Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.     

Proteome profiling of peripheral mononuclear cells from human blood

Peripheral blood mononuclear cells (MNCs) are accessible through blood collection and represent a useful source for investigations on disease mechanisms and treatment response. Aiming to build a reference proteome database, we generated three proteome data sets from MNCs using a combination of SDS‐PAGE and nanoflow LC‐MS. Experiments were performed in triplicates and 514 unique proteins were identified by at least two non‐redundant peptides with 95% confidence for all replicates. Identified proteins are associated with a range of dermatologic, inflammatory and neurological conditions as well as molecular processes, such as free radical scavenging and cellular growth and proliferation. Mapping the MNC proteome provides a valuable resource for studies on disease pathogenesis and the identification of therapeutic targets.     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients

Introduction

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Methods

Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.

Results

Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.

Conclusions

Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.     

A pattern of protein expression in peripheral blood mononuclear cells distinguishes rheumatoid arthritis patients from healthy individuals

We compared the expression levels of proteins in peripheral blood mononuclear cells (PBMCs) of healthy control individuals to those of patients diagnosed with rheumatoid arthritis (RA) using a proteomics approach. Using two-dimensional electrophoresis we identified 18 proteins that were 2-fold or more highly expressed in patients than in controls, and 11 proteins that were 2-fold or more highly expressed in controls than in patients. Some of these differentially expressed proteins, identified by MALDI-TOF spectrometry, have previously been shown to play a potential role in the pathogenesis of RA. Hierarchical cluster analyses of the data segregated the samples into two groups, one which contained only controls and the other which contained only patients, and was used to compare the expression pattern of these 29 proteins in individual samples with the median expression pattern determined in the healthy control and in the RA patient groups. This analyses was able to predict whether a sample was derived from a rheumatoid arthritis patient or from a healthy individual, suggesting that a comparison of such protein expression patterns may be of diagnostic value.     

Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells

Background

Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.

Methodology/Principal Findings

We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.

Conclusions/Significance

Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.     

Immune complexes from rheumatoid arthritis synovial fluid induce FcgammaRIIa dependent and rheumatoid factor correlated production of tumour necrosis factor-alpha by peripheral blood mononuclear cells

Immune complexes (ICs) can induce production of cytokines by peripheral blood mononuclear cells via Fc receptors. Rheumatoid factor (RF) develop in response to ICs in many clinical and experimental settings. We investigated whether and how polyethylene glycol (PEG) precipitated ICs from rheumatoid arthritis (RA) sera and synovial fluid (SF) can influence cytokine production by peripheral blood mononuclear cells. We also examined the relationship between RF and IC induced cytokine production. Parallel sera and SF from 47 RA patients and sera from 15 healthy control individuals were PEG precipitated. The precipitates were added to serum-free peripheral blood mononuclear cell cultures and tumour necrosis factor (TNF)-alpha levels were measured after 20 hours. In separate cell culture experiments FcgammaRIIa and FcgammaRIII were blocked and monocytes were depleted or enriched. RF in serum was determined by nephelometry, and IgG levels in precipitates and anti-cyclic citrullinated peptide antibodies in serum were measured using ELISA. Clinical data were collected from the patients' charts. In two separate investigations, we demonstrated a correlation between RF, PEG-precipitated IgG levels and induction of the proinflammatory cytokine TNF-alpha by PEG-precipitated SF ICs. No such correlation was found for serum ICs. TNF-alpha levels induced by SF precipitates, but not serum precipitates, correlated with the number of swollen and tender joints. Monocytes/macrophages were shown to be the main responder cells, and blockade of FcgammaRIIa, but not blockade of FcgammaRIII, inhibited TNF-alpha production in cultures stimulated with precipitated ICs. Anti-cyclic citrullinated peptide correlated with RF but exhibited no association with IgG content in PEG precipitates or with precipitate-induced TNF-alpha levels. These findings support the hypothesis that SF ICs and correlated RF production are directly linked to cytokine-dependent inflammation in RA. Suppression of monocytes/macrophages in RA joints or blockade of the primate-specific activating FcgammaRIIa receptor might be ways to reduce IC-induced TNF-alpha production in the joints of seropositive RA patients.     

类风湿性关节炎患者外周血单个核细胞 IL-10 基因启动子甲基化状态研究

白细胞介素-10（interleukin-10,IL-10）是在类风湿性关节炎中发挥重要免疫调节作用的细胞因子,其基因失活与已分化的Th1和Th2细胞染色质结构重塑有关。为了探讨IL-10基因启动子甲基化及基因失活在类风湿性关节炎(Rheumatoid Arthritis,RA)发病和进展中的作用,采用逆转录聚合酶链反应(RT-PCR)、酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)及甲基化特异性聚合酶链反应(MSP), 分别检测34例类风湿性关节炎患者和30例健康人外周血单个核细胞 IL-10 mRNA、蛋白表达水平及基因启动子甲基化状态。结果显示,病例组IL-10 mRNA及蛋白表达均低于健康对照组,无统计学差异（P>0.05）。病例组甲基化率(85.29%)高于健康对照组(43.33%), 具有统计学差异(x² =12.439,P=0.000)。IL-10基因启动子甲基化状态与其mRNA表达呈显著负相关(r=-0.579, P=0.001), 与所累关节数显著相关,但与血沉（ESR）、C反应蛋白（CRP）、类风湿因子(RF)、年龄均无相关性(P>0.05)。IL-10 mRNA表达与年龄、所累关节数、ESR、CRP及RF均无相关性(P>0.05)。上述结果提示,启动子甲基化可能是IL-10基因失活的重要机制,IL-10基因异常高甲基化状态可能参与了RA的发生发展。     

The restoration of proliferation and differentiation of peripheral blood mononuclear non-adherent cells into immunoglobulin-secreting cells by autologous synovial adherent cells from patients with rheumatoid arthritis

The ability of enzyme-dissociated synovial adherent cells (SAC) obtained from patients with active rheumatoid arthritis to restore the proliferation and differention of peripheral blood mononuclear non-adherent cells (NAC) into immunoglobulin-secreting cells (ISC) was investigated. Autologous combinations of cells were used in this study to eliminate allogeneic reactions. Peripheral blood NAC, prepared by glass adherence and leucine methylester treatment to remove monocytes, almost completely lost their capacity to proliferate and differentiate into ISC in response to pokeweed mitogen. The response of NAC was restored by adding 12.5% of 'fresh SAC', which was obtained by glass-adherence after an overnight culture of non-rosette forming, enzyme-dissociated rheumatoid synovial cells. Although the response was also restorable by adding more than 25% fresh SAC, this was less satisfactory than adding 12.5% SAC. 'Old SAC', obtained by glass-adherence after 7 days culture of enzyme-dissociated synovial cells, did not restore the response of NAC. Immunohistochemical studies showed that 55% of fresh SAC and 3% of old SAC expressed HLA-DR antigens. When 100 units/ml of interferon gamma was present, 25% of old SAC remained HLA-DR-positive and some of these cells retained a dendritic morphology after 7 days culture. The results indicate that rheumatoid synovia contain macrophage-like cells that can effectively support the ultimate differentiation of lymphocytes to ISC.     

Aberrant splicing of the hRasGRP4 transcript and decreased levels of this signaling protein in the peripheral blood mononuclear cells in a subset of patients with rheumatoid arthritis

Introduction  

An unidentified population of peripheral blood mononuclear cells (PBMCs) express Ras guanine nucleotide releasing protein 4 (RasGRP4). The aim of our study was to identify the cells in human blood that express hRasGRP4, and then to determine if hRasGRP4 was altered in any patient with rheumatoid arthritis (RA).     

Proteomic analysis of peripheral blood mononuclear cells: selective protein processing observed in patients with rheumatoid arthritis

In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases.     

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.     

Kinetics of selenite uptake by mononuclear cells from peripheral human blood

The present study deals with the kinetics and thermodynamics of the uptake of⁷⁵Se-labeled SeO ₃ ^2? from incubation media to lymphocytes cultivated from eight normal individuals (14–55 years of age, two females). The uptake of SeO ₃ ^2? was evaluated on the assumption of pseudo-first-order kinetics with regard to a reacting cellular receptor pool. On the basis of the experimental observations, it was assumed that the suggested pool of receptor molecules-symbolically represented by “£H₄”—reacts with SeO ₃ ^2? in the hypothetical reaction: $$\pounds H_4 + SeO_3^{2 - } + 2H^ + \underset{{ - k_1 }}{\overset{{k_1 }}{\longleftrightarrow}}\pounds Se + 3H_2 O$$ The mean value of the change in standard free energy at 25°C was calculated to be ΔG ^o=?141.6±1.3 kJ/mol, while the corresponding mean value of the free energy of activation at 25°C was calculated to be ΔG ²⁺=?7.8±0.9 kJ/mol for the forward reaction. The calculated values of the corresponding individual changes in the respective standard enthalpies and entropies were mutually interdependent for all eight donors. ΔH ^o=?152+315ΔS ^o(kJ/mol) corresponding to the common value ΔG ^o??152 kJ/mol at 315°K. These mutual interdependencies are possibly the effect of variable conformational states (e.g., the macromolecular compactness) of the cellular receptor pools. This suggestion may furthermore be supported by the correlation traced between ΔH ^o vs the biological age in years of the donors: △H ^°?76.7?1.0 (age)kJ/mol (r = ?0.92) The calculated values of activation enthalpy ΔH ²⁺ kJ/mol and activation entropy ΔS ²⁺ (kJ/mol K) also mutually correlated linearly (r=0.998); the regression line was: △H ²⁺ = ?8.9 + 305△S²⁺ (kJ/mol) corresponding to the common value △H ²⁺ △ ?8.9 (kJ/mol) at 305°K Similarly the activation enthalpy ΔH ²⁺ vs the biological age in years correlated linearly: ΔH ²⁺=67.4?0.73(age) (kJ/mol) (r=?0.76) The range of ΔH ²⁺ studied was from 13.8 to 53.9 kJ/mol with a linearly corresponding range in ΔS ²⁺ from 73 to 205 J/mol K. The thermodynamic data reveal the selenite uptake during the hypothetical standard reaction to be exergonic and endothermic. Critical pH dependencies of the selenite uptake were explained.