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1.  HSP: Bystander Antigen in Atopic Diseases? 
Over the last years insight in the complex interactions between innate and adaptive immunity in the regulation of an inflammatory response has increased enormously. This has revived the interest in stress proteins; proteins that are expressed during cell stress. As these proteins can attract and trigger an immunological response they can act as important mediators in this interaction. In this respect, of special interest are proteins that may act as modulators of both innate and adaptive immunity. Heat shock proteins (HSPs) are stress proteins that have these, and more, characteristics. More than two decades of studies on HSPs has revealed that they are part of intrinsic, “natural” mechanisms that steer inflammation. This has provoked comprehensive explorations of the role of HSPs in various human inflammatory diseases. Most studies have focused on classical autoimmune diseases. This has led to the development of clinical studies with HSPs that have shown promise in Phase II/III clinical trials. Remarkably, only very little is yet known of the role of HSPs in atopic diseases. In allergic disease a number of studies have investigated the possibility that allergen-specific regulatory T cell (Treg) function is defective in individuals with allergic diseases. This raises the question whether methods can be identified to improve the Treg repertoire. Studies from other inflammatory diseases have suggested HSPs may have such a beneficial effect on the T cell repertoire. Based on the immune mechanisms of atopic diseases, in this review we will argue that, as in other human inflammatory conditions, understanding immunity to HSPs is likely also relevant for atopic diseases. Specifically, we will discuss why certain HSPs such as HSP60 connect the immune response to environmental antigens with regulation of the inflammatory response. Thus they provide a molecular link that may eventually even help to better understand the immune pathological basis of the hygiene hypothesis.
PMCID: PMC3364480  PMID: 22666223
allergic disease; atopic disease; stress proteins; heat shock protein; HSP60; regulatory T cells; hygiene hypothesis; inflammation
2.  Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross‐sectional study 
Annals of the Rheumatic Diseases  2006;66(5):589-598.
Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of disorders with, for the most part, an unknown immunopathogenesis. Although onset and disease course differ, the subtypes of JIA share the occurrence of chronic inflammation of the joints, with infiltrations of immunocompetent cells that secrete inflammatory mediators.
To identify a panel of cytokines specifically related to the inflammatory process in JIA.
Using a new technology, the multiplex immunoassay, 30 cytokines were measured in plasma of 65 patients with JIA , of which 34 were paired with synovial fluid. These data were compared with plasma of 20 healthy controls and 9 patients with type I diabetes, a chronic inflammatory disease.
Patients with JIA had, irrespective of their subclassification, significantly higher levels of tumour necrosis factor α, macrophage inhibitory factor (MIF), CCL2, CCL3, CCL11, CCL22 and CXCL9 in plasma than controls. In paired plasma and synovial fluid samples of patients with JIA, significantly higher levels of interleukin (IL)6, IL15, CCL2, CCL3, CXCL8, CXCL9 and CXCL10 were present in synovial fluid. Cluster analysis in all patients with JIA revealed a predominant pro‐inflammatory cytokine cluster during active disease and a regulatory/anti‐inflammatory‐related cytokine cluster during remission. Whether a discrimination profile of various cytokines could help in the determination of disease classification was tested.
It is suggested that several cytokines (IL18, MIF, CCL2, CCL3, CCL11, CXCL9 and CXCL10) may correspond to the activation status during inflammation in JIA and could be instrumental in monitoring disease activity and outcomes of (new) immunotherapies.
PMCID: PMC1954617  PMID: 17170049
3.  T cell recognition of naturally presented epitopes of self-heat shock protein 70 
Cell Stress & Chaperones  2014;19(4):569-578.
Self-reactive T cells have shown to have a potential role as regulators of the immune system preventing or even suppressing autoimmunity. One of the most abundant proteins that can be eluted from human HLA molecules is heat shock protein 70 (HSP70). The aims of the current study are to identify HSP70 epitopes based on published HLA elution studies and to investigate whether T cells from healthy individuals may respond to such self-epitopes. A literature search and subsequent in silico binding prediction based on theoretical MHC binding motifs resulted in the identification of seven HSP70 epitopes. PBMCs of healthy controls proliferated after incubation with two of the seven peptides (H167 and H290). Furthermore H161, H290, and H443 induced CD69 expression or production of cytokines IFNγ or TNFα in healthy controls. The identification of these naturally presented epitopes and the response they elicit in the normal immune system make them potential candidates to study during inflammatory conditions as well as in autoimmune diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-013-0484-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4041940  PMID: 24425585
Heat shock protein 70; HSP70; Naturally processed T cell epitopes; Human HSP70 peptides; Autoreactive T cells
4.  Increased Presence of FOXP3+ Regulatory T Cells in Inflamed Muscle of Patients with Active Juvenile Dermatomyositis Compared to Peripheral Blood 
PLoS ONE  2014;9(8):e105353.
Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4+CD25+FOXP3+ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne’s Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne’s muscular dystrophy. Both in JDM and Duchenne’s muscular dystrophy the proportion of FOXP3+ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.
PMCID: PMC4144849  PMID: 25157414
5.  Stabilization of the Transcription Factor Foxp3 by the Deubiquitinase USP7 Increases Treg-Cell-Suppressive Capacity 
Immunity  2013;39(2):259-271.
Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.
PMCID: PMC4133784  PMID: 23973222
6.  Autologous stem cell transplantation restores immune tolerance in experimental arthritis by renewal and modulation of the T effector compartment 
Autologous stem cell transplantation (aSCT) induces long-term drug free disease remission in patient with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunological mechanism underlying aSCT by using a mouse model of proteoglycan (PG)- induced arthritis (PGIA).
PGIA was induced in BALB/c mice by two intraperitoneal injections of human proteoglycan in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, mice received 7.5 Gy total body irradiation and (un)manipulated bone marrow grafts of PGIA mice. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production and intracellular cytokine expression were determined following autologous bone marrow transplantation.
Autologous bone marrow transplantation (aBMT) induced amelioration and stabilisation of arthritis scores. Bone marrow containing T cells gave the same clinical benefit as T cell depleted grafts, with similar reduction in PG-induced T cell proliferation and PG-specific autoantibodies. In vivo re-exposure to PG did not result in disease exacerbation. Following aBMT, basal levels of disease associated pro-inflammatory cytokines (IFNγ, IL-17 and TNFα) were reduced. In addition, T cell re-stimulation with the disease antigen showed a strong reduction in disease-associated pro-inflammatory cytokine production. Finally, while remaining host T cells displayed a pro-inflammatory phenotype following aBMT, IFNγ, IL-17 and TNFα cytokine production by the newly reconstituted donor derived T cells was significantly lower.
Together our data suggest that aBMT restores immune tolerance by renewal and modulation of the T effector compartment, leading to a strong reduction in pro-inflammatory (self antigen-specific) T cell cytokine production.
PMCID: PMC4131556  PMID: 24504807
7.  Hydroxychloroquine preferentially induces apoptosis of CD45RO+ effector T cells by inhibiting autophagy: A possible mechanism for therapeutic modulation of T cells 
Although hydroxychloroquine is used for treatment of numerous autoimmune disorders the mechanism is unclear. We here demonstrate that hydroxychloroquine preferentially induces apoptosis of CD45RO+ memory and effector T cells by inhibiting the survival pathway of autophagy.
PMCID: PMC3955204  PMID: 23541328
Hydroxychloroquine; autoimmunity; autophagy; apoptosis; T cells
8.  Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut 
One of the IL-17 family members, IL-25, has been implicated with the initiation and amplification of Th2 responses in animal models and has been associated with airway hyper-reactivity. The involvement of IL-25 and also IL-17 in food allergic disease remains to be investigated.
In this study thirty children suspected of peanut allergic disease underwent a double-blind placebo controlled food challenge (DBPCFC) and IL-25 and IL-17 plasma levels were determined before and after challenge. IL-25 was highly elevated only in subgroup of children with a positive DBPCFC outcome. Plasma IL-25 was absent in children with a negative DBPCFC outcome and in healthy controls.
This study shows that IL-25, an IL-17 family member, is highly elevated only in children with a clinical response to peanut. This suggests a role for IL-25 in the pathogenesis of peanut allergy and elevated plasma IL-25 may be a sign of a severe atopic phenotype.
PMCID: PMC4176502  PMID: 24295226
DBPCFC; IL-17 family; IL-25; Peanut allergy
9.  Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis 
Cell Stress & Chaperones  2012;18(1):87-95.
Heat shock protein 60 (hsp60) is a highly conserved stress protein and target of self-reactive T cells in various inflammatory diseases. Not much is known about a possible role in atopic disease. As atopic diseases are considered to be the result of a disturbance in the balance between T helper cells type 2 and regulatory T cells, it is of interest to know whether hsp60 acts as a bystander antigen in atopic disease. Our aim was to investigate whether hsp60 is involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expression of hsp60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood mononuclear cells (PBMC) of children with AD were cultured with hsp60 and proliferative responses, cytokine secretion, surface markers, and functional assays were compared to responses of PBMC of healthy controls (HC). Hsp60 was detected more in lesional skin of AD patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated more strongly in response to hsp60 compared to HC. hsp60-reactive T cells of atopic children produced high levels of IFNγ and low levels of IL-10. In vitro activation with hsp60 leads to the induction of CD4+CD25bright T cells expressing FOXP3 in both HC as well as in atopic children. However, despite their regulatory phenotype, hsp60-induced CD4+CD25brightCD127−FOXP3+ T cells of AD patients were incapable of suppressing effector T cells in vitro. hsp60 is recognized by proinflammatory (IFNγ high, IL-10 low) T cells in atopic patients and is more present in lesional AD skin. This suggests that hsp60-specific T cell responses contribute to local inflammation in AD.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-012-0361-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3508125  PMID: 22869467
Atopy; Atopic dermatitis; Regulatory T cells; Human heat shock protein 60
10.  Cerebral ischemia initiates an immediate innate immune response in neonates during cardiac surgery 
A robust inflammatory response occurs in the hours and days following cerebral ischemia. However, little is known about the immediate innate immune response in the first minutes after an ischemic insult in humans. We utilized the use of circulatory arrest during cardiac surgery to assess this.
Twelve neonates diagnosed with an aortic arch obstruction underwent cardiac surgery with cardiopulmonary bypass and approximately 30 minutes of deep hypothermic circulatory arrest (DHCA, representing cerebral ischemia). Blood samples were drawn from the vena cava superior immediately after DHCA and at various other time points from preoperatively to 24 hours after surgery. The innate immune response was assessed by neutrophil and monocyte count and phenotype using FACS, and concentrations of cytokines IL-1β, IL-6, IL-8, IL-10, TNFα, sVCAM-1 and MCP-1 were assessed using multiplex immunoassay. Results were compared to a simultaneously drawn sample from the arterial cannula. Twelve other neonates were randomly allocated to undergo the same procedure but with continuous antegrade cerebral perfusion (ACP).
Immediately after cerebral ischemia (DHCA), neutrophil and monocyte counts were higher in venous blood than arterial (P = 0.03 and P = 0.02 respectively). The phenotypes of these cells showed an activated state (both P <0.01). Most striking was the increase in the ‘non-classical’ monocyte subpopulations (CD16intermediate; arterial 6.6% vs. venous 14%; CD16+ 13% vs. 22%, both P <0.01). Also, higher IL-6 and lower sVCAM-1 concentrations were found in venous blood (both P = 0.03). In contrast, in the ACP group, all inflammatory parameters remained stable.
In neonates, approximately 30 minutes of cerebral ischemia during deep hypothermia elicits an immediate innate immune response, especially of the monocyte compartment. This phenomenon may hold important clues for the understanding of the inflammatory response to stroke and its potentially detrimental consequences.
Trial registration NCT01032876
PMCID: PMC3599234  PMID: 23390999
Cardiac surgery; Cerebral blood flow; Hypothermia; Inflammation; Neonatal ischemia; Randomized controlled trials
12.  STAT3 Regulates Monocyte TNF-Alpha Production in Systemic Inflammation Caused by Cardiac Surgery with Cardiopulmonary Bypass 
PLoS ONE  2012;7(4):e35070.
Cardiopulmonary bypass (CPB) surgery initiates a controlled systemic inflammatory response characterized by a cytokine storm, monocytosis and transient monocyte activation. However, the responsiveness of monocytes to Toll-like receptor (TLR)-mediated activation decreases throughout the postoperative course. The purpose of this study was to identify the major signaling pathway involved in plasma-mediated inhibition of LPS-induced tumor necrosis factor (TNF)-α production by monocytes.
Methodology/Principal Findings
Pediatric patients that underwent CPB-assisted surgical correction of simple congenital heart defects were enrolled (n = 38). Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated at consecutive time points. Patient plasma samples were added back to monocytes obtained pre-operatively for ex vivo LPS stimulations and TNF-α and IL-6 production was measured by flow cytometry. LPS-induced p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation by patient plasma was assessed by Western blotting. A cell-permeable peptide inhibitor was used to block STAT3 signaling. We found that plasma samples obtained 4 h after surgery, regardless of pre-operative dexamethasone treatment, potently inhibited LPS-induced TNF-α but not IL-6 synthesis by monocytes. This was not associated with attenuation of p38 MAPK activation or IκB-α degradation. However, abrogation of the IL-10/STAT3 pathway restored LPS-induced TNF-α production in the presence of suppressive patient plasma.
Our findings suggest that STAT3 signaling plays a crucial role in the downregulation of TNF-α synthesis by human monocytes in the course of systemic inflammation in vivo. Thus, STAT3 might be a potential molecular target for pharmacological intervention in clinical syndromes characterized by systemic inflammation.
PMCID: PMC3323636  PMID: 22506067
16.  Cord Blood CD4+ T Cells Respond to Self Heat Shock Protein 60 (HSP60) 
PLoS ONE  2011;6(9):e24119.
To prevent harmful autoimmunity most immune responses to self proteins are controlled by central and peripheral tolerance. T cells specific for a limited set of self-proteins such as human heat shock protein 60 (HSP60) may contribute to peripheral tolerance. It is not known whether HSP60-specific T cells are present at birth and thus may play a role in neonatal tolerance. We studied whether self-HSP60 reactive T cells are present in cord blood, and if so, what phenotype these cells have.
Methodology/Principal Findings
Cord blood mononuclear cells (CBMC) of healthy, full term neonates (n = 21), were cultured with HSP60 and Tetanus Toxoid (TT) to study antigen specific proliferation, cytokine secretion and up-regulation of surface markers. The functional capacity of HSP60-induced T cells was determined with in vitro suppression assays. Stimulation of CBMC with HSP60 led to CD4+ T cell proliferation and the production of various cytokines, most notably IL-10, Interferon-gamma, and IL-6. HSP60-induced T cells expressed FOXP3 and suppressed effector T cell responses in vitro.
Self-reactive HSP60 specific T cells are already present at birth. Upon stimulation with self-HSP60 these cells proliferate, produce cytokines and express FOXP3. These cells function as suppressor cells in vitro and thus they may be involved in the regulation of neonatal immune responses.
PMCID: PMC3172234  PMID: 21931651
17.  Treating arthritis by immunomodulation: is there a role for regulatory T cells? 
Rheumatology (Oxford, England)  2010;49(9):1632-1644.
The discovery of regulatory T cells almost 15 years ago initiated a new and exciting research area. The growing evidence for a critical role of these cells in controlling autoimmune responses has raised expectations for therapeutic application of regulatory T cells in patients with autoimmune arthritis. Here, we review recent studies investigating the presence, phenotype and function of these cells in patients with RA and juvenile idiopathic arthritis (JIA) and consider their therapeutic potential. Both direct and indirect methods to target these cells will be discussed. Arguably, a therapeutic approach that combines multiple regulatory T-cell-enhancing strategies could be most successful for clinical application.
PMCID: PMC3203406  PMID: 20463189
Rheumatoid arthritis; Juvenile idiopathic arthritis; Regulatory T cells; Treatment; Pro-inflammatory cytokines; Effector T cells; Combination therapy
18.  Heat shock protein 60 reactive T cells in juvenile idiopathic arthritis: what is new? 
Juvenile idiopathic arthritis (JIA) is a disease characterized by chronic joint inflammation, caused by a deregulated immune response. In patients with JIA, heat shock proteins (HSPs) are highly expressed in the synovial lining tissues of inflamed joints. HSPs are endogenous proteins that are expressed upon cellular stress and are able to modulate immune responses. In this review, we concentrate on the role of HSPs, especially HSP60, in modulating immune responses in both experimental and human arthritis, with a focus on JIA. We will mainly discuss the tolerogenic immune responses induced by HSPs, which could have a beneficial effect in JIA. Overall, we will discuss the immune modulatory capacity of HSPs, and the underlying mechanisms of HSP60-mediated immune regulation in JIA, and how this can be translated into therapy.
PMCID: PMC2714101  PMID: 19519922
19.  Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays 
BMC Immunology  2009;10:52.
Growing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.
Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles.
Since under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80°C. After 4 years several cytokines (IL-1α, IL-1β, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays.
All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.
PMCID: PMC2761376  PMID: 19785746
20.  Human Regulatory T Cell Suppressive Function Is Independent of Apoptosis Induction in Activated Effector T Cells 
PLoS ONE  2009;4(9):e7183.
CD4+CD25+FOXP3+ Regulatory T cells (Treg) play a central role in the immune balance to prevent autoimmune disease. One outstanding question is how Tregs suppress effector immune responses in human. Experiments in mice demonstrated that Treg restrict effector T cell (Teff) responses by deprivation of the growth factor IL-2 through Treg consumption, resulting in apoptosis of Teff.
Principal Findings
In this study we investigated the relevance of Teff apoptosis induction to human Treg function. To this end, we studied naturally occurring Treg (nTreg) from peripheral blood of healthy donors, and, to investigate Treg function in inflammation in vivo, Treg from synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients (SF-Treg). Both nTreg and SF-Treg suppress Teff proliferation and cytokine production efficiently as predicted. However, in contrast with murine Treg, neither nTreg nor SF-Treg induce apoptosis in Teff. Furthermore, exogenously supplied IL-2 and IL-7 reverse suppression, but do not influence apoptosis of Teff.
Our functional data here support that Treg are excellent clinical targets to counteract autoimmune diseases. For optimal functional outcome in human clinical trials, future work should focus on the ability of Treg to suppress proliferation and cytokine production of Teff, rather than induction of Teff apoptosis.
PMCID: PMC2746309  PMID: 19779623
21.  Hsp70 and cardiac surgery: molecular chaperone and inflammatory regulator with compartmentalized effects 
Cell Stress & Chaperones  2008;14(2):117-131.
Open heart surgery is a unique model to study the interplay between cellular injury, regulation of inflammatory responses and tissue repair. Stress-inducible heat shock protein 70-kDa (Hsp70) provides a molecular link between these events. In addition to molecular chaperoning, Hsp70 exerts modulatory effects on endothelial cells and leukocytes involved in inflammatory networks. Hsp70 residing in the intracellular compartment is part of an inhibitory feedback loop that acts on nuclear factor kappaB (NF-κB). In contrast, extracellular Hsp70 is recognized by multiple germline-encoded immune receptors, e.g., Toll-like receptor (TLR) 2, TLR4, LOX-1, CD91, CD94, CCR5 and CD40. Hsp70 is thereby able to enhance chemotaxis, phagocytosis and cytolytic activity of innate immune cells and stimulate antigen-specific responses. These apparent contradictory pro- and anti-inflammatory effects of endogenous Hsp70 in the context of cardiac surgery are still not fully understood. An all-embracing model of the compartmentalized effects of endogenous Hsp70 in the orchestration of inflammatory responses in cardiac surgery is proposed.
PMCID: PMC2727984  PMID: 18668350
Cardiac surgery; Heat-shock protein 70-kDa; Inflammation; Innate immunity; Nuclear Factor kappa B; Toll-like receptors
22.  Modulation of T Cell Function by Combination of Epitope Specific and Low Dose Anticytokine Therapy Controls Autoimmune Arthritis 
PLoS ONE  2006;1(1):e87.
Innate and adaptive immunity contribute to the pathogenesis of autoimmune arthritis by generating and maintaining inflammation, which leads to tissue damage. Current biological therapies target innate immunity, eminently by interfering with single pro-inflammatory cytokine pathways. This approach has shown excellent efficacy in a good proportion of patients with Rheumatoid Arthritis (RA), but is limited by cost and side effects. Adaptive immunity, particularly T cells with a regulatory function, plays a fundamental role in controlling inflammation in physiologic conditions. A growing body of evidence suggests that modulation of T cell function is impaired in autoimmunity. Restoration of such function could be of significant therapeutic value. We have recently demonstrated that epitope-specific therapy can restore modulation of T cell function in RA patients. Here, we tested the hypothesis that a combination of anti-cytokine and epitope-specific immunotherapy may facilitate the control of autoimmune inflammation by generating active T cell regulation. This novel combination of mucosal tolerization to a pathogenic T cell epitope and single low dose anti-TNFα was as therapeutically effective as full dose anti-TNFα treatment. Analysis of the underlying immunological mechanisms showed induction of T cell immune deviation.
PMCID: PMC1762388  PMID: 17183718
23.  Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health 
Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index ≥2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-γ (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.
PMCID: PMC1794523  PMID: 17129378
24.  Expression of the inflammatory chemokines CCL5, CCL3 and CXCL10 in juvenile idiopathic arthritis, and demonstration of CCL5 production by an atypical subset of CD8+ T cells 
This study focuses upon three chemokines, namely CCL5, CXCL10 and CCL3, which are potential novel therapeutic targets in arthritis. The aim of the study was to analyse the expression and production of these three chemokines within the joints of children with juvenile idiopathic arthritis (JIA) of the oligoarticular and polyarticular subtypes. All three of these chemokines are highly expressed at the level of mRNA, with the most significant increase in mRNA levels being demonstrated for CCL5 when compared with matched peripheral blood samples and controls. We show that high levels of all three chemokines are present in synovial fluid of children with JIA. We investigate the major source of CCL5 from inflammatory synovial cells, which we show to be CD8+ T cells. This CD8+ synovial T cell population has an unexpected phenotype that has not been described previously, being CCR7- yet predominantly CD28+ and CD45RA-. These cells contain high levels of stored intracellular CCL5, and rapid release of CCL5 takes place on T cell stimulation, without requiring new protein synthesis. In addition, we demonstrate that CCL5 is present in synovial biopsies from these patients, in particular on the endothelium of small and medium sized vessels. We believe this to be the first in depth analysis of these mediators of inflammation in JIA.
PMCID: PMC1526593  PMID: 16507178
25.  Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells 
Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.
PMCID: PMC145264  PMID: 12522051

Results 1-25 (25)