[Biomarkers and imaging for diagnosis and stratification of rheumatoid arthritis and spondylarthritis in the BMBF consortium ArthroMark]. / Biomarker und Bildgebung zur Diagnose und Stratifizierung der rheumatoiden Arthritis und Spondylarthritis im BMBF-Verbund ArthroMark.

Rheumatic diseases are among the most common chronic inflammatory disorders. Besides severe pain and progressive destruction of the joints, rheumatoid arthritis (RA), spondyloarthritides (SpA) and psoriatic arthritis (PsA) impair working ability, reduce quality of life and if treated insufficiently may enhance mortality. With the introduction of biologics to treat these diseases, the demand for biomarkers of early diagnosis and therapeutic stratification has been growing continuously. The main goal of the consortium ArthroMark is to identify new biomarkers and to apply modern imaging technologies for diagnosis, follow-up assessment and stratification of patients with RA, SpA and PsA. With the development of new biomarkers for these diseases, the ArthroMark project contributes to research in chronic diseases of the musculoskeletal system. The cooperation between different national centers will utilize site-specific resources, such as biobanks and clinical studies for sharing and gainful networking of individual core areas in biomarker analysis. Joint data management and harmonization of data assessment as well as best practice characterization of patients with new imaging technologies will optimize quality of marker validation.

[Relevance of big data for molecular diagnostics]. / Bedeutung von Big Data für die molekulare Diagnostik.

Big data analysis raises the expectation that computerized algorithms may extract new knowledge from otherwise unmanageable vast data sets. What are the algorithms behind the big data discussion? In principle, high throughput technologies in molecular research already introduced big data and the development and application of analysis tools into the field of rheumatology some 15 years ago. This includes especially omics technologies, such as genomics, transcriptomics and cytomics. Some basic methods of data analysis are provided along with the technology, however, functional analysis and interpretation requires adaptation of existing or development of new software tools. For these steps, structuring and evaluating according to the biological context is extremely important and not only a mathematical problem. This aspect has to be considered much more for molecular big data than for those analyzed in health economy or epidemiology. Molecular data are structured in a first order determined by the applied technology and present quantitative characteristics that follow the principles of their biological nature. These biological dependencies have to be integrated into software solutions, which may require networks of molecular big data of the same or even different technologies in order to achieve cross-technology confirmation. More and more extensive recording of molecular processes also in individual patients are generating personal big data and require new strategies for management in order to develop data-driven individualized interpretation concepts. With this perspective in mind, translation of information derived from molecular big data will also require new specifications for education and professional competence.

[New aspects on the pathogenesis of myositis]. / Neues zur Pathogenese der Myositiden.

Idiopathic inflammatory myopathies (IIM) are chronic inflammatory diseases of muscle characterized by proximal muscle weakness. There are three main groups of diseases, dermatomyositis, polymyositis and inclusion body myositis. The muscle tissue is invaded by the humoral autoantibody producing immune system (B-cells) and by the cellular immune system with autoaggressive and inflammation modulating cells (e.g. dendritic cells, monocytes/macrophages, CD4 + and CD8 + T-cells and natural killer cells). The presence of specific or associated autoantibodies and inflammatory cellular infiltrates with cytotoxic and immune autoreactive properties are characteristic for IIM diseases. The pathogenesis is still unknown; nevertheless, there are several hints that exogenic factors might be involved in initiation and disease progression and bacterial, fungal and viral infections are thought to be possible initiators. Up to now information on prognostic markers to help with decision-making for individual treatment are limited. In addition, there has been only limited therapeutic success including conventional or novel drugs and biologicals and comparative validation studies are needed using similar outcome measurements. Moreover, to facilitate the use and development of novel therapies, elaboration of intracellular and cell-specific regulation could be useful to understand the etiopathogenesis and allow a better diagnosis, prognosis and possibly also a prediction for individualized subgroup treatment.

[Biomarkers in rheumatology. Biomarkers and imaging for the diagnosis and stratification of rheumatoid arthritis and spondylarthritis in the ArthroMark network funded by the Federal Ministry of Education and Research]. / Biomarker in der Rheumatologie. Biomarker und Bildgebung zur Diagnose und Stratifizierung der rheumatoiden Arthritis und Spondylarthritis im BMBF-Verbund ArthroMark.

The introduction of biologics has continuously increased the demand for biomarkers for early diagnosis and therapeutic stratification. ArthroMark, a research network funded by the Federal Ministry of Education and Research, aims to establish such biomarkers for rheumatoid arthritis and spondyloarthritides. Biobanks and previous work on genotyping, gene expression and autoreactivity profiling build the basis. Bioinformatic networks will help to harmonize the investigations and a clinical study with modern imaging techniques to characterize the functional relevance of the new biomarkers as effectively as possible. To validate the markers for diagnostic application the network aims to expand gradually.

CD11c as a transcriptional biomarker to predict response to anti-TNF monotherapy with adalimumab in patients with rheumatoid arthritis.

We performed transcription profiling using monocytes to identify predictive markers of response to anti-tumor necrosis factor (anti-TNF) therapy in patients with rheumatoid arthritis (RA). Several potential predictors of response were identified, including CD11c. Validation in samples from independent cohorts (total of n = 27 patients) using reverse transcription-PCR confirmed increased expression of CD11c in responders to adalimumab (100% sensitivity; 91.7% specificity, power 99.6%; alpha = 0.01). Pretherapy CD11c levels significantly correlated with the response criteria as defined by the American College of Rheumatology (ACR) (r = 0.656, P < 0.0001). However, CD11c was neither predictive of response to methotrexate (MTX) alone (n = 34) nor to MTX in combination with adalimumab (n = 16). Clinical responders revealed a reset to a normal expression pattern of resident/inflammatory monocyte markers, which was absent in nonresponders. Therefore, an analysis of key cell types identifies potentially predictive biomarkers that may help to restrict the use of adalimumab to therapy responders. Larger studies, including studies of monotherapy with other drugs, are now needed to confirm and validate the specificity of CD11c for anti-TNF biologics.

Does gene expression analysis inform us in rheumatoid arthritis?

Transcription profiling has become a standard technology in research. It is mainly applied in the search for biomarkers to improve diagnostic and prognostic classification, to quantify disease activity and to predict or indicate response to therapy. This review will focus on rheumatoid arthritis and discuss considerations for sample selection, prerequisites for functional interpretation of data and the current status of information deduced in the field of biomarkers for the various clinical questions. In the next few years, prediction of response to treatment is the most important aim of biomarker research. With the growing number of new biological agents, there is increasing pressure to identify molecular parameters that will not only guide the therapeutic decision but also help to define the most important targets for which new biological agents should be tested in clinical studies.

[New aspects of molecular biology diagnosis. Array technology and expression profile for characterization of rheumatic diseases]. / Neue Aspekte der molekularbiologischen Diagnostik. Array-Technologie und Expressionsprofile für die Charakterisierung rheumatischer Erkrankungen.

Increasing availability of high throughput technologies, exponentially growing information about the human genome and gene expression, and the growing global network of databases on systematic biomedical information will change our view on inflammatory rheumatic diseases in a revolutionary way. Several research laboratories have already generated initial extensive datasets on gene expression analysis. Application of this technique has demonstrated that in addition to a precise analysis, verification and validation of the results also on the level of cell populations, a meticulous characterization of the patients according to current conventional clinical, laboratory, imaging and histological standards is essential. For functional interpretation, in vitro tests, animal models and therapeutic studies will provide further information. Bioinformatic structuring and development is needed for the large amounts of data. After an initial genome-wide screening, the objective is to identify those genes, which will allow characterization of the disease, classification according to molecular pathophysiology, evaluation of the prognosis and prediction of the correct and most potent therapeutic regimen. Derived from the improved knowledge about the molecular mechanisms, new and--as to expect--the crucial approaches for an effective therapy against chronic inflammation, organ destruction and pathological immune response in rheumatic diseases will emerge.

Macrophages in rheumatoid arthritis.

The abundance and activation of macrophages in the inflamed synovial membrane/pannus significantly correlates with the severity of rheumatoid arthritis (RA). Although unlikely to be the 'initiators' of RA (if not as antigen-presenting cells in early disease), macrophages possess widespread pro-inflammatory, destructive, and remodeling capabilities that can critically contribute to acute and chronic disease. Also, activation of the monocytic lineage is not locally restricted, but extends to systemic parts of the mononuclear phagocyte system. Thus, selective counteraction of macrophage activation remains an efficacious approach to diminish local and systemic inflammation, as well as to prevent irreversible joint damage.

Identification of known and novel genes in activated monocytes from patients with rheumatoid arthritis.

OBJECTIVE: To define gene activation patterns of monocytes (MO) in patients with rheumatoid arthritis (RA). METHODS: A complementary DNA (cDNA) library was constructed from first-leukapheresis MO obtained from an RA patient with active disease; 32P-labeled cDNA from first-leukapheresis MO (activated pool) and third-leukapheresis MO (nonactivated pool) were used as probes for differential hybridization. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess gene activation in MO from an additional 26 RA patients and 6 normal controls. RESULTS: Subtraction of genes from first- and third-leukapheresis MO resulted in 482 differentially expressed clones. In first-leukapheresis MO, these clones included the following: 1) interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, tumor necrosis factor alpha, growth-related oncogene alpha (GROalpha)/melanoma growth-stimulatory activity, macrophage inflammatory protein 2/GRObeta, ferritin, alpha1-antitrypsin, lysozyme, transaldolase, Epstein-Barr virus-encoded RNA 1 (EBER-1)/EBER-2-associated-protein, thrombospondin 1, an angiotensin receptor II (ATRII) C-terminal homolog, and RNA polymerase II elongation factor (elongin); 2) two clones homologous to functionally undefined genes (BSK-67 and BSK-83); and 3) three unknown cDNA sequences (BSK-66, 80, 89). In third-leukapheresis MO, the clones included differentiation genes (HOX-B3, thymosin-beta4, PU.1, glucocerebrosidase, MEL-18, and glucose-6-phosphate dehydrogenase) and 3 unknown/functionally undefined sequences. Differential expression of most genes from the activated pool was confirmed in leukapheresis samples from 2 additional RA patients. In MO from RA patients, not only were IL-1beta and the ATRII homolog significantly overexpressed (maximum 36-fold), but also 4 of the unknown/functionally undefined genes (maximum 102-fold). Notably, messenger RNA levels of BSK-89 correlated positively with the erythrocyte sedimentation rate (ESR), whereas those of BSK-83 correlated negatively with the ESR and C-reactive protein level. CONCLUSION: The combined strategy of gene subtraction and semiquantitative RT-PCR may allow the definition of MO activation patterns during different disease phases (including therapy-induced remission) and the identification of novel MO genes with pathogenetic relevance in RA.

Preactivated peripheral blood monocytes in patients with essential hypertension.

The purpose of this study was to investigate the possible involvement of human peripheral blood monocytes in the pathology of hypertensive disease. We determined the in vitro secretion patterns of proinflammatory cytokines obtained from isolated peripheral monocytes from normal controls and from hypertensive patients either after in vitro stimulation with angiotensin II (Ang II) with or without preincubation with an Ang II type 1 receptor antagonist (losartan) or after stimulation with lipopolysaccharide. Blood samples were obtained from 22 patients with essential hypertension (before any drug administration or after interruption of antihypertensive therapy) and from 24 normotensive healthy individuals used as a control group. Peripheral blood monocytes were isolated by density gradient centrifugation and plastic adherence. The state of monocyte activity was determined by the capacity to secrete tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6, (IL-6) either spontaneously or after stimulation. Cytokine concentrations were determined in culture supernatants by specific ELISA. Proinflammatory cytokine levels were assessed by semiquantitative reverse transcribed polymerase chain reaction. After stimulation with Ang II, the IL-1beta secretion of peripheral blood monocytes was significantly increased in hypertensive patients versus healthy individuals (P<0.05). In contrast, in monocytes preincubated with losartan before exposure to Ang II, IL-1beta secretion was diminished in both groups to comparable levels. The secretion of IL-1beta and TNF-alpha was significantly increased in peripheral blood monocytes from hypertensive patients versus healthy individuals after stimulation with lipopolysaccharide (TNF-alpha, P<0.02; IL-1beta, P<0.05). Upregulation of IL-1beta and TNF-alpha secretion in peripheral blood monocytes from hypertensive patients was also seen at the RNA level. Our results indicate preactivated peripheral blood monocytes in hypertensive patients. Ang II may be directly involved in the process of monocyte activation.

p205 is a major target of autoreactive T cells in rheumatoid arthritis.

OBJECTIVE: The p205 autoantigen and interleukin-2 (IL-2) function synergistically to stimulate T lymphocytes from patients with rheumatoid arthritis (RA), and a p205-derived amino acid sequence is identical to an immunoglobulin sequence located within a domain that is reactive with rheumatoid factors (RF). This study was conducted to analyze in detail the T cell immune response against p205 and to investigate whether immunity to p205 may play a role in T cell-mediated immunopathology in active RA. METHODS: Cibachron blue, protein A-Sepharose, and gel filtration on Sephacryl were used successively to enrich p205 from synovial fluid (SF). T lymphocytes from RA patients were isolated from the peripheral blood (PB), lymph nodes, and SF, and p205 and peptides derived from known sequences were assessed by T cell proliferation assays in the presence of IL-2. RESULTS: P205-specific proliferation of T cells was observed in PB as well as in SF. When p205 was isolated from RA SF, proliferation of RA T cells peaked on day 3. With p205 purified from SF from trauma patients, there was a significant shift of the maximum T cell proliferation to day 8. T cells were of CD4 or CD8 phenotype, and B cells did not proliferate to a significant degree. The T cell response to p205 was always higher for SF mononuclear cells (SFMC) compared with PBMC (P < 0.001). In 1 RA patient who underwent repeated leukapheresis, this led to a reproducible decline in p205-specific T cell proliferation to control levels. PB T cells specifically proliferating in response to p205 were detected in 20 of 32 RA patients (63%). Of 26 patients with other inflammatory rheumatic diseases, only 1 showed a minor response to p205, while normal donors did not demonstrate a significant T cell proliferation. A synthetic p205-derived peptide, with an amino acid sequence identical to an immunoglobulin sequence located in the area where RF binds, was reactive with T cells from RA patients. CONCLUSION: P205 appears to be a major target of autoreactive T cells in RA. P205-specific T cells are primed and more abundant at the site of inflammation. As a T cell target in RA, p205 may well be an antigen involved in the initiation of RF production.

Novel autoantibodies against muscle-cell membrane proteins in patients with myositis.

OBJECTIVE: To search for autoantibodies against muscle cell-specific surface membrane antigens in patients with inflammatory myopathies. METHODS: A cell enzyme-linked immunosorbent assay (cell ELISA) using a human rhabdomyosarcoma cell line (TE-671) was developed and performed in serial dilutions with either nonfixed or fixed cells. A total of 141 different patient sera were tested: 90 from patients with various rheumatic diseases, 12 from patients with cardiomyopathies, 25 from patients with other muscular diseases, and 14 from patients who had undergone major surgery or who had other noninflammatory diseases. As controls, 20 sera were obtained from healthy donors. Results were correlated using immunofluorescence staining and flow cytometry. RESULTS: Using the nonfixed cell ELISA, the proportions of positive sera from the patient groups with rheumatic diseases were 71% with polymyositis (PM), 15% with dermatomyositis (DM), 18% with systemic sclerosis (SSc), 15% with systemic lupus erythematosus (SLE), and 7% with rheumatoid arthritis. Sera from healthy donors, as well as sera from patients with nonrheumatic diseases, did not show significant reactivities. When other cell lines, including a chondrosarcoma, a bladder carcinoma, a pancreas carcinoma, and human foreskin fibroblasts, were used as substrates, positive sera did not react in the cell ELISA. Results obtained with the cell ELISA system using nonfixed cells were confirmed by flow cytometry and immunofluorescence staining. A strong protein band of 50 kd was detected on plasma membrane preparations from TE-671 muscle cells in 33% of PM sera (n = 12). CONCLUSION: In most sera from patients with PM, DM, and some other rheumatic diseases (i.e., SSc and SLE), autoantibodies directed against muscle-cell surface antigens can be detected. Since these molecules are localized in the muscle-cell surface membrane, autoantibodies directed against these antigens could play a major role in the pathogenesis of PM.

Biochemical characterization and microsequencing of a 205-kDa synovial protein stimulatory for T cells and reactive with rheumatoid factor containing sera.

Synovial fluid (SF) was found to possess stimulatory capacity for the proliferation of T cell clones derived from patients with rheumatoid arthritis (RA) when cultured together with IL-2. Using chromatography technique and gel electrophoresis, a synovial fluid protein with an apparent m.w. of 205 kDa (p205) was isolated that demonstrated a bioactivity analogous to that obtained with native synovial fluid. After electroelution, p205 dissociated into 70-kDa fragment(s). Upon IEF, it appeared as a single band with an isoelectric point of 6.5, suggesting a noncovalently bound trimer complex. Amino acid sequences of the whole protein and of tryptic peptides were determined by N terminal sequencing. The N terminal amino acid sequence of the 70-kDa fragment and of the tryptic peptides showed no identity to recently described protein sequences. One peptide matched, in 11 amino acid residues, with the human IgG1-4 constant heavy chain and rheumatoid factor (RF) binding region. The p205 induced the proliferation of peripheral blood T cells and long term T cell cultures that had been raised by alternate stimulation with IL-2 and p205. In a similar approach, synovial lining cells were shown to release a protein with biochemical characteristics similar to the synovial fluid-derived p205. Western blot analysis revealed the binding of RF-containing sera to p205, which was diminished by absorption with an RF reagent. These observations suggest that p205 is expressed by synovial cells and may be a target for T and B cells in RA.

Characterization of a 58- and a 78-kD monocytic membrane protein with affinity to the acetylcholine receptor in myasthenia gravis patients.

The autoimmune disease myasthenia gravis (MG), caused by the effect of specific antibodies, directed towards the nicotinic acetylcholine receptor, is triggered by autoantigen-specific T cells. In order to investigate cellular parts of the immune response in MG, the authors investigated the binding of the nicotinic acetylcholine receptor (AChR) to peripheral blood mononuclear cells (PBMC) from MG patients. AChR binding cells were identified by rosetting experiments using AChR-coated fluorescein beads. Applying this technique, a significant percentage of PBMC (21.2 +/- 7.65%) from MG patients formed rosettes with AChR-coated beads. Membrane preparations of nycodenz- or percoll-separated monocytes from MG patients or T-cell depleted monocytic subpopulations were applied to SDS-PAGE under reducing conditions. Ligand-blotting studies with biotinylated AChRs revealed two cell-membrane proteins with molecular weights of 58- and 78-kD. In parallel the same results were obtained by affinity chromatography of monocytic membrane proteins using AChR-sepharose. A possible interference of anti-AChR IgG was excluded. The 58- and the 78-kD proteins are detectable under reducing conditions by ligand blotting with AChR-biotin, while under non-reducing conditions only the 58-kD protein can be detected. Furthermore, in experiments using Endoglycosidase-H, the 58-kD protein appears to be non-glycosylated, while the 78-kD protein bears carbohydrates. These findings suggest that monocytes which bind the AChR via specific membrane proteins on their surface might act as antigen-presenting cells and may lead to an induction of the T-cell response, in the early phase of the disease.

Immunological and functional properties of the acetylcholine receptor expressed on the human cell line TE671.

In the first part of the present study we compared the antigenicity of affinity-purified acetylcholine receptors from the cell line TE671 and from human skeletal muscle. The reactivities of the two acetylcholine receptor preparations showed a strong correlation (r = 0.96) in a radioimmunoassay using sera from myasthenia gravis patients. In additional functional studies, carbamylcholine stimulated cAMP production in TE671 cells to 130%. This increase was even more pronounced when TE671 cells were grown in the presence of dexamethasone. alpha-Bungarotoxin completely blocked this carbamylcholine-induced cAMP increase. Using the Ca2+ indicator, indo-1, it was shown that intracellular Ca2+ concentrations ([Ca2+]i) were elevated in TE671 cells after stimulation with carbamylcholine. This effect was also completely blocked by alpha-bungarotoxin. To test the functional activity of autoantibodies against the acetylcholine receptor, TE671 cells were preincubated with sera from myasthenia gravis patients. In one third of sera a significant inhibition of the agonist-stimulated [Ca2+]i increase was detected, possibly caused by antibodies directed to functionally important areas of the acetylcholine receptor. There was no correlation between the inhibition rate of [Ca2+]i and anti-acetylcholine receptor antibody titres in these patient sera.

Modulation of monocyte activation in patients with rheumatoid arthritis by leukapheresis therapy.

One of the hallmarks in rheumatoid arthritis (RA) is the intense activation of the monocyte-macrophage system. In the present investigation, the modulation of blood monocyte activation was studied with regard to the secretion of cytokines and inflammatory mediators, and to the expression of cytokine receptors. Patients with severe active RA underwent repeated leukapheresis procedures that removed all circulating monocytes. Highly enriched monocyte preparations from the first and third leukapheresis were studied. There were striking differences between the two monocyte populations. Cells obtained from the first leukapheresis constitutively released large amounts of prostaglandin E2 (PGE2), neopterin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). In particular, IL-1 beta and neopterin production were further enhanced by stimulation with either interferon-gamma (IFN-gamma) or TNF-alpha without a synergistic effect. In contrast, cells derived from the third leukapheresis procedure showed a close to normal activation status with only low levels of cytokine and mediator production as well as a reduced response to cytokine stimulation. The number of the receptors for IFN-gamma and TNF-alpha was not changed between first and third leukapheresis. However, TNF-binding capacity was only detectable upon acid treatment of freshly isolated monocytes. A further upregulation was noted upon 24 h in vitro culture, suggesting occupation of membrane receptors and receptor down-regulation by endogenously produced TNF-alpha. Northern blot analysis of cytokine gene expression was in good correlation with the amount of mediators determined on the protein level. These data indicate that cells of the monocyte-macrophage system are already highly activated in the peripheral blood in RA patients with active disease. These cells can be efficiently removed by repeated leukapheresis and are replenished by monocytes that have, with respect to cytokine and mediator production, a considerably lower activation status.