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1.  An In Silico Approach for Modelling T-Helper Polarizing iNKT Cell Agonists 
PLoS ONE  2014;9(1):e87000.
Many analogues of the glycolipid alpha-galactosylceramide (α-GalCer) are known to activate iNKT cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and Th2 cytokines. Because of iNKT cell involvement and associated Th1/Th2 cytokine changes in a broad spectrum of human diseases, the design of iNKT cell ligands with selective Th1 and Th2 properties has been the subject of extensive research. This search for novel iNKT cell ligands requires refined structural insights. Here we will visualize the chemical space of 333 currently known iNKT cell activators, including several newly tested analogues, by more than 3000 chemical descriptors which were calculated for each individual analogue. To evaluate the immunological responses we analyzed five different cytokines in five different test-systems. We linked the chemical space to the immunological space using a system biology computational approach resulting in highly sensitive and specific predictive models. Moreover, these models correspond with the current insights of iNKT cell activation by α-GalCer analogues, explaining the Th1 and Th2 biased responses, downstream of iNKT cell activation. We anticipate that such models will be of great value for the future design of iNKT cell agonists.
doi:10.1371/journal.pone.0087000
PMCID: PMC3909045  PMID: 24498010
2.  Microbes, the gut and ankylosing spondylitis 
It is increasingly clear that the interaction between host and microbiome profoundly affects health. There are 10 times more bacteria in and on our bodies than the total of our own cells, and the human intestine contains approximately 100 trillion bacteria. Interrogation of microbial communities by using classic microbiology techniques offers a very restricted view of these communities, allowing us to see only what we can grow in isolation. However, recent advances in sequencing technologies have greatly facilitated systematic and comprehensive studies of the role of the microbiome in human health and disease. Comprehensive understanding of our microbiome will enhance understanding of disease pathogenesis, which in turn may lead to rationally targeted therapy for a number of conditions, including autoimmunity.
doi:10.1186/ar4228
PMCID: PMC4060176  PMID: 23750937
3.  Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci 
Nature genetics  2013;45(7):730-738.
Ankylosing spondylitis is a common, highly heritable inflammatory arthritis affecting primarily the spine and pelvis. In addition to HLA-B*27 alleles, 12 loci have previously been identified that are associated with ankylosing spondylitis in populations of European ancestry, and 2 associated loci have been identified in Asians. In this study, we used the Illumina Immunochip microarray to perform a case-control association study involving 10,619 individuals with ankylosing spondylitis (cases) and 15,145 controls. We identified 13 new risk loci and 12 additional ankylosing spondylitis–associated haplotypes at 11 loci. Two ankylosing spondylitis–associated regions have now been identified encoding four aminopeptidases that are involved in peptide processing before major histocompatibility complex (MHC) class I presentation. Protective variants at two of these loci are associated both with reduced aminopeptidase function and with MHC class I cell surface expression.
doi:10.1038/ng.2667
PMCID: PMC3757343  PMID: 23749187
4.  Preclinical Evaluation of Invariant Natural Killer T Cells in the 5T33 Multiple Myeloma Model 
PLoS ONE  2013;8(5):e65075.
Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.
doi:10.1371/journal.pone.0065075
PMCID: PMC3669090  PMID: 23741460
5.  Divergent synthetic approach to 6”-modified α-GalCer analogues 
Organic & biomolecular chemistry  2011;9(24):8413-8421.
A synthetic approach is presented for the synthesis of galacturonic acid and d-fucosyl modified KRN7000. The approach allows for late-stage functionalisation of both the sugar 6”-OH and the sphingosine amino groups, which enables convenient synthesis of promising 6”-modified KRN7000 analogues.
doi:10.1039/c1ob06235b
PMCID: PMC3246390  PMID: 22042483
6.  Natural killer T cells in adipose tissue prevent insulin resistance 
The Journal of Clinical Investigation  2012;122(9):3343-3354.
Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell–deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue–resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue–resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.
doi:10.1172/JCI62739
PMCID: PMC3428087  PMID: 22863618
7.  Quantification of IFNγ- and IL17-producing cells after stimulation with citrullinated proteins in healthy subjects and RA patients 
Rheumatology International  2012;33:2661-2664.
Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA) and are currently used as a diagnostic marker. In this study, we wanted to quantify the numbers of T cells that react to a wide range of citrullinated proteins in a wide range of HLA-DR subtypes in order to investigate whether citrullination might create T-cell neo-epitopes and could initiate a universal T-cell response. Therefore, PBMCs from healthy volunteers and RA patients were stimulated with a citrullinated and non-citrullinated cell extract on IFNγ-ELISpot. We found a significantly higher number of IFNγ-secreting cells after stimulation with citrullinated proteins compared to non-citrullinated proteins in RA patients (1:14,441 cells vs. 1:32,880 cells) as well as in healthy subjects (1:6,261 reactive cells compared to 1:16,212 cells). Additionally, a higher number of IL17-secreting cells were found after stimulation with citrullinated proteins compared to their non-citrullinated counterparts. Our data indicate that citrulline-dependent T-cell response is not restricted to RA patients but that citrullination as such gives rise to a universal break in tolerance.
doi:10.1007/s00296-012-2470-9
PMCID: PMC3782635  PMID: 22825303
Citrullination; Rheumatoid arthritis; ELISpot; T cells
8.  Activated iNKT Cells Promote Memory CD8+ T Cell Differentiation during Viral Infection 
PLoS ONE  2012;7(5):e37991.
α-galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8+ T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8+ T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8+ T cells, as a consequence of reduced inflammation.
doi:10.1371/journal.pone.0037991
PMCID: PMC3359346  PMID: 22649570
9.  Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease? 
PLoS ONE  2011;6(10):e25589.
Background
Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.
Methodology/Principal Findings
Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn's disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.
Conclusions/Significance
Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.
doi:10.1371/journal.pone.0025589
PMCID: PMC3196494  PMID: 22028783
10.  Polymorphic Variants of LIGHT (TNF Superfamily-14) Alter Receptor Avidity and Bioavailability1 
The TNF superfamily member, LIGHT (TNFSF14) is a key cytokine that activates T cells and dendritic cells, and is implicated as a mediator of inflammatory, metabolic and malignant diseases. LIGHT engages the Lymphotoxin-β receptor (LTβR) and herpesvirus entry mediator (HVEM, TNFRSF14), but is competitively limited in activating these receptors by soluble decoy receptor-3 (DcR3, TNFRSF6B). Two variants in the human LIGHT alter the protein at E214K (rs344560) in the receptor-binding domain and S32L (rs2291667) in the cytosolic domain, however, the functional impact of these polymorphisms is unknown. A neutralizing antibody failed to bind the LIGHT-214K variant indicating this position as a part of the receptor-binding region. Relative to the predominant reference variant S32/E214, the other variants showed altered avidity with LTβR, and less with HVEM. Heterotrimers of the LIGHT variants decreased binding avidity to DcR3, and minimized the inhibitory effect of DcR3 towards LTβR-induced activation of NF-κB. In patients with immune-mediated inflammatory diseases, such as rheumatoid arthritis, DcR3 protein levels were significantly elevated.
Immunohistochemistry revealed synoviocytes as a significant source of DcR3 production, and DcR3 hyperexpression is controlled by post-transcriptional mechanisms. The increased potential for LTβR signaling, coupled with increased bioavailability due to lower DcR3 avidity, provides a mechanism of how polymorphic variants in LIGHT could contribute to the pathogenesis of inflammatory diseases.
doi:10.4049/jimmunol.1001159
PMCID: PMC2921593  PMID: 20592286
11.  Synthesis and Evaluation of Amino-Modified α-GalCer Analogues 
Organic letters  2010;12(13):2928-2931.
α-GalCer analogues featuring a phytoceramide 3- and 4-amino group have been synthesized. A Mitsunobu reaction involving phthalimide was employed for the introduction of the amino groups at the 3- and 4-positions of suitable phytosphingosine-derived precursors. The influence of these modifications on the interaction with the T-cell receptor of NKT cells was investigated, as well as the capacity of the amino-modified analogues to induce a cytokine response after in vivo administration.
doi:10.1021/ol100934z
PMCID: PMC2903208  PMID: 20518554
12.  Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis 
EMBO Molecular Medicine  2011;3(4):222-234.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
doi:10.1002/emmm.201000125
PMCID: PMC3377067  PMID: 21328541
Mycobacterium; SapM; tuberculosis; vaccine; BCG
13.  Histopathological cutaneous alterations in systemic sclerosis: a clinicopathological study 
Introduction
The aims of the present study were to identify histopathological parameters which are linked to local clinical skin disease at two distinct anatomical sites in systemic sclerosis (SSc) patients with skin involvement (limited cutaneous systemic sclerosis (lcSSc) or diffuse cutaneous systemic sclerosis (dcSSc)) and to determine the sensitivity of SSc specific histological alterations, focusing on SSc patients without clinical skin involvement (limited SSc (lSSc)).
Methods
Histopathological alterations were systematically scored in skin biopsies of 53 consecutive SSc patients (dorsal forearm and upper inner arm) and 18 controls (upper inner arm). Clinical skin involvement was evaluated using the modified Rodnan skin score. In patients with lcSSc or dcSSc, associations of histopathological parameters with local clinical skin involvement were determined by generalised estimation equation modelling.
Results
The hyalinised collagen score, the myofibroblast score, the mean epidermal thickness, the mononuclear cellular infiltration and the frequency of focal exocytosis differed significantly between biopsies with and without local clinical skin involvement. Except for mononuclear cellular infiltration, all of the continuous parameters correlated with the local clinical skin score at the dorsal forearm. Parakeratosis, myofibroblasts and intima proliferation were present in a minority of the SSc biopsies, but not in controls. No differences were found between lSSc and controls.
Conclusions
Several histopathological parameters are linked to local clinical skin disease. SSc-specific histological alterations have a low diagnostic sensitivity.
doi:10.1186/ar3267
PMCID: PMC3241379  PMID: 21356083
14.  Optimized alkylated cyclodextrin polysulphates with reduced risks on thromboembolic accidents improve osteoarthritic chondrocyte metabolism 
Rheumatology (Oxford, England)  2011;50(7):1226-1235.
Objectives. To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro.
Methods. OA chondrocytes were cultured in alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-CDPS (CE-CDPS), (2-hydroxypropyl)-β-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-β-CDPS (MA-CDPS) or β-CDPS for 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism were assayed by analysis of the accumulation of aggrecan in the interterritorial matrix, IL-6 secretion and qPCR. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analysed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible cross-reactivity with heparin-induced thrombocytopenia (HIT) antibodies.
Results. The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 µg/ml concentrations, on the other hand, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. aPTT and PT for all derivatives were lengthened for polysaccharide concentrations >50 µg/ml. Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT. Furthermore, CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate platelets, suggesting a minimal potential to induce HIT thromboembolic accidents in vivo.
Conclusions. CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.
doi:10.1093/rheumatology/keq396
PMCID: PMC3116210  PMID: 21345936
Chondroprotection; Osteoarthritis; Cyclodextrin polysulphates
15.  Differential mucosal expression of Th17-related genes between the inflamed colon and ileum of patients with inflammatory bowel disease 
BMC Immunology  2010;11:61.
Background
Immunological and genetic findings implicate Th17 effector cytokines in the pathogenesis of inflammatory bowel disease (IBD). Expression of Th17 pathway-associated genes is mainly studied in colonic disease. The present study assessed the mRNA expression levels of Th17 effector cytokines (IL17A, IL17F, IL21, IL22 and IL26) and genes involved in differentiation (IL6, IL1B, TGFB1, IL23A and STAT3) and recruitment of Th17 cells (CCR6 and CCL20) by quantitative real-time PCR analysis of colonic and ileal biopsies from 22 healthy control subjects, 26 patients with Crohn's disease (CD) and 12 patients with ulcerative colitis (UC). Inflammation was quantified by measuring expression of the inflammatory mediators IL8 and TNF.
Results
Evaluation of mRNA expression levels in colonic and ileal control samples revealed that TNF, TGFB1, STAT3 and CCR6 were expressed at higher levels in the ileum than in the colon. Expression of all the Th17 pathway-associated genes was increased in inflamed colonic samples. The increased expression of these genes was predominantly observed in samples from UC patients and was associated with more intense inflammation. Although increased expression of IL17A, IL17F, IL21 and IL26 was detected in inflamed ileal samples, expression of the indispensable Th17 cell differentiation factors TGFB1 and IL23A, the signaling molecule STAT3 and the Th17 recruitment factors CCR6 and CCL20 were unchanged.
Conclusions
Our findings suggest that immune regulation is different in colonic and ileal disease, which might have important consequences for therapeutic intervention.
doi:10.1186/1471-2172-11-61
PMCID: PMC3016394  PMID: 21144017
16.  Linking Crohn's Disease and Ankylosing Spondylitis: It's All about Genes! 
PLoS Genetics  2010;6(12):e1001223.
doi:10.1371/journal.pgen.1001223
PMCID: PMC2996322  PMID: 21152009
17.  Evidence for Significant Overlap between Common Risk Variants for Crohn's Disease and Ankylosing Spondylitis 
PLoS ONE  2010;5(11):e13795.
Background
A multicenter genome-wide association scan for Crohn's Disease (CD) has recently reported 40 CD susceptibility loci, including 29 novel ones (19 significant and 10 putative). To gain insight into the genetic overlap between CD and ankylosing spondylitis (AS), these markers were tested for association in AS patients.
Principal Findings
Two previously established associations, namely with the MHC and IL23R loci, were confirmed. In addition, rs2872507, which maps to a locus associated with asthma and influences the expression of the ORMDL3 gene in lymphoblastoid cells, showed a significant association with AS (p = 0.03). In gut biopsies of AS and CD patients, ORMDL3 expression was not significantly different from controls and no correlation was found with the rs2872507 genotype (Spearman's rho: −0.067). The distribution of p-values for the remaining 36 SNPs was significantly skewed towards low p-values unless the top 5 ranked SNPs (ORMDL3, NKX2–3, PTPN2, ICOSLG and MST1) were excluded from the analysis.
Conclusions
Association analysis using risk variants for CD led to the identification of a new risk variant associated with AS (ORMDL3), underscoring a role for ER stress in AS. In addition, two known and five potentially relevant associations were detected, contributing to common susceptibility of CD and AS.
doi:10.1371/journal.pone.0013795
PMCID: PMC2970560  PMID: 21072187
18.  A multiparameter approach to monitor disease activity in collagen-induced arthritis 
Arthritis Research & Therapy  2010;12(4):R160.
Introduction
Disease severity in collagen-induced arthritis (CIA) is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Although this is an accurate approach, it is also labour-intensive and the application of less invasive and less time-consuming methods is of great interest. However, it is still unclear which of these methods represents the most discriminating measure of disease activity.
Methods
We undertook a comparative analysis in which different measurements of inflammation and tissue damage in CIA were studied on an individual mouse level. We compared the current gold standard methods - clinical scoring and histological examination - with alternative methods based on scoring of X-ray or micro-computed tomography (CT) images and investigated the significance of systemically expressed proteins, involved in CIA pathogenesis, that have potential as biomarkers.
Results
Linear regression analysis revealed a marked association of serum matrix metalloproteinase (MMP)-3 levels with all features of CIA including inflammation, cartilage destruction and bone erosions. This association was improved by combined detection of MMP-3 and anti-collagen IgG2a antibody concentrations. In addition, combined analysis of both X-ray and micro-CT images was found to be predictive for cartilage and bone damage. Most remarkably, validation analysis using an independent data set proved that variations in disease severity, induced by different therapies, could be accurately represented by predicted values based on the proposed parameters.
Conclusions
Our analyses revealed that clinical scoring, combined with serum MMP-3, anti-collagen IgG2a measurement and scoring of X-ray and micro-CT images, yields a comprehensive insight into the different aspects of disease activity in CIA.
doi:10.1186/ar3119
PMCID: PMC2945063  PMID: 20731827
19.  Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins 
Arthritis Research & Therapy  2010;12(4):R132.
Introduction
Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients.
Methods
IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining.
Results
Circulating IC in the serum of RA patients and healthy controls contain fibrinogenβ and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogenγ and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogenβ were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients.
Conclusions
Citrullinated fibrinogenβ and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA.
doi:10.1186/ar3070
PMCID: PMC2945022  PMID: 20609218
20.  LTβR Signaling Induces Cytokine Expression and Up-Regulates Lymphangiogenic Factors in Lymph Node Anlagen1 
The formation of lymph nodes is a complex process crucially controlled through triggering of LTβR on mesenchymal cells by LTα1β2 expressing lymphoid tissue inducer (LTi) cells. This leads to the induction of chemokines to attract more hematopoietic cells and adhesion molecules to retain them. In this study, we show that the extravasation of the first hematopoietic cells at future lymph node locations occurs independently of LTα and that these cells, expressing TNF-related activation-induced cytokine (TRANCE), are the earliest LTi cells. By paracrine signaling the first expression of LTα1β2 is induced. Subsequent LTβR triggering on mesenchymal cells leads to their differentiation to stromal organizers, which now also start to express TRANCE, IL-7, as well as VEGF-C, in addition to the induced adhesion molecules and chemokines. Both TRANCE and IL-7 will further induce the expression of LTα1β2 on newly arrived immature LTi cells, resulting in more LTβR triggering, generating a positive feedback loop. Thus, LTβR triggering by LTi cells during lymph node development creates a local environment to which hematopoietic precursors are attracted and where they locally differentiate into fully mature, LTα1β2 expressing, LTi cells. Furthermore, the same signals may regulate lymphangiogenesis to the lymph node through induction of VEGF-C.
doi:10.4049/jimmunol.0801165
PMCID: PMC2703443  PMID: 19380791
21.  Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus–infected mice 
The Journal of Experimental Medicine  2009;206(11):2339-2349.
Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c+ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11chi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.
doi:10.1084/jem.20090410
PMCID: PMC2768850  PMID: 19808255
22.  Progress in spondylarthritis. Immunopathogenesis of spondyloarthritis: which cells drive disease? 
Spondyloarthritides, or SpA, form a cluster of chronic inflammatory diseases with the axial skeleton as the most typical disease localisation, although extra-articular manifestations such as intestinal inflammation may frequently occur during the course of the disease. This review summarises recent progress in our understanding of the immunopathogenesis of SpA with special emphasis on the cellular constituents considered to be responsible for the initiation and/or perpetuation of inflammation. There are several arguments favouring a role for haematopoietic cells in the pathophysiology of spondyloarthritis, including HLA-B27-associated dendritic cell disturbances, HLA-B27 misfolding properties and T helper 17 cells. In addition, recent studies have pointed toward a pivotal role for stromal cells. A major challenge, however, remains to determine how recently identified genetic associations such as interleukin-23 receptor polymorphisms may influence cellular targets in spondyloarthritis.
doi:10.1186/ar2722
PMCID: PMC2714138  PMID: 19591637
23.  Morbid anatomy of ‘erosive osteoarthritis’ of the interphalangeal finger joints: an optimised scoring system to monitor disease progression in affected joints 
Annals of the Rheumatic Diseases  2009;69(5):862-867.
Objectives
To develop and validate a quantitative radiographic scoring system, the Ghent University Scoring System (GUSS), with better ability to detect progression over a shorter period of time in erosive osteoarthritis (OA) of the interphalangeal (IP) finger joints compared with the existing anatomic phase scoring system.
Methods
Thirty IP finger joints showing erosive features at baseline or follow-up were selected from 18 patients with erosive hand OA. Posteroanterior radiographs of these joints obtained at baseline, 6 and 12 months—totalling 90 images—were used for the study. All joints were first scored according to the original anatomic phase scoring system. Erosive progression and signs of repair or remodelling were then scored by indicating the proportion of normal subchondral bone, subchondral plate and joint space on an 11-point rating scale (range 0–100 with 10 unit increases). Inter- and intrareader reproducibility was studied using intraclass correlation coefficients (ICCs). Based on the within-variance of two readers, the smallest detectable change (SDC) was calculated and allowed identification of joints with changes above the SDC as ‘progressors’.
Results
Longitudinal inter-reader ICC scores rated well for all variables and the total score (ICC 0.86–0.93). To identify ‘real’ change over background noise, a change of at least 40 units on the total score (range 0–300) over 12 months (SDC 0–12:36.0), and 50 units over 6 months (SDC 0–6:47.6) had to be present. 60% of the 30 joints were identified as ‘progressors’ over 6 months compared with 33.3% with the classical anatomical scoring system, and 70% versus 56.6%, respectively, over 12 months.
Conclusion
GUSS, is a reliable method to score radiographic change over time in erosive IP OA and detects more progression over a shorter period of time than the classical scoring system.
doi:10.1136/ard.2009.112714
PMCID: PMC2925149  PMID: 19948521
24.  Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases 
Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic–spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial–gut axis in human disease.
doi:10.1084/jem.20070906
PMCID: PMC2271010  PMID: 18250193
25.  Diagnostic value of anti-human citrullinated fibrinogen ELISA and comparison with four other anti-citrullinated protein assays 
We studied the diagnostic performance of the anti-human citrullinated fibrinogen antibody (AhFibA) ELISA for rheumatoid arthritis (RA) in a consecutive cohort (population 1) and evaluated the agreement between the AhFibA ELISA and four other assays for anti-citrullinated protein/peptide antibodies (ACPAs) as well as rheumatoid factor in patients with longstanding RA (population 2). Population 1 consisted of 1024 patients with rheumatic symptoms; serum samples from these patients were sent to our laboratory for ACPA testing within the context of a diagnostic investigation for RA. Ninety-two of these patients were classified as having RA according to the American College of Rheumatology criteria and 463 were classified as non-RA patients. Population 2 consisted of 180 patients with longstanding RA and was used to assess agreement and correlations between five ACPA assays: anti-cyclic citrullinated peptide (CCP)1 and anti-CCP2 antibodies were detected using a commercially available ELISA, AhFibA using ELISA, and anti-PepA and anti-PepB antibodies using line immunoassay. Applying previously proposed cut-offs for AhFibA, we obtained a sensitivity of 60.9% and a specificity of 98.7% in population 1. Receiver operating characteristic curve analysis could not detect a significant difference in diagnostic performance between the AhFibA ELISA and anti-CCP2 assay. Performing a hierarchical nearest neighborhood cluster analysis of the five different ACPA assays in population 2, we identified two clusters: a cluster of anti-pepA, anti-pepB and anti-CCP1, and a cluster of AhFibA and anti-CCP2. In conclusion, we found that AhFibA and anti-CCP2 antibodies had similar diagnostic performance. However, disagreement between ACPA tests may occur.
doi:10.1186/ar2011
PMCID: PMC1779401  PMID: 16859515

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