Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy--a single centre, open-label study.

INTRODUCTION: With the development of increasing numbers of potential therapeutic agents in inflammatory disease comes the need for effective biomarkers to help screen for drug efficacy and optimal dosing regimens early in the clinical trial process. This need has been recognized by the Outcome Measures in Rheumatology Clinical Trials (OMERACT) group, which has established guidelines for biomarker validation. To seek a candidate synovial biomarker of treatment response in psoriatic arthritis (PsA), we determined whether changes in immunohistochemical markers of synovial inflammation correlate with changes in disease activity scores assessing 28 joints (ΔDAS28) or magnetic resonance imaging synovitis scores (ΔMRI) in patients with PsA treated with a biologic agent. METHODS: Twenty-five consecutive patients with PsA underwent arthroscopic synovial biopsies and MRI scans of an inflamed knee joint at baseline and 12 weeks after starting treatment with either anakinra (first 10 patients) or etanercept (subsequent 15 patients) in two sequential studies of identical design. DAS28 scores were measured at both time points. Immunohistochemical staining for CD3, CD68 and Factor VIII (FVIII) was performed on synovial samples and scored by digital image analysis (DIA). MRI scans performed at baseline and at 12 weeks were scored for synovitis semi-quantitatively. The ΔDAS28 of the European League Against Rheumatism good response definition (>1.2) was chosen to divide patients into responder and non-responder groups. Differences between groups (Mann Whitney U test) and correlations between ΔDAS28 with change in immunohistochemical and MRI synovitis scores (Spearman's rho test) were calculated. RESULTS: Paired synovial samples and MRI scans were available for 21 patients (8 anakinra, 13 etanercept) and 23 patients (8 anakinra, 15 etanercept) respectively. Change in CD3 (ΔCD3) and CD68 expression in the synovial sublining layer (ΔCD68sl) was significantly greater in the disease responders compared to non-responders following treatment (P = 0.005 and 0.013 respectively). ΔCD3, but not ΔCD68 or ΔFVIII, correlated with both ΔDAS28 (r = 0.49, P = 0.025) and ΔMRI (r = 0.58, P = 0.009). CONCLUSIONS: The correlation of ΔCD3 with ΔDAS28 and ΔMRI following biologic treatment in this cohort contributes to the validation of ΔCD3 as a synovial biomarker of disease response in PsA, and supports the further evaluation of ΔCD3 for predictive properties of future clinical outcomes.

Synovial tissue sublining CD68 expression is a biomarker of therapeutic response in rheumatoid arthritis clinical trials: consistency across centers.

OBJECTIVE: To determine whether the correlation between the mean change in disease activity and the mean change in synovial sublining (sl) CD68 expression could be demonstrated across different academic centers. METHODS: Synovial biopsies obtained at arthroscopy from patients with rheumatoid arthritis before and 160 days after rituximab therapy were selected and coded. Paired sections were processed independently at Amsterdam Medical Center (AMC) and at St. Vincent's University Hospital (SVUH), Dublin. Digital image analysis (DIA) was employed at both centers to quantify sublining CD68 expression. RESULTS: After analysis of CD68sl expression at centers in 2 different countries, high levels of intracenter and intercenter agreement were observed. For the pooled sections stained at AMC, the correlation between 2 investigators was R = 0.942, p = 0.000, and for sections stained at SVUH, R = 0.899, p = 0.001. Similarly, the intracenter correlations for DeltaCD68sl expression after treatment were R = 0.998, p = 0.000, for sections stained at AMC and R = 0.880, p = 0.000, for sections stained at SVUH. The intercenter correlation for the pooled scores of sections stained at AMC was R = 0.85, p = 0.000, and for the sections stained at SVUH, R = 0.62, p = 0.001. The consistent correlation between DeltaDAS (Disease Activity Score) and DeltaCD68sl expression across different studies (Pearson correlation = 0.895, p < 0.001) was confirmed. The standardized response mean values for DeltaCD68sl, calculated from analyses at both AMC and SVUH, were consistently 0.5 or greater, indicating a moderate to high potential to detect change. CONCLUSION: The correlation between mean DeltaDAS and mean DeltaCD68sl expression was confirmed across 2 centers. Examination of serial biopsy samples can be used reliably to screen for interesting biological effects at the site of inflammation at an early stage of drug development.

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis.

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3+ T cells, CD38+ plasma cells and CD68+ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland-Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.

Random or selective neuroanatomical connectivity. Study of the distribution of fibers over two populations of identified interneurons in cerebral cortex.

We present a neuroanatomical tracing method in a stereological approach to study the proportional distribution of fibers of a particular projection over two chemically different populations of neurons. The fiber projection from the presubiculum to the medial division of the entorhinal cortex of the rat serves as a model projection. Potential target interneurons express calcium binding proteins, either parvalbumin or calretinin. The three markers were simultaneously stained in one and the same histological section. The procedure is according to a three-phase procedure, i.e., in vivo tracer injection phase, histology phase, laserscanning phase. Steps involved are: (1) Surgical application to the presubiculum (injection) of the neuroanatomical tracer, biotinylated dextran amine (BDA), with the purpose of labeling fibers innervating the entorhinal cortex. After surgery, transport of the tracer takes place during the one-week survival period; (2) Fluorescence detection of the labeled fibers through staining with fluorochromated avidin (avidin-Alexa Fluor 488 [green fluorescence]); (3) Simultaneous Immunofluorescence detection of two interneuron markers (using the appropriate primary antibodies and secondary antibodies conjugated to the fluorochromes Alexa Fluor 594 [red fluorescence] and Alexa Fluor 633 [infrared fluorescence]); (4) Acquisition of low-magnification images in a confocal laserscanning microscope and the preparation on a computer of a montage image covering the entire entorhinal cortex; (5) Overlaying this montage with a sampling grid; (6) Acquisition at high magnification of Z-series of confocal images in a statistical valid way based on this grid. Each marker was visualized in its own laser excitation/emission channel: 488, 568 and 647 nm; (7) Image processing and 3D reconstruction followed by evaluation of the results. The present approach can be used to examine whether or not a particular class of chemically identified neurons receives preferential innervation by a particular fiber projection.

Tracing tools to resolve neural circuits.

In this paper we summarize neuroanatomical tracing methods, in particular combinations of methods designed to achieve the combined goals of tracing connectivity and extracting extra information from the projection or the target neurons. These combinations include techniques that identify projection fibres together with the neurons from which they originate, methods which establish the morphological or chemical identity of the target neurons and techniques to verify the presence of contacts between the terminal boutons on fibres and prospective target neurons. In the second part of this paper we describe a method recently developed in our laboratory, which consists of fluorescence tracing (introducing marker #1) combined with immunofluorescence with different fluorochromes (markers #2 and #3). This method enables us to observe the distribution of terminals of a particular set of projection fibres in contact with neurons belonging to two chemically different populations of interneuron. Multifluorescence confocal laser scanning is used for image acquisition of fibres and processes of presumed target neurons, and follow-up is by three-dimensional computer reconstruction. These reconstructions are used to determine that the contacts between the differentially labelled structures are real, i.e. with no optically empty space in between regardless of the angle of inspection. The specific experiments reported in this paper comprise the tracing with biotinylated dextran amine of fibres in the rat brain running from the presubiculum to layer III of the medial division of the entorhinal cortex. We studied the possibility of contacts in the latter area between the terminal boutons of these projecting fibres in association with presumed target neurons, i.e. parvalbumin and calretinin expressing interneurons.