Quantitative real-time RT-PCR using hybridization probes and imported standard curves for cytokine gene expression analysis

Quantitative real-time or kinetic RT-PCR is increasingly used for the quantification of specific mRNA targets, especially in clinical applications. To quantify the mRNA of cytokines and their receptors, which play important roles in the pathogenesis of autoimmune diseases such as multiple sclerosis, we have developed quantitative two-step RT-PCR assays for IL-4, IL-4R, IFN-gamma, IFN-beta, and the housekeeping gene porphobilinogen deaminase (PBGD). The LightCycler system was used to quantify the copy numbers with the sequence-specific hybridization probe detection format. The quantification was carried out on the basis of standard curves generated with external homologous plasmids for each different parameter in relation to the gene expression of PBGD. Therefore, this procedure represents a relative quantification method with external standards, as the standard curves were used to obtain an absolute value for the copy numbers of the targets and the reference (PBGD). The new software version 3.5 of the LightCycler system allows the construction of a single parameter-dependent plasmid standard curve for the quantification of unknown samples from different runs. Here we demonstrate how to achieve precise and reproducible quantification, even when using measurements from different PCR runs.     

Interleukin-29 modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis

Introduction

The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.

Methods

In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.

Results

A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.

Conclusions

Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.     

Quantitative trait associated microarray gene expression data analysis

Selection on phenotypes may cause genetic change. To understand the relationship between phenotype and gene expression from an evolutionary viewpoint, it is important to study the concordance between gene expression and profiles of phenotypes. In this study, we use a novel method of clustering to identify genes whose expression profiles are related to a quantitative phenotype. Cluster analysis of gene expression data aims at classifying genes into several different groups based on the similarity of their expression profiles across multiple conditions. The hope is that genes that are classified into the same clusters may share underlying regulatory elements or may be a part of the same metabolic pathways. Current methods for examining the association between phenotype and gene expression are limited to linear association measured by the correlation between individual gene expression values and phenotype. Genes may be associated with the phenotype in a nonlinear fashion. In addition, groups of genes that share a particular pattern in their relationship to phenotype may be of evolutionary interest. In this study, we develop a method to group genes based on orthogonal polynomials under a multivariate Gaussian mixture model. The effect of each expressed gene on the phenotype is partitioned into a cluster mean and a random deviation from the mean. Genes can also be clustered based on a time series. Parameters are estimated using the expectation-maximization algorithm and implemented in SAS. The method is verified with simulated data and demonstrated with experimental data from 2 studies, one clusters with respect to severity of disease in Alzheimer's patients and another clusters data for a rat fracture healing study over time. We find significant evidence of nonlinear associations in both studies and successfully describe these patterns with our method. We give detailed instructions and provide a working program that allows others to directly implement this method in their own analyses.     

Purine modulation of cytokine release during diuretic therapy of rheumatoid arthritis

Since free radicals are implicated in rheumatoid arthritis (RA) and since uric acid is a free radical scavenger, we examined the effects of treating RA patients with with the diuretic bumetanide to try to improve their arthritic control. Seventy patients, aged 18-75 years, were randomised to receive bumetanide 4 mg/day or placebo. Uric acid levels increased, but not that of other purines, in the blood of drug-treated patients compared with placebo-treated controls. There were no significant changes in clinical measurements of disease activity or in ESR or CRP levels. There were no over all differences in the blood levels of the cytokines, nor in the basal or stimulated production of cytokines from the blood cultures. The adenosine receptor agonist 5'N-ethylcarboxamido-adenosine (NECA) used to modify cytokine release in cultures of whole blood taken from the patients, depressed the release of tumour necrosis factor-alpha (TNFalpha), but failed to depress the release of interleukin-1b (IL-1b) or interleukin-6 (IL-6), a difference from earlier studies of healthy control subjects and, thus, a difference which may contribute to the disease activity.     

Validation study of existing gene expression signatures for anti-TNF treatment in patients with rheumatoid arthritis

So far, there are no means of identifying rheumatoid arthritis (RA) patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF), prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non) response.     

Quantitative analysis of gene expression in living adult neural stem cells by gene trapping

The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses beta-lactamase-induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.     

Quantitative biomarker analysis of synovial gene expression by real-time PCR

Synovial biomarker analysis in rheumatoid arthritis can be used to evaluate drug effect in clinical trials of novel therapeutic agents. Previous studies of synovial gene expression for these studies have mainly relied on histological methods including immunohistochemistry and in situ hybridization. To increase the reliability of mRNA measurements on small synovial tissue samples, we developed and validated real time quantitative PCR (Q-PCR) methods on biopsy specimens. RNA was isolated from synovial tissue and cDNA was prepared. Cell-based standards were prepared from mitogen-stimulated peripheral blood mononuclear cells. Real time PCR was performed using TaqMan chemistry to quantify gene expression relative to the cell-based standard. Application of the cellular standard curve method markedly reduced intra- and inter-assay variability and corrected amplification efficiency errors compared with the C(t) method. The inter-assay coefficient of variation was less than 25% over time. Q-PCR methods were validated by demonstrating increased expression of IL-1β and IL-6 expression in rheumatoid arthritis synovial samples compared with osteoarthritis synovium. Based on determinations of sampling error and coefficient of variation, twofold differences in gene expression in serial biopsies can be detected by assaying approximately six synovial tissue biopsies from 8 to 10 patients. These data indicate that Q-PCR is a reliable method for determining relative gene expression in small synovial tissue specimens. The technique can potentially be used in serial biopsy studies to provide insights into mechanism of action and therapeutic effect of new anti-inflammatory agents.     

Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts

Introduction  

Rheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation.     

IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium.

IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.     

Enhancement of lymphocyte migration and cytokine production by ephrinB1 system in rheumatoid arthritis

Although the etiology of early events in rheumatoid arthritis (RA) remains undefined, an anomaly in T cell homeostasis and hyperproliferation of synovial-lining cells are involved in the disease process. Since it has been reported that the ephrin/Eph receptor system plays important signaling roles in inflammation processes, we attempted to examine ephrinB molecules in T cells and synovial cells derived from RA in this study. The expression level of ephrinB1 was significantly high in synovial fibroblasts and CD3-positive exudate lymphocytes in synovial tissues derived from patients with RA compared with those in osteoarthritis (OA). Protein and mRNA levels of ephrinB1 were also higher in peripheral blood lymphocytes (PBLs) prepared from patients with RA than those from normal controls. Similar results were obtained from an animal model of human RA, collagen antibody-induced arthritis mice. Moreover, a recombinant ephrinB1/Fc fusion protein stimulated normal PBLs to exhibit enhanced migration and production of TNF-alpha. EphrinB1/Fc also activated synovial cells established from patients with RA to produce IL-6. Tyrosine phosphorylation of EphB1 was induced in these cells by ephrinB1/Fc. The CpG islands in the 5' upstream regulatory region of the ephrinB1 gene were hypomethylated in RA patients compared with those of normal donors. These results suggest that ephrinB1 and EphB1 receptors play an important role in the inflammatory states of RA, especially by affecting the population and function of T cells. Inhibition of the ephrinB/EphB system might be a novel target for the treatment of RA.     

Genetic and expression deregulation of immunoregulatory genes in rheumatoid arthritis

Molecular Biology Reports - Rheumatoid arthritis (RA) is one of the most common autoimmune diseases globally, and is an important public health concern, associating with early death and systemic...