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UnlabelledArticular destruction by invading synovial fibroblasts is a typical feature in rheumatoid arthritis (RA),. Recent data support the hypothesis that key players in this scenario are transformed-appearing synovial fibroblasts at the site of invasion into articular cartilage and bone. They maintain their aggressive phenotype toward cartilage, even when first cultured and thereafter coimplanted together with normal human cartilage into severe combined immunodeficient mice for and extended period of time. However, little is known about the upregulation of genes that leads to this aggressive fibroblast phenotype. To inhibit this progressive growth without interfering with pathways of physiological matrix remodelling, identification of pathways that operate specifically in RA synovial fibroblasts is required. In order to achieve this goal, identification of genes showing upregulation restricted to RA synovial fibroblasts is essential.AimsTo identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with RA.MethodsRNA was extracted from cultured synovial fibroblasts from 10 patients with RA, four patients with osteoarthritis (OA), and one patient with psoriatic arthritis. RAP-PCR was performed using different arbitrary primers for first-strand and second-strand synthesis. First-strand and second-strand synthesis were performed using arbitrary primers: US6 (5'-GTGGTGACAG-3') for first strand, and Nuclear 1+ (5'ACGAAGAAGAG-3'), OPN28 (5'GCACCAGGGGG-3'), Kinase A2+(5'-GGTGCCTTTGG-3') and OPN24 (5'AGGGGCACCA-3') for second strand synthesis. PCR reactions were loaded onto 8 mol/l urea/6% polyacrylamide-sequencing gels and electrophoressed. Gel slices carrying the target fragment were then excised with a razor blade, eluated and reamplified. After verifying their correct size and purity on 4% agarose gels, the reamplified products derived from the single-strand confirmation polymorphism gel were cloned, and five clones per transcript were sequenced. Thereafter, a genbank analysis was performed. Quantitative reverse transcription PCRj of the segments was performed using the PCR MIMIC technique. In-situ expression of centromere kinesin-like protein-E (CENP-E) messenger (m)RNA in RA synovium was assessed using digoxigenin-labelled riboprobes, and CENP-E protein expression in fibroblasts and synovium was performed by immunogold-silver immunohistochemistry and cytochemistry. Functional analysis of CENP-E was done using different approaches (eg glucocorticoid stimulation, serum starvation and growth rate analysis of synovial fibroblasts that expressed CENP-E).ResultsIn RA, amplification of a distinct PCR product suitable for sequencing could be observed. The indicated complementary DNA fragment of 434 base pairs from RA mRNA corresponded to nucleotides 6615-7048 in the human centromere kinesin-like protein CENP-E mRNA (GenBank accession No. emb/Z15005). The isolated sequence shared greater than 99% nucleic acid (P=2.9e(-169)) identify with the human centromere kinesin-like protein CENP-E. Two base changes at positions 6624 (A to C) and 6739 (A to G) did not result in alteration in the amino acid sequence, and therefore 100% amino acid identity could be confirmed. The amplification of 10 clones of the cloned RAP product revealed the presence of CENP-E mRNA in every fibroblast culture examined, showing from 50% (271.000 +/- 54.000 phosphor imager arbitrary units) up to fivefold (961.000 +/- 145.000 phosphor image arbitrary units) upregulation when compared with OA fibroblasts. Neither therapy with disease-modifying antirheumatic drugs such as methotrexate, gold, resochine or cyclosporine A, nor therapy with oral steroids influenced CENP-E expression in the RA fibroblasts. Of the eight RA fibroblast populations from RA patients who were receiving disease-modifying antirheumatic drugs, five showed CENP-E upregulation; and of the eight fibroblast populations from RA patients receiving steroids, four showed CENP-E upregulation. Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely.DiscussionSince its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G2 phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA.The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and transactivation processes resembling the situation in AP-1 (Jun/Fos)-dependent DNA-binding in rheumatoid synovium. Most interestingly, these coiled-coil motifs are crucial for the assembly of viral proteins, and the upregulation of CENP-E might reflect the influence of infectious agents in RA synovium. We also performed experiments showing that serum starvation decreased, but did not completely inhibit CENP-E mRNA expression. This shows that CENP-E is related to, but does not completely depend on proliferation of these cells. In addition, we determined the growth rate of CENP-E high and low expressors, showing that it was independent from the amount of CENP-E expression. supporting the statement that upregulation of CENP-E reflects an activated RA fibroblast phenotype. In summary, the results of the present study support the hypothesis that CENP-E, presumably independently from medication, may not only be upregulated, but may also be involved in RA pathophysiology.

Different approaches of engineering cartilage to treat defects in the articulating surfaces of the joints have been designed, which mainly use mesenchymal stem cells or autologous chondrocytes for in situ transplantation. However, these cells are poorly characterized with respect to viability, degree of differentiation and morphology or production of extracellular matrix. At present, one of the key approaches to generate chondrocytes is the stimulation of stem cells with transforming growth factor (TGF) β1. To characterize the molecular alterations occurring during the cellular transformation induced by TGF-β1 exposure, the differentiation process of bone marrow-derived stem cells into chondrocytes was investigated using an in vitro chondrogenesis model and the RNA arbitrarily primed PCR (RAP-PCR) fingerprinting technique. Distinct genes were found to be differentially regulated during chondrocyte development beginning on day 1: collagen type I, non-muscle myosin MYH9, followed by manganese superoxide dismutase and sodium-potassium ATPase on day 7. The results suggest that using RAP-PCR for differential display fingerprinting is a useful tool to investigate the differentiation process of bone marrow-derived stem cells following TGF-β1-stimulation.

Microarrays and high-throughput sequencing methods can be used to measure the expression of thousands of genes in a biological sample in a few days, whereas PCR-based methods can be used to measure the expression of a few genes in thousands of samples in about the same amount of time. These methods become more costly as the number of biological samples increases or as the number of genes of interest increases, respectively, and these factors constrain experimental design. To address these issues, we introduced 'vertical arrays' in which RNA from each biological sample is converted into multiple, overlapping cDNA subsets and spotted on glass slides. These vertical arrays can be queried with single gene probes to assess the expression behavior in thousands of biological samples in a single hybridization reaction. The spotted subsets are less complex than the original RNA from which they derive, which improves signal-to-noise ratios. Here, we demonstrate the quantitative capabilities of vertical arrays, including the sensitivity and accuracy of the method and the number of subsets needed to achieve this accuracy for most expressed genes.

Objective: To identify genes that are differentially expressed in systemic sclerosis (SSc) fibroblasts of clinically involved and noninvolved skin compared to normal dermal fibroblasts, using RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) for differential display., Methods: We examined 12 fibroblast cultures derived from clinically involved skin, 3 fibroblast cultures from noninvolved skin, and 4 fibroblast cultures from healthy skin. After extraction of total RNA, the first step of RAP-PCR was performed using different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling using different arbitrary 10-base primers, followed by sequence analysis of the amplified fingerprint products. The resulting sequences were aligned to the GenBank database using Blast Search. Confirmation of differential expression was performed with specific primers using real-time PCR., Results: Using 8 different primer combinations, in total 48 cDNA were differentially expressed between SSc and healthy dermal fibroblasts. Sequence analysis identified distinct PCR products, which were overexpressed in SSc as highly homologous to gene segments of gremlin protein, lysyl oxidase, c-cbl proto-oncogene, an estrogen-responsive element, fibronectin, and collagen type XIIa1 precursor., Conclusion: Our results show that RAP-PCR is a suitable method to identify differentially expressed genes in SSc fibroblasts. Further, we identified genes that have not yet been described in the pathophysiology of SSc and that may be involved in matrix synthesis and cellular interaction.

Evaluation of differentially regulated genes is essential for the development of novel therapeutic approaches in multifactorial diseases such as rheumatoid arthritis (RA). RA synovial fibroblasts (RASF) are key players in inflammation and cartilage destruction. Therefore, RASF are important cellular targets for the analysis of gene expression profiles. Such analyses may include a comparison of SF from nontreated RA patients with those from treated RA patients, and may also be used to evaluate which genes and intra-cellular processes can be modulated through genetic modification, gene transfer, and drug treatment for the identification of the pathways driving the destructive behavior of these cells. This chapter reports the combination of RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and cDNA array with defined genes for a highly sensitive analysis of gene expression profiles in RASF using small amounts of total RNA. RNA can be extracted from cultured SF, isolated, and analyzed using RAP-PCR with different arbitrary primers for first-and second-strand synthesis to generate a radioactive labeled probe which can be used for cDNA array hybridization. Visualization and evaluation of gene expression can be performed by phosphorimaging in combination with an array-specific software analyzing system followed by statistic evaluation of the generated data. In summary, the combination of RAP-PCR combined with cDNA arrays is a sensitive method to identify differentially expressed genes in RASF with high specificity, especially for low abundant mRNAs.

Most current approaches used to analyze gene expression in tissue samples are based on RNA isolated either from cultured synovial cells or from synovial biopsies. However, this strategy does not distinguish between specific gene expression profiles of cells originating from discrete tissue areas. Therefore, we established the combination of laser-mediated microdissection and RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display to analyze profiles of gene expression in histologically defined areas of arthritic tissue. Cryosections derived from synovial tissue were used to obtain cell samples from different tissue areas of both rheumatoid arthritis (RA) and osteoarthritis (OA) patients using a microbeam laser microscope. RNA was isolated and analyzed using nested RNA arbitrarily primed PCR to generate a fingerprint of the expressed gene sequences. Differentially expressed bands were isolated, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry.

Chemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture. Monocytes/macrophages, whose survival was almost doubled in the presence of CCL18 compared with controls, were the primary cells mediating this effect. Conditioned media from CCL18-treated mature monocytes fostered colony-promoting activity that increased the number of colonies formed by hematopoietic progenitor cells. Gene expression profiling of CCL18-stimulated monocytes demonstrated more than 200 differentially expressed genes, including those regulating apoptosis (caspase-8) and proliferation (IL-6, IL-15, stem cell factor [SCF]). Up-regulation of these cytokines was confirmed on the protein expression level. The contribution of SCF and IL-6 in CCL18-mediated stimulatory activity for hematopoiesis was confirmed by SCF- and IL-6-blocking antibodies that significantly inhibited the colony-promoting activity of CCL18-stimulated conditioned medium. In addition to the effect on monocytes, CCL18 facilitated the formation of the adherent layer in LTBMCs and increased the proliferation of stromal fibroblast-like cells.

Vertical arrays are microarrays that have complex mixtures of nucleic acids as array elements, and that are hybridized with single sequence probes. Like dot blots, many different experiments can be spotted on a single vertical array, allowing single genes to be compared across many conditions. Vertical arrays have two additional advantages over dot blots. First, they are printed on glass slides, allowing the use of low-volume, high-concentration hybridization reactions. Second, they can be made using low-complexity representations of the original nucleic acid population. This increases signal-to-noise relative to the usual use of dot blots, wherein the entire complexity of the population is usually spotted. Whereas standard microarrays achieve horizontal coverage of many genes and are repeated to cover many experiments, vertical arrays achieve vertical coverage of many experiments and are repeated to cover many genes. In cases where the number of genes is limited, but the number of experiments is very large, vertical arrays may be advantageous.

Low abundance mRNAs are more difficult to examine using microarrays than high abundance mRNAs due to the effect of concentration on hybridization kinetics and signal-to-noise ratios. This report describes the use of low complexity representations (LCRs) of mRNA as the targets for cDNA microarrays. Individual sequences in LCRs are more highly represented than in the mRNA populations from which they are derived, leading to favorable hybridization kinetics. LCR targets permit the measurement of abundance changes that are difficult to measure using oligo(dT) priming for target synthesis. An oligo(dT)-primed target and three LCRs detect twice as many differentially regulated genes as could be detected by the oligo(dT)-primed target alone, in an experiment in which serum-starved fibroblasts responded to the reintroduction of serum. Thus, this target preparation strategy considerably increases the sensitivity of cDNA microarrays.

Most current approaches to the analysis of gene expression in arthritic tissue samples are based on RNA isolated either from cultured synovial cells or from synovial biopsies. However, this strategy does not distinguish between specific gene expression profiles of cells originating from separate tissue areas. Therefore, we established the combination of laser-mediated microdissection and RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display to analyze profiles of gene expression in histologically defined areas of arthritic tissue. Cryosections derived from synovial tissue were used to obtain cell samples from different tissue areas using a microbeam laser microscope. RNA was isolated and analyzed using nested RAP-PCR to generate a fingerprint of the expressed gene sequences. Differentially expressed bands were isolated, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry.

Objective: Complement has been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA) based on studies showing reduced levels of native complement components and increased levels of complement metabolites in plasma, synovial fluid (SF), and synovial tissue (ST) of RA patients. However, there is limited information on local production and activation of key factors of the complement cascade in RA synovium and their potential modulation by novel anticytokine therapies. This study was undertaken to characterize the expression of complement proteins and receptors in RA SF and ST., Methods: Using in situ hybridization, immunohistochemistry, and Western blot techniques, we assessed the presence of complement proteins C3, factor B (FB), and C5b-9, as well as the expression of complement receptors C3aR and C5aR in rheumatoid synovium. C3 and FB levels in SF were determined by enzyme-linked immunosorbent assay. Functional assessment was performed by examining the effects of soluble tumor necrosis factor receptor (sTNFR) p55 gene transfer in the SCID mouse model of RA., Results: Complement proteins and receptors could be localized in all RA synovial specimens, whereas in osteoarthritis (OA) synovium, only a few, single cells expressed complement proteins and receptors. No differences were noted in the concentration of C3 between RA and OA in SF; however, FB levels were markedly reduced in RA versus OA SF. In RA synovium, in contrast to OA synovium, local expression of complement factor and complement receptor messenger RNA was found throughout the various ST compartments, suggesting that activation of the complement cascade occurs in all parts of the rheumatoid synovium. Moreover, C5aR expression was up-regulated following overexpression of sTNFR p55 by adenovirus-based gene transfer., Conclusion: In summary, local complement production and activation may play an important role in RA, and specific modulation and inhibition of local complement production could be an attractive therapeutic target for RA.

STATs act as second messenger after binding of a signaling molecule to its receptor. IL-4 STAT is directly involved in the IL-4-dependent gene transcription in the nucleus. We examined the expression and activation of IL-4 STAT and its related kinase Jak-1 in rheumatoid synovium. Rheumatoid arthritis (RA) synovial frozen sections of patients with short-term (<1 year) and long-term disease (>2 years) were examined using in situ hybridization and immunohistochemistry. IL-4 STAT mRNA could be detected in synovium of patients with short-term and long-term RA. The most intensive expression of IL-4 STAT mRNA could be seen in follicular inflammatory infiltrates. In the synovial lining, both fibroblasts and macrophages expressed IL-4 STAT mRNA. IL-4 STAT and Jak-1 protein was expressed by synoviocytes, and up-regulation could be induced after stimulation with IL-4. Activation of IL-4 STAT was reflected by phosphorylation of IL-4 STAT. The results indicate that IL-4 STAT is involved in key pathomechanisms in RA synovium and that IL-4 STAT-dependent pathways operate in early and late stages of the disease and presumably contribute to inhibitory immune mechanisms in RA synovium.

Objective: To determine whether mutations in the tumor suppressor gene p53 may contribute to the transformed-appearing phenotype of rheumatoid arthritis (RA) synovial fibroblasts., Methods: We performed p53 gene mutation analysis using different molecular approaches. Synovial fibroblasts of 10 patients with RA were cultured and RNA and DNA were harvested after 3-5 passages in cell culture. Sequence analysis of all exons of the p53 gene was performed using 3 different techniques: 1) single-strand conformational polymorphism, 2) nonisotopic RNase cleavage assay, and 3) base excision sequence scanning T-scan, followed by sequence analysis of specific gene segments., Results: Although p53 antigen could be detected by immunocytochemistry in numerous cultured fibroblasts, gel electrophoresis analysis of products obtained using all 3 methods and subsequent sequence analysis showed no specific mutation pattern in the genome of the synovial fibroblasts from patients in Germany, including the known "hot spots" within the p53 genome. However, p53 mutations were identified in different clones of 3 additional RA synovial fibroblast populations from the United States. Sequence analysis of the p53 promoter did not reveal mutational base changes., Conclusion: The findings of the study support the hypothesis that the majority of the mutations of the p53 gene observed in RA synovium are not derived from the genome of RA synovial fibroblasts, and that the variability of the mutation pattern reflects, in part, the heterogeneity of the disease.

Objective: Destruction of articular cartilage and bone by invading synovial fibroblasts is a typical histopathologic feature in rheumatoid arthritis (RA). However, little is known about specific up- or downregulation of genes leading to this aggressive phenotype. Thus, our aim was to identify genes, which are differentially expressed in RA synovial fibroblasts as compared to synovial fibroblasts derived from patients with osteoarthritis (OA) using RAP-PCR for differential display., Methods: After extraction of total RNA, the first step of RAP-PCR was performed using various different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling at low stringency conditions for 35 cycles using different arbitrary 10-base primers, followed by electrophoretic separation and sequence analysis of the amplified fingerprint products., Results: On average, approximately 70 different RNAs were obtained per primer, of which most were expressed both by RA and OA synovial fibroblasts. Using 26 different primer combinations, in total 12 cDNAs were differentially expressed between RA and OA synovial fibroblasts. In the RA group strong amplification of distinct PCR products suitable for sequencing could be observed. Sequence analysis identified these PCR products as highly homologous to various genes involved in regulation of cell cycle and metabolism., Conclusion: The data indicate that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts potentially involved in the specific pathophysiology of RA.

Genomes of various hyperthermophilic and extremely thermophilic prokaryotes were analyzed with respect to size, physical organization, and 16S rDNA copy number. Our results show that all the genomes are circular, and they are in the size range of 1.6-1.8 Mb for Pyrodictium abyssi, Methanococcus igneus, Pyrobaculum aerophilum, Archaeoglobus fulgidus, Archaeoglobus lithotrophicus, and Archaeoglobus profundus (the two bacteria Fervidobacterium islandicum and Thermosipho africanus possess genomes of 1.5-Mb size). A systematic study of all validly described species of the order Sulfolobales revealed the existence of two classes of genome size for these archaea, correlating with phylogenetic analyses. The Metallosphaera-Acidianus group, plus Sulfolobus metallicus, have genomes of ca. 1.9 Mb; the other members of the order Sulfolobales group possess genome > 2.7 Mb. The special case of stygiolobus azoricus is discussed.