An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.

The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

Use of MRP8/14 in clinical practice as a predictor of outcome after methotrexate withdrawal in patients with juvenile idiopathic arthritis.

The objective of this study was to determine the effectiveness of MRP8/14 as a predictor of disease flare in patients with juvenile idiopathic arthritis (JIA) following the withdrawal of methotrexate (MTX) in a routine clinical setting. All MRP8/14 tests performed at a single centre in a 27-month period were considered for analysis. Patients were assessed against criteria for inactive disease and subsequent disease flare. Decisions on whether or not to stop treatment were recorded. MRP8/14 results were assessed in conjunction with clinical information. Clinicians were also surveyed to investigate if MRP8/14 influenced their decision to discontinue MTX where this was available at that time point. One hundred four cases met the inclusion criteria during the study period. Although there was no significant difference in flares between patients with an elevated or low MRP8/14 value, in those who stopped MTX (n = 22), no patients with a low MRP8/14 (≤ 4000 ng/ml) result flared (follow-up time 12 months). Clinicians reported that for patients with clinically inactive disease and an elevated MRP8/14 result (> 4000 ng/ml), none would advise withdrawal of MTX. Low MRP8/14 was interpreted favourably when considering stopping MTX treatment in patients with JIA. Implementation of MRP8/14 testing has changed clinical practice at this centre. However, the observation that some patients in our cohort who had an elevated MRP8/14 value did not flare after stopping MTX for non-disease-related reasons highlights the need for further biomarkers to predict the risk of flare off medication in JIA and aid clinicians in treatment decisions. Key Points • First study of serum MRP8/14 measurement in clinical practice to inform treatment decisions in patients with JIA. • No patients with a low MRP8/14 test result went on to suffer a disease flare in 12 months of follow follow-up. • Further biomarkers are needed to predict the risk of flare off medication in JIA and treatment decisions.

Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations.

DNA damage occurs constantly in every cell triggered by endogenous processes of replication and metabolism, and external influences such as ionizing radiation and intercalating chemicals. Large sets of proteins are involved in sensing, stabilizing and repairing this damage including control of cell cycle and proliferation. Some of these factors are phosphorylated upon activation and can be used as biomarkers of DNA damage response (DDR) by flow and mass cytometry. Differential survival rates of lymphocyte subsets in response to DNA damage are well established, characterizing NK cells as most resistant and B cells as most sensitive to DNA damage. We investigated DDR to low dose gamma radiation (2Gy) in peripheral blood lymphocytes of 26 healthy donors and 3 patients with ataxia telangiectasia (AT) using mass cytometry. γH2AX, p-CHK2, p-ATM and p53 were analyzed as specific DDR biomarkers for functional readouts of DNA repair efficiency in combination with cell cycle and T, B and NK cell populations characterized by 20 surface markers. We identified significant differences in DDR among lymphocyte populations in healthy individuals. Whereas CD56+CD16+ NK cells showed a strong γH2AX response to low dose ionizing radiation, a reduced response rate could be observed in CD19+CD20+ B cells that was associated with reduced survival. Interestingly, γH2AX induction level correlated inversely with ATM-dependent p-CHK2 and p53 responses. Differential DDR could be further noticed in naïve compared to memory T and B cell subsets, characterized by reduced γH2AX, but increased p53 induction in naïve T cells. In contrast, DDR was abrogated in all lymphocyte populations of AT patients. Our results demonstrate differential DDR capacities in lymphocyte subsets that depend on maturation and correlate inversely with DNA damage-related survival. Importantly, DDR analysis of peripheral blood cells for diagnostic purposes should be stratified to lymphocyte subsets.

SARS-CoV-2 infection is associated with a pro-thrombotic platelet phenotype.

Novel coronavirus disease 2019 (COVID-19) is associated with a hypercoagulable state, characterized by abnormal coagulation parameters and by increased incidence of cardiovascular complications. With this study, we aimed to investigate the activation state and the expression of transmembrane proteins in platelets of hospitalized COVID-19 patients. We investigated transmembrane proteins expression with a customized mass cytometry panel of 21 antibodies. Platelets of 8 hospitalized COVID-19 patients not requiring intensive care support and without pre-existing conditions were compared to platelets of healthy controls (11 donors) with and without in vitro stimulation with thrombin receptor-activating peptide (TRAP). Mass cytometry of non-stimulated platelets detected an increased surface expression of activation markers P-Selectin (0.67 vs. 1.87 median signal intensity for controls vs. patients, p = 0.0015) and LAMP-3 (CD63, 0.37 vs. 0.81, p = 0.0004), the GPIIb/IIIa complex (4.58 vs. 5.03, p < 0.0001) and other adhesion molecules involved in platelet activation and platelet-leukocyte interactions. Upon TRAP stimulation, mass cytometry detected a higher expression of P-selectin in COVID-19 samples compared to controls (p < 0.0001). However, we observed a significantly reduced capacity of COVID-19 platelets to increase the expression of activation markers LAMP-3 and P-Selectin upon stimulation with TRAP. We detected a hyperactivated phenotype in platelets during SARS-CoV-2 infection, consisting of highly expressed platelet activation markers, which might contribute to the hypercoagulopathy observed in COVID-19. In addition, several transmembrane proteins were more highly expressed compared to healthy controls. These findings support research projects investigating antithrombotic and antiplatelet treatment regimes in COVID-19 patients, and provide new insights on the phenotypical platelet expression during SARS-CoV-2 infection.

S100A12 Is Associated with Response to Therapy in Juvenile Idiopathic Arthritis.

OBJECTIVE: Around one-third of patients with juvenile idiopathic arthritis (JIA) fail to respond to first-line methotrexate (MTX) or anti-tumor necrosis factor (TNF) therapy, with even fewer achieving ≥ American College of Rheumatology Pediatric 70% criteria for response (ACRpedi70), though individual responses cannot yet be accurately predicted. Because change in serum S100-protein myeloid-related protein complex 8/14 (MRP8/14) is associated with therapeutic response, we tested granulocyte-specific S100-protein S100A12 as a potential biomarker for treatment response. METHODS: S100A12 serum concentration was determined by ELISA in patients treated with MTX (n = 75) and anti-TNF (n = 88) at baseline and followup. Treatment response (≥ ACRpedi50 score), achievement of inactive disease, and improvement in Juvenile Arthritis Disease Activity Score (JADAS)-10 score were recorded. RESULTS: Baseline S100A12 concentration was measured in patients treated with anti-TNF [etanercept n = 81, adalimumab n = 7; median 200, interquartile range (IQR) 133-440 ng/ml] and MTX (median 220, IQR 100-440 ng/ml). Of the patients in the anti-TNF therapy group, 74 (84%) were also receiving MTX. Responders to MTX (n = 57/75) and anti-TNF (n = 66/88) therapy had higher baseline S100A12 concentration compared to nonresponders: median 240 (IQR 125-615) ng/ml versus 150 (IQR 87-233) ng/ml, p = 0.021 for MTX, and median 308 (IQR 150-624) ng/ml versus 151 (IQR 83-201) ng/ml, p = 0.002, for anti-TNF therapy. Followup S100A12 could be measured in 44/75 MTX-treated patients (34/44 responders) and 39/88 anti-TNF-treated patients (26/39 responders). Responders had significantly reduced S100A12 concentration (MTX: p = 0.031, anti-TNF: p < 0.001) at followup versus baseline. Baseline serum S100A12 in both univariate and multivariate regression models for anti-TNF therapy and univariate analysis alone for MTX therapy was significantly associated with change in JADAS-10. CONCLUSION: Responders to MTX or anti-TNF treatment can be identified by higher pretreatment S100A12 serum concentration levels.

Elastography in the diagnosis of breast lesions: comparison of different elastographic features.

Background Breast elastography is a non-invasive and widely accessible method used in the differential diagnostic procedure in addition to B-mode imaging. Purpose To assess the role of elastographic features in the differentiation of breast lesions and to evaluate the importance of depth in the choice of reference fat tissue for the calculation of strain ratio. Material and Methods From January to August 2015, 242 breast lesions were evaluated using elastographic assessment based on qualitative and semi-quantitative parameters (color map, strain ratio, length ratio) as well as elastographic analysis. Histological findings were considered as gold-standard. Results Sensitivity and specificity of B-mode ultrasound imaging were 90% and 60%, respectively. Color map sensitivity was 98% and specificity 46%; strain ratio sensitivity 81% and specificity 70%; length ratio sensitivity 64% and specificity 76%. Combined analysis of these three elastographic features improved the overall diagnostic performance of any of the three parameters alone, yielded sensitivity similar to that of color map (95%) and specificity comparable to strain ratio and length ratio (70%). There was no significant difference in strain ratio obtained from the reference fat tissue at the same depth as the lesion and at a different depth (sensitivity 77% versus 84%; specificity 70% versus 68%; P < 0.001). Conclusion In our experience, elastography can improve ultrasound characterization of the lesion, particularly if elastographic analysis is performed.

[Cardiovascular diseases and cancer incidence in general population: results from MATISS cohort]. / Incidenza di malattie cardiovascolari maggiori e tumori nella coorte longitudinale del Progetto MATISS in persone con differente profilo di rischio cardiovascolare.

OBJECTIVES: to assess the incidence of fatal and non-fatal major cardiovascular diseases and cancers in people with different cardiovascular risk profile of a longitudinal cohort. DESIGN: longitudinal study carried out in a large sample of general population, with a median follow-up of 17.7 years. SETTING AND PARTICIPANTS: MATISS longitudinal cohort, 7,491 men and women aged 20-75 years, free of cancer or cardiovascular diseases at baseline. Two cardiovascular risk groups (low-intermediate cardiovascular risk and high cardiovascular risk) and two educational levels (low level: primary school; middle/high level: middle/high school, university) have been considered. MAIN OUTCOME MEASURES: for both cancer and cardiovascular events the standardized incidence rates have been calculated, taking into account the cardiovascular risk profile and the educational level. RESULTS: people with a high-risk profile have higher incidence of major cardiovascular diseases and cancers. Furthermore, incidence rates are higher in people with lower educational level, except for cardiovascular diseases in men. CONCLUSIONS: this study shows that, in the considered population, cancer incidence rates are higher than cardiovascular diseases rates; moreover, cancer incidence is higher in people with high-risk cardiovascular profile. The increase of prevalence of favourable risk profile in the general population could represent effective community strategies for prevention of cardiovascular diseases and cancer. Major attention should be dedicated towards people at lower socioeconomical level.

MRP8/14 serum levels as a predictor of response to starting and stopping anti-TNF treatment in juvenile idiopathic arthritis.

INTRODUCTION: Approximately 30% of juvenile idiopathic arthritis (JIA) patients fail to respond to anti-TNF treatment. When clinical remission is induced, some patients relapse after treatment has been stopped. We tested the predictive value of MRP8/14 serum levels to identify responders to treatment and relapse after discontinuation of therapy. METHODS: Samples from 88 non-systemic JIA patients who started and 26 patients who discontinued TNF-blockers were analyzed. MRP8/14 serum levels were measured by in-house MRP8/14 ELISA and by Bühlmann Calprotectin ELISA at start of anti-TNF treatment, within 6 months after start and at discontinuation of etanercept in clinical remission. Patients were categorized into responders (ACRpedi ≥ 50 and/or inactive disease) and non-responders (ACRpedi < 50) within six months after start, response was evaluated by change in JADAS-10. Disease activity was assessed within six months after discontinuation. RESULTS: Baseline MRP8/14 levels were higher in responders (median MRP8/14 of 1466 ng/ml (IQR 1045-3170)) compared to non-responders (median MRP8/14 of 812 (IQR 570-1178), p < 0.001). Levels decreased after start of treatment only in responders (p < 0.001). Change in JADAS-10 was correlated with baseline MRP8/14 levels (Spearman's rho 0.361, p = 0.001). Patients who flared within 6 months after treatment discontinuation had higher MRP8/14 levels (p = 0.031, median 1025 ng/ml (IQR 588-1288)) compared to patients with stable remission (505 ng/ml (IQR 346-778)). Results were confirmed by Bühlmann ELISA with high reproducibility but different overall levels. CONCLUSION: High levels of baseline MRP8/14 are associated with good response to anti-TNF treatment, whereas elevated MRP8/14 levels at discontinuation of etanercept are associated with higher chance to flare.

Correlation of low CD73 expression on synovial lymphocytes with reduced adenosine generation and higher disease severity in juvenile idiopathic arthritis.

OBJECTIVE: To investigate the expression and adenosine-generating activity of the ecto-5'-nucleotidase CD73 on synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from children with juvenile idiopathic arthritis (JIA). METHODS: Given the role of CD73 protein in the production of antiinflammatory adenosine and its intersection with inflammatory biologic pathways, the expression of CD73 on SF and PB lymphocytes from patients with JIA and PB lymphocytes from healthy control subjects was determined by flow cytometry. The AMPase activity of CD73 on PBMCs and SFMCs was measured by high-performance liquid chromatography. The effects of cell activation on CD73 expression were examined by in vitro culture of PBMCs. RESULTS: CD8+ and CD19+ SFMCs from patients with JIA expressed decreased levels of CD73 when compared to paired PBMCs from JIA patients and PBMCs from healthy controls. When the percentages of CD73+ synovial lymphocytes were compared between the 2 clinical forms of oligoarthritis, children with extended oligoarthritis showed lower CD73 expression compared to those with the milder form of the disease. CD8+ SFMCs had a lower ability to produce adenosine from etheno-AMP compared to CD8+ PBMCs. T cell activation through the T cell receptor (TLR) of CD8+CD73+ cells and B cell activation through TLR-9 resulted in reduced expression of CD73. This down-regulation occurred on dividing cells. CONCLUSION: These findings show that low CD73 expression on T and B cells in the inflamed site is related to cell proliferation and is correlated with the clinical severity of oligoarticular JIA. The decreased CD73 expression on SFMCs, in turn, results in reduced adenosine production, which leads to a decreased potential for antiinflammatory activity.

Hypomethylation at the regulatory T cell-specific demethylated region in CD25hi T cells is decoupled from FOXP3 expression at the inflamed site in childhood arthritis.

The maintenance of FOXP3 expression in CD25(hi) regulatory T cells (Tregs) is crucial to the control of inflammation and essential for successful Treg transfer therapies. Coexpression of CD25 and FOXP3 in combination with a hypomethylated region within the FOXP3 gene, called the Treg-specific demethylated region (TSDR), is considered the hallmark of stable Tregs. The TSDR is an epigenetic motif that is important for stable FOXP3 expression and is used as a biomarker to measure Treg lineage commitment. In this study, we report that, unlike in peripheral blood, CD4(+) T cell expression of CD25 and FOXP3 is frequently dissociated at the inflamed site in patients with juvenile idiopathic arthritis, which led us to question the stability of human Tregs in chronic inflammatory environments. We describe a novel CD4(+)CD127(lo)CD25(hi) human T cell population that exhibits extensive TSDR and promoter demethylation in the absence of stable FOXP3 expression. This population expresses high levels of CTLA-4 and can suppress T conventional cell proliferation in vitro. These data collectively suggest that this population may represent a chronically activated FOXP3(lo) Treg population. We show that these cells have defects in IL-2 signaling and reduced expression of a deubiquitinase important for FOXP3 stability. Clinically, the proportions of these cells within the CD25(hi) T cell subset are increased in patients with the more severe courses of disease. Our study demonstrates, therefore, that hypomethylation at the TSDR can be decoupled from FOXP3 expression in human T cells and that environment-specific breakdown in FOXP3 stability may compromise the resolution of inflammation in juvenile idiopathic arthritis.

A subgroup of juvenile idiopathic arthritis patients who respond well to methotrexate are identified by the serum biomarker MRP8/14 protein.

OBJECTIVES: In JIA there is an unmet need for biomarkers with which to identify patients who will respond well to MTX. The aim of this study was to define the prognostic value of baseline serum proteins and clinical variables in response to MTX to help inform the clinician at time of diagnosis whether the patient is likely to respond well to MTX. METHODS: JIA patients were recruited into the Childhood Arthritis Response to Medication Study (CHARMS). Clinical data and venous blood were collected before administration of MTX and at follow-up. MRP8/14 and inflammatory cytokines were measured by ELISA and multiplex immunoassay, respectively. CRP and ESR were measured as part of routine clinical assessment. To explore which baseline factors might predict successful treatment, binary logistic regression models were fitted for outcome. RESULTS: High disease activity (high serum MRP8/14, active joint count or physician's score) pre-MTX was observed in a subgroup of patients with a better response to therapy. In a multivariable analysis, after accounting for MRP8/14 at baseline, no other factors were independently significantly associated with outcome. Patients with baseline MRP8/14 >3000 ng/ml were more likely to respond to MTX at ACR50 or better: odds ratio 16.07 (95% CI 2.00, 129.3). CONCLUSION: We have demonstrated that high levels of baseline serum MRP8/14 have prognostic value in predicting a subgroup of patients whose arthritis will improve on MTX. Routine collection of serum prior to the start of medication would be a valuable step in collaborative validation of such biomarkers.

CD161 defines the subset of FoxP3+ T cells capable of producing proinflammatory cytokines.

Regulatory FoxP3+CD4+ T cells (Treg) are vital for maintaining the balance between tolerance, adequate immune response, and autoimmunity. Despite this immunoregulatory role, it has been shown that Treg may also produce proinflammatory cytokines. Here we present a distinct population of Treg, defined by CD161 expression, as the major source of FoxP3+ Treg-derived proinflammatory cytokines. CD161+ Treg can be followed throughout development, from thymus and cord blood to healthy child and adult samples. CD161+ Treg display anergy, are suppressive in cocultures with conventional T cells (Tconv), and possess a predominantly demethylated Treg-specific demethylated region of the FOXP3 locus. In addition to the production of interleukin (IL) 17A, interferon γ, and IL-2, CD161+FoxP3+ cells share markers with Tconv, including expression of the transcription factors retinoic acid-related orphan receptor Cv2 (RORCv2) and T-cell-specific T-box transcription factor (Tbet). Expression of CD161 and enrichment for cytokine production are stable characteristics of CD161+ Treg upon both short- and longer-term culture in vitro. Additionally, CD161+ Treg are highly enriched within the inflammatory environment of childhood arthritis, suggesting a role in disease. Our data therefore demonstrate that CD161+FoxP3+ T cells are a novel Treg subset, found in health and disease, which display high proinflammatory potential but also exhibit hallmark Treg characteristics.

Association of the IL-10 gene family locus on chromosome 1 with juvenile idiopathic arthritis (JIA).

BACKGROUND: The cytokine IL-10 and its family members have been implicated in autoimmune diseases and we have previously reported that genetic variants in IL-10 were associated with a rare group of diseases called juvenile idiopathic arthritis (JIA). The aim of this study was to fine map genetic variants within the IL-10 cytokine family cluster on chromosome 1 using linkage disequilibrium (LD)-tagging single nucleotide polymorphisms (tSNPs) approach with imputation and conditional analysis to test for disease associations. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-three tSNPs were tested for association between Caucasian paediatric cohorts [219 systemic JIA (sJIA), 187 persistent oligoarticular JIA (pOJIA), and 139 extended OJIA (eOJIA) patients], and controls (Wellcome Trust control cohort, WTCCC2). Significant association with sJIA was detected at rs1400986 in the promoter of IL-20 (odds ratio 1.53; 95% CI 1.21-1.93; p = 0.0004), but in no other subtypes. Imputation analysis identified additional associated SNPs for pOJIA at IL-20 and IL-24, including a rare, functional, missense variant at IL-24 with a p = 0.0002. Penalised logistic regression analysis with HyperLasso and conditional analysis identified several further associations with JIA subtypes. In particular, haplotype analysis refined the sJIA association, with a joint effect at rs1400986 and rs4129024 in intron 1 of MAPKAPK2 (p = 3.2E-5). For pOJIA, a 3-SNP haplotype including rs1878672 in intron 3 of IL-10 showed evidence for association (p = 0.0018). In eOJIA, rs10863962 (3'UTR of FCAMR) and rs12409577 (intron of IL-19) haplotype showed some evidence of association (p = 0.0003). CONCLUSIONS: This study supports previous association of IL-20 with sJIA. Haplotype analyses provided stronger association signals than single point analyses, while a penalised logistic regression approach also suggested multiple independent association signals. Replication studies are required to confirm or refute these findings. The results indicate that combined effects with unknown/rare variants remain to be characterised in JIA, and represent a possible example of synthetic association in this region.

ClC1 chloride channel in myotonic dystrophy type 2 and ClC1 splicing in vitro.

Myotonic dystrophy type 2 (DM2) is caused by CCTG-repeat expansions. Occurrence of splicing and mutations in the muscle chloride channel gene CLCN1 have been reported to contribute to the phenotype. To examine the effect of CLCN1 in DM2 in Germany, we determined the frequency of a representative ClC1 mutation, R894X, and its effect on DM2 clinical features. Then, we examined CLCN1 mRNA splice variants in patient muscle functionally expressed the most abundant variant, and determined its subcellular localization. Finally, we established a cellular system for studying mouse clcn1 pre-mRNA splicing and tested effects of expression of (CCUG)18, (CUG)24 and (AAG)24 RNAs. The R894X mutation was present in 7.7% of DM2 families. DM2 R894X-carriers had more myotonia and myalgia than non-carriers. The most abundant CLCN1 splice variant in DM2 (80% of all transcripts) excluded exons 6-7 and lead to a truncated ClC1(236X) protein. Heterologous ClC1(236X) expression did not yield functional channels. Co-expression with ClC1 did not show a dominant negative effect, but a slightly suppressive effect. In C2C12 cells, the clc1 splice variants generated by (CCUG)18-RNA resembled those in DM2 muscle and differed from those generated by (CUG)24 and (AAG)24. We conclude that ClC1 mutations exert gene dose effects and enhance myotonia and pain in DM2 in Germany. Additionally, the ClC1(236X) splice variant may contribute to myotonia in DM2. Since splice variants depend on the types of repeats expressed in the cellular C2C12 model, similar cell models of other tissues may be useful for studying repeatdependent pathogenetic mechanisms more easily than in transgenic animals.

Th1 and Th17 cell subpopulations are enriched in the peripheral blood of patients with systemic juvenile idiopathic arthritis.

OBJECTIVE: The role of the adaptive immune system has not been explored in detail compared with the innate immune system in systemic JIA (sJIA) pathogenesis. The aim of this study was to examine the phenotype of circulating peripheral blood CD4(+) T-cell subpopulations in a cross-sectional study of sJIA patients during disease remission on medication and during acute flare of the disease. METHODS: Flow cytometry was used to examine the phenotype and cytokine production of IFNγ-, IL-4- and IL-17-producing CD4(+) T cells in the peripheral blood of 10 sJIA patients with active disease, 9 sJIA with inactive disease, 14 JIA patients with oligoarticular onset, 10 adult control subjects and 10 age-matched control subjects. In parallel, we examined the proportion of FoxP3(+) Tregs. RESULTS: IFNγ- and IL-17-producing CD4(+) T cells and IL-17-producing CD3(+)CD4(-) T cells were present at higher proportions in the peripheral blood of sJIA patients, irrespective of their disease status. Our data also confirm the known increase of the proportions of IFNγ-producing Th1 cells with increasing age and suggest an increase with age in the IL-17-producing CD4(+) T-cell population. CONCLUSION: This study is the first to describe significantly higher proportions of Th1 and Th17 T helper cell subsets in the peripheral blood of sJIA patients. These proinflammatory cells may play a pathogenic role in sJIA. Our data also emphasize the importance of using paediatric age-matched control subjects when evaluating the T-cell cytokine profile in JIA.

The Toll-like receptor 4 agonist MRP8/14 protein complex is a sensitive indicator for disease activity and predicts relapses in systemic-onset juvenile idiopathic arthritis.

BACKGROUND: Analysis of myeloid-related protein 8 and 14 complex (MRP8/14) serum concentrations is a potential new tool to support the diagnosis of systemic-onset juvenile idiopathic arthritis (SJIA) in the presence of fever of unknown origin. OBJECTIVE: To test the ability of MRP8/14 serum concentrations to monitor disease activity in patients with SJIA and stratify patients at risk of relapse. METHODS: Serum concentrations of MRP8/14 in 52 patients with SJIA were determined by a sandwich ELISA. The monitoring of therapeutic regimens targeting interleukin 1 and tumour necrosis factor α, and methotrexate treatment was analysed and diagnostic power to predict flares was tested. RESULTS: MRP8/14 levels were clearly raised in active disease and decreased significantly in response to successful treatments. Serum concentrations of MRP8/14 increased significantly (p<0.001) (mean±95% CI 12.030±3.090 ng/ml) during disease flares compared with patients with inactive disease (864±86 ng/ml). During clinical remission MRP8/14 serum levels of >740 ng/ml predicted disease flares accurately (sensitivity 92%, specificity 88%). MRP8/14 levels correlated well with clinical disease activity, as assessed by physician's global assessment of disease activity (r=0.62), Childhood Health Assessment Questionnaire (r=0.56), active joint count (r=0.46) and with C-reactive protein (r=0.71) and erythrocyte sedimentation rate (r=0.72) (for all p<0.001). CONCLUSION: MRP8/14 serum concentrations correlate closely with response to drug treatment and disease activity and therefore might be an additional measurement for monitoring anti-inflammatory treatment of individual patients with SJIA. MRP8/14 serum concentrations are the first predictive biomarker indicating subclinical disease activity and stratifying patients at risk of relapse during times of clinically inactive disease.

Association of the 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene with response to methotrexate in juvenile idiopathic arthritis.

OBJECTIVES: Methotrexate (MTX) is the mainstay treatment for juvenile idiopathic arthritis (JIA), however approximately 30% of children will fail to respond to the drug. Identification of genetic predictors of response to MTX would be invaluable in developing optimal treatment strategies for JIA. Using a candidate gene approach, single nucleotide polymorphisms (SNPs) within genes in the metabolic pathway of MTX, were investigated for association with response to treatment in JIA cases. METHODS: Tagging SNPs were selected across 13 MTX metabolic pathway genes and were genotyped using Sequenom genotyping technology in subjects recruited from the Sparks Childhood Arthritis Response to Medication Study. Response to MTX was defined using the American College of Rheumatology (ACR) paediatric response criteria and SNP genotype frequencies were compared between the worst and best responders (ACR-Ped70) to MTX. An independent cohort of US JIA cases was available for validation of initial findings. RESULTS: One SNP within the inosine triphosphate pyrophosphatase gene (ITPA) and two SNPs within 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene (ATIC) were significantly associated with a poor response to MTX. One of the ATIC SNPs showed a trend towards association with MTX response in an independent cohort of US JIA cases. Meta-analysis of the two studies strengthened this association (combined p value=0.002). CONCLUSIONS: This study presents association of a SNP in the ATIC gene with response to MTX in JIA. There is now growing evidence to support a role of the ATIC gene with response to MTX treatment. These results could contribute towards a better understanding of and ability to predict MTX response in JIA.

Generation of novel pharmacogenomic candidates in response to methotrexate in juvenile idiopathic arthritis: correlation between gene expression and genotype.

OBJECTIVES: Little is known about the mechanisms of efficacy of methotrexate (MTX) in childhood arthritis, or genetic influences upon response to MTX. The aims of this study were to use gene expression profiling to identify novel pathways/genes altered by MTX and then investigate these genes for genotype associations with response to MTX treatment. METHODS: Gene expression profiling before and after MTX treatment was performed on 11 children with juvenile idiopathic arthritis (JIA) treated with MTX, in whom response at 6 months of treatment was defined. Genes showing the most differential gene expression after the treatment were selected for single nucleotide polymorphism (SNP) genotyping. Genotype frequencies were compared between nonresponders and responders (ACR-Ped70). An independent cohort was available for validation. RESULTS: Gene expression profiling before and after MTX treatment revealed 1222 differentially expressed probes sets (fold change >1.7, P<0.05) and 1065 when restricted to full responder cases only. Six highly differentially expressed genes were analyzed for genetic association in response to MTX. Three SNPs in the SLC16A7 gene showed significant association with MTX response. One SNP showed validated association in an independent cohort. CONCLUSION: This study is the first, to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyze genetic variation in differentially expressed genes. We have identified a gene, which may contribute to genetic variability in MTX response in JIA, and established as proof of principle that genes that are differentially expressed at mRNA level after drug administration may also be good candidates for genetic analysis.

Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment.

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17-secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells "convert" to Th17/1 under conditions that mimic the disease site, namely low TGFbeta and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.