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1.  Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo. 
Molecular Medicine  2000;6(2):114-125.
BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.
PMCID: PMC1949938  PMID: 10859028
2.  Increased circulating advanced glycation endproducts (AGEs) in acute trauma patients 
Circulating levels of pro-inflammatory advanced glycation end products (AGEs) are increased in diabetes and other conditions characterized by chronically elevated oxidant stress (OS). OS also increases after acute trauma and is implicated in the development of complications such as multiple organ failure. Herein, we assess the effect of acute OS on circulating levels of AGEs in a cohort of acute trauma victims.
An observational study was performed at a large Level 1 Trauma Center. Blood samples for measurement of two AGEs, carboxymethyllysine (CML) and methylglyoxal (MG), were obtained at admission, and serially afterwards in patients admitted to the ICU. Demographics, dietary history, markers of injury severity and ICU morbidity and mortality data were collected.
One hundred and fifty-six trauma patients (TP) (age: 39±17 years, 83% males, injury severity score: 18±14) were included in the study. TP had significantly higher serum AGE levels than normal healthy controls (CML, TP 12.4±8.2 U/mL vs. controls 8.9±5.3 U/mL, p<0.001; MG, TP 2.1±1.4 nmol/mL vs. controls 0.79±0.3 nmol/mL, p< 0.001). Admission serum AGE levels in 49 severe TP admitted to the ICU were lower than those who were not. However, among the ICU patients, serum AGEs increased further for about 7 days in patients with an uncomplicated course, and remained markedly elevated in those with a complicated course.
Circulating AGEs are transiently increased after acute trauma and persistently elevated AGE levels are associated with greater severity of injury.
PMCID: PMC3795826  PMID: 23612541
dietary AGEs; glycation; oxidative stress; nutrition
3.  Association between carotid diameter and the advanced glycation endproduct Nε-Carboxymethyllysine (CML) 
Nε-Carboxymethyllysine (CML) is the major non-cross linking advanced glycation end product (AGE). CML is elevated in diabetic patients and apparent in atherosclerotic lesions. AGEs are associated with hypertension and arterial stiffness potentially by qualitative changes of elastic fibers. We investigated whether CML affects carotid and aortic properties in normoglycemic subjects.
Hundred-two subjects (age 48.2 ± 11.3 years) of the FLEMENGHO study were stratified according to the median of the plasma CML level (200.8 ng/ml; 25th percentile: 181.6 ng/ml, 75th percentile: 226.1 ng/ml) into "high CML" versus "low CML" as determined by ELISA. Local carotid artery properties, carotid intima media thickness (IMT), aortic pulse wave velocity (PWV), blood pressure and fetuin-A were analyzed. In 26 patients after carotidectomy, CML was visualized using immunohistochemistry.
According to the CML median, groups were similar for anthropometric and biochemical data. Carotid diameter was enlarged in the "high" CML group (485.7 ± 122.2 versus 421.2 ± 133.2 μm; P < 0.05), in particular in participants with elevated blood pressure and with "high" CML ("low" CML: 377.9 ± 122.2 μm and "high" CML: 514.5 ± 151.6 μm; P < 0.001). CML was associated fetuin-A as marker of vascular inflammation in the whole cohort (r = 0.28; P < 0.01) and with carotid diameter in hypertensive subjects (r = 0.42; P < 0.01). CML level had no effect on aortic stiffness. CML was detected in the subendothelial space of human carotid arteries.
In normoglycemic subjects CML was associated with carotid diameter without adaptive changes of elastic properties and with fetuin-A as vascular inflammation marker, in particular in subjects with elevated blood pressure. This may suggest qualitative changes of elastic fibers resulting in a defective mechanotransduction, in particular as CML is present in human carotid arteries.
PMCID: PMC2733133  PMID: 19660101
4.  Immunological detection of a novel advanced glycation end-product. 
Molecular Medicine  2001;7(11):783-791.
BACKGROUND: The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. RESULTS: Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. CONCLUSION: This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.
PMCID: PMC1950006  PMID: 11788793
5.  Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures 
The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.
RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.
Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.
These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.
PMCID: PMC3241377  PMID: 21345222
6.  Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration* 
Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein Nε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors.
PMCID: PMC2722770  PMID: 19435712
7.  Decreased levels of soluble receptor for advanced glycation end products in patients with rheumatoid arthritis indicating deficient inflammatory control 
Arthritis Research & Therapy  2005;7(4):R817-R824.
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily being expressed as a cell surface molecule and binding a variety of ligands. One of these ligands is high-mobility group box chromosomal protein 1, a potent proinflammatory cytokine, expression of which is increased in synovial tissue and in synovial fluid of rheumatoid arthritis (RA) patients. The interaction of high-mobility group box chromosomal protein 1 with cell-surface RAGE leads to an inflammatory response. In contrast, the presence of soluble RAGE (sRAGE) may abrogate cellular activation since the ligand is bound prior to interaction with the surface receptor.
Our aim was to analyse to what extent sRAGE is present in patients with chronic joint inflammation (RA) as compared with patients with non-inflammatory joint disease and with healthy subjects, and to assess whether there is an association between sRAGE levels and disease characteristics.
Matching samples of blood and synovial fluid were collected from 62 patients with RA with acute joint effusion. Blood from 45 healthy individuals, synovial fluid samples from 33 patients with non-inflammatory joint diseases and blood from six patients with non-inflammatory joint diseases were used for comparison. sRAGE levels were analysed using an ELISA.
RA patients displayed significantly decreased blood levels of sRAGE (871 ± 66 pg/ml, P < 0.0001) as compared with healthy controls (1290 ± 78 pg/ml) and with patients with non-inflammatory joint disease (1569 ± 168 pg/ml). Importantly, sRAGE levels in the synovial fluid of RA patients (379 ± 36 pg/ml) were lower than in corresponding blood samples and correlated significantly with blood sRAGE. Interestingly, a significantly higher sRAGE level was found in synovial fluid of RA patients treated with methotrexate as compared with patients without disease-modifying anti-rheumatic treatment.
We conclude that a decreased level of sRAGE in patients with RA might increase the propensity towards inflammation, whereas treatment with methotrexate counteracts this feature.
PMCID: PMC1175032  PMID: 15987483
8.  Elevated autoantibody content in rheumatoid arthritis synovia with lymphoid aggregates and the effect of rituximab 
Arthritis Research & Therapy  2008;10(5):R105.
The purpose of this study was to quantitatively evaluate the contribution of synovial lymphoid aggregates to autoantibody (rheumatoid factor [RF] and anti-cyclic citrullinated peptide [anti-CCP]) and total immunoglobulin (IgG and IgM) production in rheumatoid arthritis (RA) patients and the effect thereon of the B-cell-depleting antibody, rituximab, in the ARISE (Assessment of Rituximab's Immunomodulatory Synovial Effects) trial.
Autoantibodies as well as total IgM and IgG were quantified by enzyme-linked immunosorbent assay in extracts of synovial tissues and matched serum from patients with RA or osteoarthritis (OA). Synovial biopsies and serum were obtained at baseline and 8 weeks following rituximab therapy in 14 RA patients. A synovial/serum index (SSI) was calculated as the ratio of synovial to serum antibody/albumin, with values above 1 representing synovial enrichment. Lymphoid aggregates were evaluated histologically.
Anti-CCP IgG, but not RF-IgM, was significantly enriched in RA synovia compared with serum. Total IgM and IgG were also enriched in RA, but not in OA. SSI correlated significantly with mRNA content for both IgM and IgG, demonstrating that it reflected synovial immunoglobulin production. RA synovia with lymphocyte aggregates contained significantly elevated RF-IgM and anti-CCP IgG compared with tissues with diffuse lymphoid infiltration. Rituximab treatment did not affect synovial autoantibody or total immunoglobulin SSI overall. However, in aggregate-containing tissues, rituximab significantly reduced total IgM and IgG SSI as well as IgM and IgG1 mRNA. Surprisingly, RF-IgM and anti-CCP IgG SSIs were unchanged by rituximab in aggregate-containing synovia.
Combined with earlier observations that synovial lymphoid aggregates are unaltered by rituximab treatment, these data suggest that lymphoid aggregates may provide a protective niche for autoantibody-producing cells.
Trial Registration
The ARISE trial is registered at as number NCT00147966.
PMCID: PMC2592782  PMID: 18761748
9.  Glycation and Carboxymethyllysine Levels in Skin Collagen Predict the Risk of Future 10-Year Progression of Diabetic Retinopathy and Nephropathy in the Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications Participants With Type 1 Diabetes 
Diabetes  2005;54(11):3103-3111.
Several mechanistic pathways linking hyperglycemia to diabetes complications, including glycation of proteins and formation of advanced glycation end products (AGEs), have been proposed. We investigated the hypothesis that skin collagen glycation and AGEs predict the risk of progression of microvascular disease. We measured glycation products in the skin collagen of 211 Diabetes Control and Complications Trial (DCCT) volunteers in 1992 who continued to be followed in the Epidemiology of Diabetes Interventions and Complications study for 10 years. We determined whether the earlier measurements of glycated collagen and AGE levels correlated with the risk of progression of retinopathy and nephropathy from the end of the DCCT to 10 years later. In multivariate analyses, the combination of furosine (glycated collagen) and carboxymethyllysine (CML) predicted the progression of retinopathy (χ2 = 59.4, P < 0.0001) and nephropathy (χ2 = 18.2, P = 0.0001), even after adjustment for mean HbA1c (A1C) (χ2 = 32.7, P < 0.0001 for retinopathy) and (χ2 = 12.8, P = 0.0016 for nephropathy). The predictive effect of A1C vanished after adjustment for furosine and CML (χ2 = 0.0002, P = 0.987 for retinopathy and χ2 = 0.0002, P = 0.964 for nephropathy). Furosine explained more of the variation in the 10-year progression of retinopathy and nephropathy than did CML. These results strengthen the role of glycation of proteins and AGE formation in the pathogenesis of retinopathy and nephropathy. Glycation and subsequent AGE formation may explain the risk of these complications associated with prior A1C and provide a rational basis for the phenomenon of “metabolic memory” in the pathogenesis of these diabetes complications.
PMCID: PMC2622724  PMID: 16249432
10.  Synovial membrane p53 protein immunoreactivity in rheumatoid arthritis patients 
Annals of the Rheumatic Diseases  2000;59(2):143-145.
OBJECTIVES—To examine the expression of the p53 protein in synovial membrane of rheumatoid arthritis (RA) patients and to compare this with the expression in normal synovial tissues in subjects without RA.
METHODS—Immunohistological expression of the p53 protein was studied using a streptavidin-biotin-peroxidase method and the monoclonal antibody DO-7, an antibody directed against both wild and mutant forms of p53 protein, in synovial tissues of RA patients (n=10) and from subjects with no known joint disease (n=4).
RESULTS—p53 protein expression was present in a small percentage of synovial cells in the majority of the RA patients (n=8; 80%) and in half of the normal control cases with no inflammatory joint disease (n=2; 50%). No sample had more than 5% cells staining with intranuclear pattern. The difference in synovial p53 immunoreactivity between the RA patients and normal controls is not statistically significant (p= 0.64; χ2 contingency test).
CONCLUSIONS—This study has shown that p53 protein is only weakly expressed in the rheumatoid synovial membrane, with a low percentage of p53 protein immunostaining cells present, with intranuclear staining. These results suggest this is wild type p53 protein rather than mutant protein. These findings suggest that synovial p53 protein expression may not be important in the pathogenesis of RA and may only represent a reactive repair process to DNA damage secondary to the immune and inflammatory reactions associated with the disease.

PMCID: PMC1753073  PMID: 10666172
11.  Infusion fluids contain harmful glucose degradation products 
Intensive Care Medicine  2010;36(7):1213-1220.
Glucose degradation products (GDPs) are precursors of advanced glycation end products (AGEs) that cause cellular damage and inflammation. We examined the content of GDPs in commercially available glucose-containing infusion fluids and investigated whether GDPs are found in patients’ blood.
The content of GDPs was examined in infusion fluids by high-performance liquid chromatography (HPLC) analysis. To investigate whether GDPs also are found in patients, we included 11 patients who received glucose fluids (standard group) during and after their surgery and 11 control patients receiving buffered saline (control group). Blood samples were analyzed for GDP content and carboxymethyllysine (CML), as a measure of AGE formation. The influence of heat-sterilized fluids on cell viability and cell function upon infection was investigated.
All investigated fluids contained high concentrations of GDPs, such as 3-deoxyglucosone (3-DG). Serum concentration of 3-DG increased rapidly by a factor of eight in patients receiving standard therapy. Serum CML levels increased significantly and showed linear correlation with the amount of infused 3-DG. There was no increase in serum 3-DG or CML concentrations in the control group. The concentration of GDPs in most of the tested fluids damaged neutrophils, reducing their cytokine secretion, and inhibited microbial killing.
These findings indicate that normal standard fluid therapy involves unwanted infusion of GDPs. Reduction of the content of GDPs in commonly used infusion fluids may improve cell function, and possibly also organ function, in intensive-care patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s00134-010-1873-x) contains supplementary material, which is available to authorized users.
PMCID: PMC2882046  PMID: 20397009
Glucose; Infusion fluids; Toxicity; Advanced glycation end products; Neutrophils; Innate defense; Cell survival
12.  Maternally Transmitted and Food-Derived Glycotoxins 
Diabetes Care  2010;33(10):2232-2237.
Proinflammatory advanced glycation end products (AGEs) found in thermally processed foods correlate with serum AGEs (sAGEs) and promote type 1 and type 2 diabetes in mice. Herein we assess the relationship of maternal blood and food AGEs to circulating glycoxidants, inflammatory markers, and insulin levels in infants up to age 1 year.
AGEs (Nε-carboxymethyllysine [CML] and methylglyoxal derivatives) were tested in sera of healthy mothers in labor (n = 60), their infants, and infant foods. Plasma 8-isoprostane, fasting glucose, insulin, leptin, and adiponectin levels were assessed in 12-month-old infants.
Significant correlations were found between newborn and maternal serum CML (sCML) (r = 0.734, P = 0.001) serum methylglyoxal derivatives (sMGs) (r = 0.593, P = 0.001), and 8-isoprostanes (r = 0.644, P = 0.001). Infant adiponectin at 12 months negatively correlated with maternal sCML (r = −0.467, P = 0.011), whereas high maternal sMGs predicted higher infant insulin or homeostasis model assessment (P = 0.027). Infant sAGEs significantly increased with the initiation of processed infant food intake, raising daily AGE consumption by ∼7.5-fold in year 1.
Maternal blood and food-derived AGEs prematurely raise AGEs in children to adult norms, preconditioning them to abnormally high oxidant stress and inflammation and thus possibly to early onset of disease, such as diabetes.
PMCID: PMC2945165  PMID: 20628088
13.  Differential expression of the urokinase receptor (CD87) in arthritic and normal synovial tissues. 
Journal of Clinical Pathology  1997;50(4):314-319.
AIM: To determine whether the urokinase plasminogen activator receptor (u-PAR; CD87) exhibits a possible pathogenic role in rheumatoid and osteoarthritis. METHODS: A semiquantitative, indirect immunoperoxidase histochemical analysis was performed on frozen synovial tissue sections. The recently characterised monoclonal antibody 10G7 recognising transfectants bearing u-PAR was used. Synovial tissue was obtained from 10 patients with rheumatoid arthritis, 10 patients with osteoarthritis, and four normal subjects. RESULTS: u-PAR was expressed on 70-90% of synovial tissue lining cells and subsynovial, interstitial macrophages from the arthritis patients, but only on a few myeloid cells from the normal subjects. It was also present on more endothelial cells from the rheumatoid and osteoarthritis patients, than from normal synovial tissue. CONCLUSIONS: Plasminogen activators are important in joint destruction underlying arthritis. The up-regulated expression of u-PAR in diseased versus normal synovial tissue suggests a role for this antigen in the inflammatory and angiogenic mechanisms underlying rheumatoid and osteoarthritis.
PMCID: PMC499882  PMID: 9215148
14.  Advanced glycation end-product (AGE)-damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis 
Arthritis Research & Therapy  2003;5(2):R82-R90.
Advanced glycation end-product (AGE)-damaged IgG occurs as a result of hyperglycemia and/or oxidative stress. Autoantibodies to IgG-AGE were previously demonstrated in patients with severe, longstanding rheumatoid arthritis (RA). We investigated whether IgG-AGE and anti-IgG-AGE antibodies were present early in the course of RA and other inflammatory arthropathies. We prospectively followed a cohort of 238 patients with inflammatory arthritis of duration less than 1 year. Patients were evaluated clinically and serologically, and radiographs were obtained at initial and 1-year visits. Sera were assayed for IgG-AGE and anti-IgG-AGE antibodies by enzyme-linked immunosorbent assay (ELISA). Rheumatoid factor (RF) was determined by nephelometry and ELISA. Of all patients, 29% had RF-positive RA, 15% had RF-negative RA, 18% had spondyloarthropathy, and 38% had undifferentiated arthritis. IgG-AGE was present in 19% of patients, and was similar in amount and frequency in all groups. Patients with elevated IgG-AGE levels had significantly higher levels of the inflammatory markers C-reactive protein and erythrocyte sedimentation rate, but there was no correlation with blood glucose levels. Overall, 27% of the patients had IgM anti-IgG-AGE antibodies. These antibodies were highly significantly associated with RFs (P < 0.0001) and with swollen joint count (P < 0.01). In early onset arthritis, IgG damaged by AGE was detected in all patient groups. The ability to make IgM anti-IgG-AGE antibodies, however, was restricted to a subset of RF-positive RA patients with more active disease. The persistence of the anti-IgG-AGE response was more specific to RA, and was transient in the patients with spondyloarthropathy and with undifferentiated arthritis who were initially found to be positive for anti-IgG-AGE antibodies.
PMCID: PMC165032  PMID: 12718751
nonenzymatic glycation; rheumatoid arthritis; rheumatoid factor
15.  RAGE Modulates Hypoxia/Reoxygenation Injury in Adult Murine Cardiomyocytes via JNK and GSK-3β Signaling Pathways 
PLoS ONE  2010;5(4):e10092.
Advanced glycation end-products (AGEs) have been implicated in diverse pathological settings including diabetes, inflammation and acute ischemia/reperfusion injury in the heart. AGEs interact with the receptor for AGEs (RAGE) and transduce signals through activation of MAPKs and proapoptotic pathways. In the current study, adult cardiomyocytes were studied in an in vitro ischemia/reperfusion (I/R) injury model to delineate the molecular mechanisms underlying RAGE-mediated injury due to hypoxia/reoxygenation (H/R).
Methodology/Principal Findings
Cardiomyocytes isolated from adult wild-type (WT), homozygous RAGE-null (RKO), and WT mice treated with soluble RAGE (sRAGE) were subjected to hypoxia for 30 minutes alone or followed by reoxygenation for 1 hour. In specific experiments, RAGE ligand carboxymethyllysine (CML)-AGE (termed “CML” in this manuscript) was evaluated in vitro. LDH, a marker of cellular injury, was assayed in the supernatant in the presence or absence of signaling inhibitor-treated cardiomyocytes. Cardiomyocyte levels of heterogeneous AGEs were measured using ELISA. A pronounced increase in RAGE expression along with AGEs was observed in H/R vs. normoxia in WT cardiomyocytes. WT cardiomyocytes after H/R displayed increased LDH release compared to RKO or sRAGE-treated cardiomyocytes. Our results revealed significant increases in phospho-JNK in WT cardiomyocytes after H/R. In contrast, neither RKO nor sRAGE-treated cardiomyocytes exhibited increased phosphorylation of JNK after H/R stress. The impact of RAGE deletion on GSK-3β phosphorylation in the cardiomyocytes subjected to H/R revealed significantly higher levels of phospho-GSK-3β/total GSK-3β in RKO, as well as in sRAGE-treated cardiomyocytes versus WT cardiomyocytes after H/R. Further investigation established a key role for Akt, which functions upstream of GSK-3β, in modulating H/R injury in adult cardiomyocytes.
These data illustrate key roles for RAGE-ligand interaction in the pathogenesis of cardiomyocyte injury induced by hypoxia/reoxygenation and indicate that the effects of RAGE are mediated by JNK activation and dephosphorylation of GSK-3β. The outcome in this study lends further support to the potential use of RAGE blockade as an adjunctive therapy for protection of the ischemic heart.
PMCID: PMC2852407  PMID: 20404919
16.  The expression of the receptor for advanced glycation end-products (RAGE) in RA-FLS is induced by IL-17 via Act-1 
Arthritis Research & Therapy  2011;13(4):R113.
The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-κB (NF-κB) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated.
RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production.
RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1β (*P <0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-α in RA-FLS. The combined stimuli of both IL-17 and IL-1β significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1β alone (P <0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL-17.
RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL-1β. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment.
PMCID: PMC3239351  PMID: 21749686
17.  Receptor activator NF-κB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis 
Annals of the Rheumatic Diseases  2002;61(12):1047-1054.
Objectives: To compare receptor activator of NF-κB ligand (RANKL) production in the synovial tissue from patients with active rheumatoid arthritis (RA), inactive RA, spondyloarthropathies (SpA), osteoarthritis, and from normal subjects. In addition, to establish the cell lineages expressing RANKL in these tissues.
Methods: Immunohistological analysis of frozen synovial tissue biopsy specimens was performed using a monoclonal antibody (mAb) to detect RANKL. Sections were evaluated by computer assisted image analysis and semiquantitative analysis to compare RANKL expression between groups. Dual and sequential labelling with mAb RANKL and cell lineage specific monoclonal antibodies were used to determine the types of cells expressing RANKL.
Results: Higher levels of RANKL were expressed in tissues from patients with active RA and SpA than in tissues from patients with inactive RA, osteoarthritis, and from normal subjects. RANKL protein was associated with CD3 antigen-positive lymphocytes and some macrophages. RANKL was predominantly associated with activated, memory T cells (CD45Ro positive cells) in patients with active RA and spondyloarthropathy (SpA).
Conclusions: The highest levels of RANKL were detected in patients with RA with active synovitis and in some patients with SpA. An increase in RANKL in the inflamed joint of patients with RA, produced by infiltrating activated T cells and macrophages, is likely to be an important cause of joint erosions in RA.
PMCID: PMC1753975  PMID: 12429533
18.  Synovial fluid beta 2 microglobulin and hydroxyproline fractions in rheumatoid arthritis and nonautoimmune arthropathies. 
Annals of the Rheumatic Diseases  1980;39(3):207-216.
Fourteen patients with classical and definite seropositive rheumatoid arthritis (RA), 5 patients with microcrystalline arthritis, and 7 patients with osteoarthrosis were studied with respect to markers of newly synthesised collagen (synovial NDOH pro levels); markers of connective tissue resorption (synovial DOH pro and NBH levels); markers of lymphoid tissue activity (synovial and plasma beta 2m levels). Higher amounts of NDOH pro in RA synovial fluid are compatible with the hypothesis of a local connective tissue production as suggested by Uitto et al. on basis of a higher protocollagen proline hydroxylase activity in RA synovial tissue. DOH pro and NBH do not differ significantly in synovial fluid from RA or gouty patients, but the correlations between these forms of OH pro and, respectively, synovial lymphocytes and polymorphonuclear leucocytes are indicative of different processes of connective tissue remodelling in the 2 conditions. Synovial beta 2m levels are a direct function of total synovial lymphocyte counts independently of the type of arthropathy being explored. The ratio of synovial to plasma beta 2m is systematically above unity in RA patients only.
PMCID: PMC1000518  PMID: 6158297
19.  Localisation of vitronectin receptor immunoreactivity and tartrate resistant acid phosphatase activity in synovium from patients with inflammatory or degenerative arthritis. 
Annals of the Rheumatic Diseases  1993;52(2):133-137.
The influx of cells into the synovial intima in rheumatoid joints may include osteoclasts and their precursors. The distribution of osteoclast markers--namely, tartrate resistant acid phosphatase activity and the expression of vitronectin receptor (shown with monoclonal antibodies 13C2 and 23C6)--was therefore examined in synovium obtained from patients with rheumatoid (RA) or degenerative (OA) arthritis. Tartrate resistant acid phosphatase positive cells were found in frozen sections of 60% (n = 30) of RA and 69% (n = 29) of OA synovial membranes. Whereas all synovia tested (four RA, four OA) showed diffuse staining of the lining cells with 13C2, 55% (n = 11) of RA and 57% (n = 14) of OA synovial membranes contained isolated cells stained with 23C6 scattered throughout the tissue. In cultures of synovial cells, tartrate resistant acid phosphatase positive, multinuclear, and 23C6 positive cells were found; these cells did not, however, form resorption pits on bone slices. The results show that fully differentiated osteoclasts are uncommon in synovium from patients with either degenerative or inflammatory arthropathies.
PMCID: PMC1004992  PMID: 7680551
20.  Synovial tissue hypoxia and inflammation in vivo 
Annals of the Rheumatic Diseases  2010;69(7):1389-1395.
Hypoxia is a microenvironmental feature in the inflamed joint, which promotes survival advantage for cells. The aim of this study was to examine the relationship of partial oxygen pressure in the synovial tissue (tPO2) in patients with inflammatory arthritis with macroscopic/microscopic inflammation and local levels of proinflammatory mediators.
Patients with inflammatory arthritis underwent full clinical assessment and video arthroscopy to quantify macroscopic synovitis and measure synovial tPO2 under direct visualisation. Cell specific markers (CD3 (T cells), CD68 (macrophages), Ki67 (cell proliferation) and terminal deoxynucleotidyl transferase dUTP nick end labelling (cell apoptosis)) were quantified by immunohistology. In vitro migration was assessed in primary and normal synoviocytes (synovial fibroblast cells (SFCs)) using a wound repair scratch assay. Levels of tumour necrosis factor α (TNFα), interleukin 1β (IL1β), interferon γ (IFNγ), IL6, macrophage inflammatory protein 3α (MIP3α) and IL8 were quantified, in matched serum and synovial fluid, by multiplex cytokine assay and ELISA.
The tPO2 was 22.5 (range 3.2–54.1) mm Hg and correlated inversely with macroscopic synovitis (r=−0.421, p=0.02), sublining CD3 cells (−0.611, p<0.01) and sublining CD68 cells (r=−0.615, p<0.001). No relationship with cell proliferation or apoptosis was found. Primary and normal SFCs exposed to 1% and 3% oxygen (reflecting the median tPO2 in vivo) induced cell migration. This was coupled with significantly higher levels of synovial fluid tumour necrosis factor α (TNFα), IL1β, IFNγ and MIP3α in patients with tPO2 <20 mm Hg (all p values <0.05).
This is the first study to show a direct in vivo correlation between synovial tPO2, inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.
PMCID: PMC2946116  PMID: 20439288
21.  Lipoxygenation of arachidonic acid as a source of polymorphonuclear leukocyte chemotactic factors in synovial fluid and tissue in rheumatoid arthritis and spondyloarthritis. 
Journal of Clinical Investigation  1980;66(5):1166-1170.
The predominant lipoxygenase products of arachidonic acid were extracted and purified from synovial fluid and sonicates of synovial tissue of patients with rheumatoid arthritis (RA), spondyloarthritis (SA), or a noninflammatory arthropathy (NIA). The concentration of 5(S),12(R)-dihydroxy-6,8,10-(trans/trans/cis)-14-cis-eicosatetraenoic acid (leukotriene B4) in synovial fluid was elevated significantly in patients with RA and a positive latex test for rheumatoid factor (P < 0.05, n = 14) and in patients with SA (P < 0.05, n = 10), compared with that of subjects with NIA (n = 9). The content of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), but not of leukotriene B4, was elevated significantly in synovial tissue of seven patients with RA in comparison with that of four subjects with NIA (P < 0.05). A single intra-articular injection of corticosteroid significantly lowered the synovial fluid level of leukotriene B4 in six patients with RA. These data suggest an involvement of the potent chemotactic factors 5-HETE and leukotriene B4 in human inflammatory disease.
PMCID: PMC371556  PMID: 6253525
22.  Are synovial biopsies of diagnostic value? 
Arthritis Research & Therapy  2003;5(6):271-278.
Synovial tissue is readily accessible by closed needle or arthroscopic biopsy. These techniques provide adequate tissue for most diagnostic requirements. Examination of synovial tissue can assist in the diagnosis of some joint infections, and in several atypical or rare synovial disorders. Histological confirmation is not normally required for diagnosis of the common forms of inflammatory arthritis, including rheumatoid arthritis (RA). In patients with either established or early RA, immunohistological measures of inflammation in synovial tissue are associated with clinical measures of disease activity, may predict the clinical outcome, and change in response to treatment. Surrogate markers of disease activity and outcome that have been identified in synovial tissue include components of the cellular infiltrate, and several mediators of inflammation and matrix degradation. There is evidence that the very early introduction of disease-modifying therapy inhibits progressive structural damage maximally. Clinicians exploiting this 'window of opportunity' therefore require very early indicators of the diagnosis and outcome in patients who present with an undifferentiated inflammatory arthritis. Some immunohistological features have been described that distinguish patients who are likely to develop progressive RA and who might benefit most from early aggressive therapeutic intervention. In this regard, the inclusion of pharmacogenomic and proteomic techniques in the analysis of synovial tissue presents some exciting possibilities for future research.
PMCID: PMC333414  PMID: 14680502
synovial biopsy; diagnosis; early arthritis; rheumatoid arthritis; undifferentiated arthritis
23.  Immunoregulatory role of interleukin 10 in rheumatoid arthritis 
The Journal of Experimental Medicine  1994;179(5):1517-1527.
The presence and the role of interleukin 10 (IL-10), a potent cytokine synthesis inhibitory factor and antiinflammatory cytokine, were investigated in rheumatoid arthritis (RA). The expression of both mRNA and protein for IL-10 could be demonstrated in RA and osteoarthritis (OA) joints. Human IL-10 mRNA could be demonstrated by polymerase chain reaction amplification of cDNA made by reverse transcription of total RNA extracted directly from synovial tissue in five out of five RA and four out of five OA patients. IL-10 protein was demonstrated by specific immunoassay and immunohistology. IL-10 protein was spontaneously produced in all 11 RA and 17 OA synovial membrane cultures investigated, and this production was sustained for up to 5 d in culture in the absence of any extrinsic stimulation. IL-10 protein could also be detected by immunohistology in all five RA and four OA synovial membrane biopsies investigated, but not three normal synovial membranes. Immunohistology revealed that the IL-10 was localized to the synovial membrane lining layer and mononuclear cell aggregates. Immunofluorescence double staining revealed that the sources of IL-10 were monocytes in the lining layer, and T cells in the mononuclear cell aggregates. We found evidence that the IL-10 expression was functionally relevant, as neutralization of endogenously produced IL-10 in the RA synovial membrane cultures resulted in a two- to threefold increase in the protein levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and IL-1 beta, although IL-6 and IL-8 levels were not affected. The addition of exogenous recombinant IL-10 to the RA synovial membrane cultures resulted in a two- to threefold decrease in the levels of TNF-alpha and IL-1 beta. IL-8 levels were reduced by day 5; however, IL-6 levels were not affected by exogenous IL-10. Neutralization of the endogenous IL-10 in two out of seven RA synovial membrane cultures resulted in the expression of detectable levels of interferon gamma (561-1,050 pg/ml). Taken together, the above findings suggest that IL-10 is spontaneously produced in RA and OA and is an important immunoregulatory component in the cytokine network of RA, regulating monocyte and in some cases T cell cytokine production.
PMCID: PMC2191503  PMID: 8163935
24.  Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients. 
Molecular Medicine  1999;5(6):393-405.
BACKGROUND: The advanced stage of the Maillard reaction, which leads to the formation of advanced glycation end products (AGE), plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. N(epsilon)-(carboxymethyl)lysine (CML) is thought to be an important epitope for many of currently available AGE antibodies. However, recent findings have indicated that a major source of CML may be by pathways other than glycation. A distinction between CML and non-CML AGE may increase our understanding of AGE formation in vivo. In the present study, we prepared antibodies directed against CML and non-CML AGE. MATERIALS AND METHODS: AGE-rabbit serum albumin prepared by 4, 8, and 12 weeks of incubation with glucose was used to immunize rabbits, and a high-titer AGE-specific antiserum was obtained without affinity for the carrier protein. To separate CML and non-CML AGE antibodies, the anti-AGE antiserum was subjected to affinity chromatography on a column coupled with AGE-BSA and CML-BSA. Two different antibodies were obtained, one reacting specifically with CML and the other reacting with non-CML AGE. Circulating levels of CML and non-CML AGE were measured in 66 type 2 diabetic patients without uremia by means of the competitive ELISA. Size distribution and clearance by hemodialysis detected by non-CML AGE and CML were assessed in serum from diabetic patients on hemodialysis. RESULTS: The serum non-CML AGE level in type 2 diabetic patients was significantly correlated with the mean fasting blood glucose level over the previous 2 months (r = 0.498, p < 0.0001) or the previous 1 month (r = 0.446, p = 0. 0002) and with HbA(1c) (r = 0.375, p = 0.0019), but the CML AGE level was not correlated with these clinical parameters. The CML and non-CML AGE were detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. The hemodialysis treatment did not affect the high-molecular-weight protein fractions. Although the low-molecular-weight peptide fractions (absorbance at 280 nm and fluorescence) were decreased by hemodialysis, there was no difference before and after dialysis in the non-CML AGE- and CML-peptide fractions (1.15 and 0.85 kD fractions). CONCLUSIONS: We propose that both CML and non-CML AGE are present in the blood and that non-CML AGE rather than CML AGE should be more closely evaluated when investigating the pathophysiology of AGE-related diseases.
PMCID: PMC2230431  PMID: 10415164
25.  Clinical Study of Advanced Glycation End Products in Egyptian Diabetic Obese and Non-Obese Patients 
Advanced glycation end products (AGEs) are complex, heterogenous molecules generated by glycation and oxidation of proteins in vivo, which are thought to markedly increase in diabetic patients. One of the recently identified AGEs is carboxy methyl lysine (CML), which is the main ligand of receptors for advanced glycation end products (RAGE). The present study aimed to assess the effect of obesity on such pathways in presence and absence of Type 2 diabetes mellitus. CML, soluble receptors for advanced glycation end products (sRAGE), HbA1C, lipid profile, liver function tests and kidney function tests were determined in 29 diabetic obese, 29 diabetic non-obese, 15 non-diabetic obese and 15 non-diabetic non-obese subjects. The study compared obese and non-obese subjects in presence and absence of type 2 diabetes. The results showed a significant increase in CML and a significant decrease in sRAGE in each of the diabetic obese group when compared with the diabetic non-obese group and the non-diabetic obese group when compared with the non-diabetic non-obese group. A significant positive correlation was found between CML and markers of obesity (body mass index and waist/hip ratio). These results suggest that obesity can increase CML independent of diabetes and support the reports that CML could be generated from both sugars and lipids. The present study suggests that treatment using glycation inhibitors like aminoguanidine or recombinant sRAGE will not only retard the diabetic complications, but may also have a prophylactic effect.
PMCID: PMC3614834  PMID: 23675236
advanced glycation end products; carboxymethyllysine; receptors for advanced glycation end products; obesity and type 2 diabetes mellitus

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