Martin Judex

Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction

OBJECTIVE There is increasing evidence that T cell-independent pathways, such as the up-regulation of protooncogenes and the production of growth factors and matrix-degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA. METHODS RNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP-PCR was performed using different arbitrary primers for first- and second-strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes. RESULTS In contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (approximately 6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT-PCR and in situ hybridization. CONCLUSION Numerous variants of the differential display method and continuous improvements, including RAP-PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP-PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.

Activation of the IL-4 STAT Pathway in Rheumatoid Synovium

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.

Transcription factor Egr-1 activates collagen expression in immortalized fibroblasts or fibrosarcoma cells.

Abstract Synovial fibroblasts from rheumatoid arthritis patients express elevated levels of the transcription factor Egr 1. The metabolic consequences of Egr-1 overexpression in fibroblasts are not known in detail. Therefore we searched for gene products that are differentially expressed in Egr-1high versus Egr-1low fibroblasts. Immortalized synovial fibroblasts were transfected with two different Egr-1 expression vectors. Expression of recombinant Egr-1 was confirmed by RTPCR and immunoblots. Random arbitrarily primed PCR revealed that Egr-1 induces enhanced transcription levels of the α1 chain of type I collagen. Increased expression of the α2 (I) chain could also be observed. We found enhanced levels of type I collagen propeptide in supernatants and stronger signals of α2 (I) protein in extracts of the Egr 1high expressing clone versus controls. Additionally, Egr-1 was transiently expressed in fibrosarcoma cells. These cells showed a pronounced elevation of type I collagen (α1) transcripts as well. Moreover, we could demonstrate that Egr-1 induces transcription of other genes including type II collagen (α1) and plateledderived growth factor β1. These data suggest that upregulation of Egr-1 might contribute to fibrosis observed in rheumatoid arthritis synovium by activation of genes encoding the α1 and α2 chains of type I collagen.

Enhanced microarray performance using low complexity representations of the transcriptome

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.

Inverse wrap: an improved implantation technique for virus-transduced synovial fibroblasts in the SCID mouse model for rheumatoid arthritis

Abstract The SCID mouse model for rheumatoid arthritis (RA) is an established and reliable approach to examining the distinct mechanisms operative in RA synovium, and evaluating novel gene therapy strategies. However, serum concentrations of circulating gene therapy products following gene transfer are frequently too low to allow detection. This problem stimulated us to develop a novel implantation technique to improve the yield of these soluble gene products. Synovial fibroblasts from patients with RA were cultured, passaged, and transduced with Ad5 sTNFRp55:Ig. sTNFRp55:Ig production was confirmed by ELISA, and then cells were implanted into SCID mice using a novel implantation strategy in which pieces of human cartilage were engrafted into a fibroblast-saturated inert sponge. Thereafter, the sponges were implanted under the skin of the mice instead of under the kidney capsule, as in the original approach, allowing co-implantation of larger pieces of cartilage together with higher numbers of adenovirus-transduced RA synovial fibroblasts. The improved implantation technique not only resulted in a reduction in the number of mice needed in each experiment by approximately 60%, and a reduction of the time taken for surgery by about 50%, but also considerably enhanced the serum concentrations of the gene product sTNFRp55-Ig, allowing detection of the soluble TNF receptor p55 by standard ELISA. In summary, the improved implantation technique for the SCID mouse model for RA results in more economic animal treatment, and facilitates the detection and quantification of circulating gene products following adenovirus-based gene transfer into synovial fibroblasts.

Laser-Mediated Microdissection As a Tool for Molecular Analysis in Arthritis

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.

Analyse des Genexpressionsmusters von synovialen Fibroblasten von Patienten mit rheumatoider Arthritis mittels RAP-PCR Differential Display

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.Zusammenfassung□ FragestellungIm rheumatischen Gelenk finden sich transformiert erscheinende Fibroblasten an der Invasionszone in den Knorpel und Knochen. Über die Faktoren, die zu diesem aggressiven Phänotyp führen, ist bisher wenig bekannt. Ziel der Untersuchungen war daher, mittels RAP-PCR (RNA arbitrarily primed PCR) zwischen rheumatoiden Fibroblasten und Osteoarthritis-Fibroblasten differentiell exprimierte Gene zu erfassen.□ MethodikDie Gesamt-RNS von synovialen Fibroblasten wurde im ersten Schritt der RAP-PCR mittels arbiträren Zehn- bis Zwölf-Basen-Primern in cDNS überschrieben. Die Amplifikation des zweiten Schrittes erfolgte dann ebenfalls mittels Zehn- bis Zwölf-Basen-Primern unter niedrigstringenten Bedingungen für insgesamt 35 Zyklen, gefolgt von elektrophoretischer Auftrennung, Klonierung und Sequenzanalyse der differentiell exprimierten RAP-PCR-Produkte.□ ErgebnisseIm Durchschnitt konnten je Primerpaar ca. 70 verschiedene RNS amplifiziert werden, wobei das Expressionsmuster der RA- und OA-Fibroblasten eine hohe Homologie aufwies. Insgesamt konnten aus den mit 26 verschiedenen Primerkombinationen durchgeführten RAP-PCR Differential Displays zwölf sehr stark differentiell exprimierte RNS nachgewiesen werden. Die Sequenzanalysen der hierbei in den rheumatoiden Fibroblasten hochregulierten und bisher identifizierten Gensegmenten zeigten sehr große Homologien zu verschiedenen in den Zellmetabolismus involvierten Gene.□ SchlußfolgerungDie Untersuchungen zeigen, daß in rheumatoiden synovialen Fibroblasten mittels RAP-PCR Differential Display spezifisch exprimierte Gene identifiziert werden können, die möglicherweise eine wichtige Rolle in der Pathophysiologie dieser Erkrankung spielen.Abstract□ ObjectiveDestruction 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.□ MethodsAfter 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.□ ResultsOn 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.□ ConclusionThe 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.

Assessment of gene expression in many samples using vertical arrays

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.

Laser Capture as a Tool for Analysis of Gene Expression in Inflamed Synovium

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.

Vertical arrays: microarrays of complex mixtures of nucleic acids.

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.