcriptase-polymerase chain reaction only showed an increased expression of CCL5 (RANTES) in lesional skin. A randomized placebo-controlled clinical trial in 32 psoriasis patients revealed no significant clinical effect and no changes at the immunohistochemical level comparing patients treated with placebo or a CCR5 inhibitor SCH351125. We conclude that although CCR5 expression is increased in psoriatic lesions, this receptor does not play a crucial role in the pathogenesis of psoriasis.

Microscopic measurement of inflammation in synovial tissue: inter-observer agreement for manual quantitative, semiquantitative and computerised digital image analysis.

OBJECTIVES: To evaluate inter-observer agreement for microscopic measurement of inflammation in synovial tissue using manual quantitative, semiquantitative and computerised digital image analysis. METHODS: Paired serial sections of synovial tissue, obtained at arthroscopic biopsy of the knee from patients with rheumatoid arthritis (RA), were stained immunohistochemically for T lymphocyte (CD3) and macrophage (CD68) markers. Manual quantitative and semiquantitative scores for sub-lining layer CD3+ and CD68+ cell infiltration were independently derived in 6 international centres. Three centres derived scores using computerised digital image analysis. Inter-observer agreement was evaluated using Spearman's Rho and intraclass correlation coefficients (ICCs). RESULTS: Paired tissue sections from 12 patients were selected for evaluation. Satisfactory inter-observer agreement was demonstrated for all 3 methods of analysis. Using manual methods, ICCs for measurement of CD3+ and CD68+ cell infiltration were 0.73 and 0.73 for quantitative analysis and 0.83 and 0.78 for semiquantitative analysis, respectively. Corresponding ICCs of 0.79 and 0.58 were observed for the use of digital image analysis. All ICCs were significant at levels of p<0.0001. At each participating centre, use of computerised image analysis produced results that correlated strongly and significantly with those obtained using manual measurement. CONCLUSION: Strong inter-observer agreement was demonstrated for microscopic measurement of synovial inflammation in RA using manual quantitative, semiquantitative and computerised digital methods of analysis. This further supports the development of these methods as outcome measures in RA.

Evidence-based review of biologic markers as indicators of disease progression and remission in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, immune-mediated inflammatory disease characterised by inflammation resulting in structural joint damage and functional disability. Tumour necrosis factor-alpha (TNFalpha) is a pivotal mediator and driver of inflammation in RA. Inflammation is closely related to the production of C-reactive protein (CRP), and a close correlation exists between serum CRP and TNFalpha levels. CRP levels are therefore a convenient, objective biomarker of disease activity. CRP correlates closely with changes in inflammation/disease activity, radiological damage and progression and functional disability. Identification of TNFalpha as a driver of RA progression has led to the introduction of TNFalpha-blocking agents and, subsequently, improvement of disease management. TNFalpha-blocking agents provide rapid, profound and sustained suppression of disease activity in correspondence with a marked reduction in CRP levels. A reduction in CRP level correlates closely with the positive clinical response to TNFalpha-blocking therapy. Thus, CRP levels can be used to predict, assess and monitor response to treatment with TNFalpha-blocking agents, and may be helpful in determining the optimal TNFalpha-blocker dosage. Given the close correlation between inflammation and disease progression and the relation between inflammation and CRP, the latter, if used effectively in clinical practice, may be means to identify patients likely to progress rapidly and who require intensive anti-TNFalpha therapy. The purpose of this review is to identify how CRP levels may be useful for monitoring the effect of therapy on halting disease progression and why monitoring CRP levels at baseline and after treatment should become a routine part of clinical practice.

Digital image analysis for the evaluation of the inflammatory infiltrate in psoriasis.

Traditionally the evaluation of the cellular infiltrate and protein expression in skin tissue sections is done by manual quantification. However, for reliable evaluation of histology in the development of new anti-psoriatic treatments there is a need for a more time-efficient and reproducible method. To test the use of digital image analysis (DIA) in this situation we compared the assessment of immunohistochemically stained skin sections with the more traditional manual quantification (MQ) and semi-quantitative analysis (SQA). The number of CD3+ T cells and the expression of E-selectin were evaluated in stained paired skin biopsies from 11 patients with chronic plaque psoriasis before and after initiation of anti-psoriasis therapy. We observed significant correlations between MQ and DIA for the number of T cells (epidermis: r=0.88, P< or =0.01, dermis r=0.87, P< or =0.01). Both DIA and MQ were equally effective in detecting reductions of T-cell numbers in active-treated patients. MQ took 20 h, compared to 6 h for DIA. We also observed significant correlations between SQA and DIA for the expression of E-selectin (r=0.88, P< or =0.01), although DIA was more sensitive than SQA to detect (early) changes. SQA took 10 h, compared to 4 h for DIA. In conclusion, the quantification of the inflammatory infiltrate in psoriatic lesional skin by DIA generated similar results as MQ and SQA in a reliable, reproducible and higher time efficient fashion.

Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes.

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.

Identification of the epidermal growth factor-TM7 receptor EMR2 and its ligand dermatan sulfate in rheumatoid synovial tissue.

OBJECTIVE: EMR2 and CD97 are closely related members of the epidermal growth factor (EGF)-TM7 family of adhesion class 7-span transmembrane (TM7) receptors. Chondroitin sulfates (CS) have recently been identified as ligands for EMR2 and CD97. CS have been implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the expression of EMR2 and the distribution of EMR2 and CD97 ligands within RA synovial tissue (ST). METHODS: ST samples were obtained by arthroscopy from 19 patients with RA, 13 patients with inflammatory osteoarthritis (OA), and 13 patients with reactive arthritis (ReA). Immunohistochemistry was performed with a monoclonal antibody against EMR2, and stained STs were analyzed by digital image analysis. Coexpression of EMR2 with cell lineage- and activation-specific markers was determined by double immunofluorescence microscopy. To evaluate the expression of EMR2 and CD97 ligands in RA synovium, binding assays were performed using EMR2- and CD97-specific multivalent fluorescent probes. RESULTS: EMR2 expression in the synovial sublining was found to be significantly higher in RA patients compared with OA and ReA control patients. Most EMR2+ cells were macrophages and dendritic cells expressing costimulatory molecules and tumor necrosis factor alpha. Dermatan sulfate was shown to be the ligand of the largest isoforms of EMR2 and CD97 in rheumatoid synovium. In addition, the smaller isoforms of CD97, but not those of EMR2, bound CD55 on fibroblast-like synoviocytes. CONCLUSION: The EGF-TM7 receptors EMR2 and CD97 are abundantly expressed on myeloid cells in ST of RA patients where their cognate ligands dermatan sulfate and CD55 are detected. These results suggest that these interactions may facilitate the retention of activated macrophages in the synovium.

Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis.

OBJECTIVE: To create greater understanding of the changes in synovial tissue parameters that occur in conjunction with clinical response by using an effective therapy, in order to facilitate the planning of future studies with therapeutic agents for rheumatoid arthritis (RA). METHODS: Twenty-one patients with active RA were randomized to receive either oral prednisolone (n = 10) or placebo (n = 11) for 2 weeks. In all patients, synovial tissue biopsy specimens were obtained by arthroscopy directly before treatment and after 14 days of treatment. Immunohistochemical analysis was performed to characterize the cell infiltrate and vascularity. Stained tissue sections were analyzed by digital imaging. Statistical analysis was performed using an analysis of covariance model. RESULTS: After treatment, the mean Disease Activity Score in 28 joints (DAS28) was 2.0 units lower (95% confidence interval [95% CI] 1.0-3.0) in patients who received prednisolone than in those who received placebo. In the prednisolone group, the mean (+/-SD) DAS28 decreased from 6.27 +/- 0.95 to 4.11 +/- 1.43 after therapy; minimal change was observed in the placebo group. For macrophages, the estimated effect of prednisolone was large. Patients receiving active treatment had fewer (mean 628 cells/mm(2) [95% CI 328-927]) macrophages after therapy compared with those receiving placebo. A reduction in the total number of CD68+ macrophages, from 1,038 +/- 283 cells/mm(2) before treatment to 533 +/- 248 cells/mm(2) after treatment, was observed in the prednisolone group. There were clear trends toward decreased infiltration by T cells, plasma cells, and fibroblast-like synoviocytes after active treatment. We observed a trend toward a reduction in alphavbeta3+ newly formed blood vessels and expression of vascular growth factors after prednisolone therapy. CONCLUSION: Prednisolone therapy in RA is associated with a marked reduction in macrophage infiltration in synovial tissue, suggesting that synovial macrophage numbers could be used as a biomarker for clinical efficacy.

The presence of citrullinated proteins is not specific for rheumatoid synovial tissue.

OBJECTIVE: Antibodies directed toward citrullinated proteins (e.g., anti-cyclic citrullinated peptide antibodies) are highly specific for rheumatoid arthritis (RA) and are produced locally at the site of inflammation. Although the presence of citrullinated proteins in rheumatoid synovium has been described in the literature, it is uncertain whether their presence is specific for RA. The present study was undertaken to investigate this. METHODS: The local production of the anti-citrullinated protein antibodies was investigated by comparing the concentration of the antibodies (corrected for the total amount of IgG present) in paired samples of serum and synovial fluid from RA patients. The presence of citrullinated proteins in the synovial tissue was investigated by immunohistochemical analysis of synovial tissue from RA patients and from patients with other arthropathies, using a variety of specific antibodies to citrullinated proteins. RESULTS: In RA patients, anti-citrullinated protein antibodies constituted a 1.4-fold higher proportion of IgG in synovial fluid compared with serum, which is indicative of a local production of the antibodies. Immunohistochemical staining of citrullinated proteins was observed in the lining layer, the sublining layer, and in extravascular fibrin deposits in inflamed synovial tissue from RA as well as non-RA patients. CONCLUSION: The presence of citrullinated proteins in the inflamed synovium is not specific for RA, but rather, it may be an inflammation-associated phenomenon. The high specificity of the anti-citrullinated protein antibodies is, therefore, most likely the result of an abnormal humoral response to these proteins.

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study.

Psoriasis and psoriatic arthritis are inflammatory diseases that respond well to anti-tumour necrosis factor-alpha therapy. To evaluate the effects of anti-tumour necrosis factor-alpha treatment on expression of adhesion molecules and angiogenesis in psoriatic lesional skin and synovial tissue, we performed a prospective single-centre study with infliximab therapy combined with stable methotrexate therapy. Eleven patients with both active psoriasis and psoriatic arthritis received infusions of infliximab (3 mg/kg) at baseline, and at weeks 2, 6, 14 and 22 in an open-label study. In addition, patients continued to receive stable methotrexate therapy in dosages ranging from 5 to 20 mg/week. Clinical assessments, including Psoriasis Area and Severity Index (PASI) and Disease Activity Score (DAS), were performed at baseline and every 2 weeks afterward. In addition, skin biopsies from a target psoriatic plaque and synovial tissue biopsies from a target joint were taken before treatment and at week 4. Immunohistochemical analysis was performed to detect the number of blood vessels, the expression of adhesion molecules and the presence of vascular growth factors. Stained sections were evaluated by digital image analysis. At week 16, the mean PASI was reduced from 12.3 +/- 2.4 at baseline to 1.8 +/- 0.4 (P