MD Alexis Mathian
email: alexis.mathian@aphp.fr.

Suzanne Mouries-Martin

Karim Dorgham

Herve Devilliers

Elyès Laura Barnabei

Ben Salah

Fleur Cohen-Aubart

Laura Garrido Castillo

Julien Haroche

Miguel Hie

Hervé Devilliers

Elyès Ben Salah

Marc Pineton De Chambrun

Makoto Miyara

Delphine Sterlin

Micheline Pha

Du Lê

Thi Huong

Frédéric Rieux-Laucat

Flore Rozenberg

Guy Gorochov

Zahir Amoura

Monitoring disease activity in systemic lupus erythematosus with single-molecule array digital ELISA

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown etiology characterized by the presence of antinuclear autoantibodies and inflammation in a wide spectrum of organs [START_REF] Tsokos | Systemic lupus erythematosus[END_REF][START_REF] Lisnevskaia | Systemic lupus erythematosus[END_REF]. The survival of SLE patients has plateaued since the middle 1990s (3). To date, anti-double-stranded DNA (anti-dsDNA) antibody titration, best achieved with the standard Supported by the AP-HP (Centre d'Investigations Biomédicales Pitié-Salpêtrière), INSERM, Sorbonne Université, the French Arthritis Foundation, the Société National Française de Médecine Interne, and the Fondation pour la Recherche Médicale. Ms Barnebai is a fellow of the ImaginePhD international program supported by the Fondation Bettencourt Schueller. Farr assay, has been used to monitor global disease activity and SLE renal involvement [START_REF] Agmon-Levin | International recommendations for the assessment of autoantibodies to cellular antigens referred to as antinuclear antibodies[END_REF][START_REF] Neogi | Anti-dsDNA antibody testing by Farr and ELISA techniques is not equivalent[END_REF][START_REF] Kavanaugh | Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests[END_REF][START_REF] Sherer | Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients[END_REF]. However, because associations with disease characteristics are at best modest, clinicians and researchers still lack reliable biomarkers of SLE activity [START_REF] Agmon-Levin | International recommendations for the assessment of autoantibodies to cellular antigens referred to as antinuclear antibodies[END_REF][START_REF] Neogi | Anti-dsDNA antibody testing by Farr and ELISA techniques is not equivalent[END_REF][START_REF] Kavanaugh | Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests[END_REF][START_REF] Sherer | Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients[END_REF]. At present, many authors consider the dysregulation of interferons (IFNs) to be a central cause of the immunologic abnormalities observed in SLE [START_REF] Theofilopoulos | Type I interferons (α/β) in immunity and autoimmunity[END_REF][START_REF] Obermoser | The interferon-α signature of systemic lupus erythematosus[END_REF][START_REF] Ronnblom | Cytokines as therapeutic targets in SLE[END_REF][START_REF] Niewold | Interferon α as a primary pathogenic factor in human lupus[END_REF][START_REF] Ronnblom | The type I interferon system in the development of lupus[END_REF][START_REF] Lauwerys | Type I interferon blockade in systemic lupus erythematosus: where do we stand?[END_REF][START_REF] Kirou | Anti-interferon α treatment in SLE[END_REF].

Early studies showed elevated serum IFNα levels in SLE patients to be associated with disease activity and severity [START_REF] Hooks | Immune interferon in the circulation of patients with autoimmune disease[END_REF][START_REF] Preble | Systemic lupus erythematosus: presence in human serum of an unusual acid-labile leukocyte interferon[END_REF][START_REF] Ytterberg | Serum interferon levels in patients with systemic lupus erythematosus[END_REF]. More recently, transcriptome analysis using microarray technology revealed up-regulation of numerous IFNstimulated genes (ISGs) in the peripheral blood mononuclear cells of patients with SLE, constituting an overall "IFN signature" [START_REF] Bennett | Interferon and granulopoiesis signatures in systemic lupus erythematosus blood[END_REF][START_REF] Baechler | Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus[END_REF]. That signature is found in almost all pediatric patients and 50-80% of adults with SLE [START_REF] Chiche | Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures[END_REF]. Thus, the close association between IFNα overexpression and SLE activity suggests that monitoring this cytokine might help physicians better evaluate disease activity [START_REF] Kanayama | Possible involvement of interferon α in the pathogenesis of fever in systemic lupus erythematosus[END_REF][START_REF] Bengtsson | Activation of type I interferon system in systemic lupus erythematosus correlates with disease activity but not with antire troviral antibodies[END_REF][START_REF] Dall'era | Type I interferon correlates with serological and clinical manifestations of SLE[END_REF][START_REF] Bauer | Interferon-regulated chemokines as biomarkers of systemic lupus erythematosus disease activity: a validation study[END_REF][START_REF] Rose | Are interferon-related biomarkers advantageous for monitoring disease activity in systemic lupus erythematosus? A longitudinal benchmark study[END_REF]. Knowing the IFNα concentration might also help select the best candidates for anti-IFNα treatment [START_REF] Khamashta | Sifalimumab, an anti-interferon-α monoclonal antibody, in moderate to severe systemic lupus erythematosus: a randomised, double-blind, placebo-controlled study[END_REF][START_REF] Furie | Anifrolumab, an anti-interferon-α receptor monoclonal antibody, in moderate-to-severe systemic lupus erythematosus[END_REF]. Unfortunately, no reliable, simple, or standardized assays to quantify IFNα in routine clinical practice are available; notably, enzyme-linked immunosorbent assays (ELISAs) to detect IFNα in human sera have been hindered by low sensitivity and low specificity [START_REF] Jabs | Failure to detect antiviral activity in serum and plasma of healthy individuals displaying high activity in ELISA for IFN-α and IFN-β[END_REF], and assays based on detecting IFNα biologic activity are difficult to standardize [START_REF] Hooks | Immune interferon in the circulation of patients with autoimmune disease[END_REF][START_REF] Preble | Systemic lupus erythematosus: presence in human serum of an unusual acid-labile leukocyte interferon[END_REF][START_REF] Ytterberg | Serum interferon levels in patients with systemic lupus erythematosus[END_REF]. Alternatively, quantification of the expression of different ISGs as "IFN scores" can be used as a surrogate to monitor IFN activity in SLE [START_REF] Feng | Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus[END_REF][START_REF] Yao | Development of potential pharmacodynamic and diagnostic markers for anti-IFN-α monoclonal antibody trials in systemic lupus erythematosus[END_REF][START_REF] Yao | Use of type I interferon-inducible mRNAs as pharmacodynamic markers and potential diagnostic markers in trials with sifalimumab, an anti-IFNα antibody, in systemic lupus erythematosus[END_REF]. Not surprisingly, IFN scores have been associated with SLE activity [START_REF] Feng | Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus[END_REF][START_REF] Kirou | Coordinate overexpression of interferon-α-induced genes in systemic lupus erythematosus[END_REF][START_REF] Kirou | Activation of the interferon-α pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease[END_REF][START_REF] Petri | Longitudinal expression of type I interferon responsive genes in systemic lupus erythematosus[END_REF]. However, the low availability and high complexity of transcriptome-microarray technology means that the IFN scores, as well, are not standardized and cannot be used in routine practice.

The new single-molecule array (Simoa) assay, also called digital ELISA, based on counting individual enzyme-labeled immunocomplexes of proteins captured on beads in single-molecule arrays, enables direct IFNα quantification at attomolar (femtogram per milliliter [fg/ml] or 5 × 10 -15 moles/ml) concentrations [START_REF] Rissin | Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations[END_REF][START_REF] Wilson | The simoa HD-1 analyzer: a novel fully automated digital immunoassay analyzer with single-molecule sensitivity and multiplexing[END_REF][START_REF] Rodero | Detection of interferon α protein reveals differential levels and cellular sources in disease[END_REF], corresponding to a 5,000-fold increased sensitivity over commercial ELISAs. We hypothesized that serum IFNα levels determined with this new standardized assay would represent a better biomarker of SLE activity than the Farr assay. Therefore, the primary objective of this study was to characterize the relationship between digital ELISA-determined serum IFNα concentrations and clinically assessed SLE activity. We also compared that assay to a functional sensitive biologic assay (bioassay), based on IFNα antiviral properties, that has been used routinely in our institution for 30 years [START_REF] Lebon | Early intrathecal synthesis of interferon in herpes encephalitis[END_REF][START_REF] Lebon | Intrathecal synthesis of interferonɑ in infants with progressive familial encephalopathy[END_REF][START_REF] Hausfater | Prevalence of viral infection markers by polymerase chain reaction amplification and interferon-α measurements among patients undergoing lumbar puncture in an emergency department[END_REF].

PATIENTS AND METHODS

Study design, patients, and controls. Serum samples were obtained from 150 consecutive patients (139 women, 11 men) diagnosed as having SLE according to the 1997 updated American College of Rheumatology criteria for SLE classification [START_REF] Hochberg | Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter][END_REF]. SLE patients were referred to our National Referral Center for SLE. SLE clinical characteristics, the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [START_REF] Bombardier | the Committee on Prognosis Studies in SLE. Derivation of the SLEDAI: a disease activity index for lupus patients[END_REF][START_REF] Buyon | The effect of combined estrogen and progesterone hormone replacement therapy on disease activity in systemic lupus erythematosus: a randomized trial[END_REF][START_REF] Petri | Combined oral contraceptives in women with systemic lupus erythematosus[END_REF], and the therapeutic regimen were recorded on the day blood was drawn (day 0). The class of lupus nephritis, according to the International Society of Nephrology/Renal Pathology Society 2003 classification [START_REF] Weening | The classification of glomerulonephritis in systemic lupus erythematosus revisited[END_REF], was recorded, and the serum sample was obtained within 3 days before or after the renal biopsy. Routine testing to determine anti-dsDNA antibody titers using the Farr assay (cutoff value 9.0 IU/ml) (Trinity Biotech) and anti-RNP antibody levels (anti-Sm, anti-SSA/Ro60, anti-Ro 52/TRIM21, and anti-SSB) (Luminex FIDIS; Theradiag) was conducted, and laboratory analyses to determine C3 and C4 levels (Optilite; The Binding Site), complete blood cell counts, serum creatinine levels, and presence of proteinuria and hematuria were performed.

According to their SELENA-SLEDAI scores, patients were divided into groups of inactive SLE (SLEDAI score <4; n = 68) or active SLE (SLEDAI score ≥4; n = 82). The presence of a severe or mild/moderate lupus flare was recorded according to the SELENA-SLEDAI flare instrument (see Supplementary Methods, available on the Arthritis & Rheumatology web site at http:// onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract) [START_REF] Buyon | The effect of combined estrogen and progesterone hormone replacement therapy on disease activity in systemic lupus erythematosus: a randomized trial[END_REF][START_REF] Petri | Combined oral contraceptives in women with systemic lupus erythematosus[END_REF]. For logistic regression analyses, the domains of activity were assigned by the SELENA-SLEDAI scores in those domains. Serum samples from 68 age-and sex-matched healthy donors (Établissement Français du Sang, Île-de-France, Pitié-Salpêtrière Hospital) were collected during the same time period.

The primary objective of the study was to assess the diagnostic performance of the IFNα digital ELISA to monitor SLE activity. The secondary goal was to compare the performances of the digital ELISA and the bioassay. Exclusion criteria were 1) known or suspected infection on day 0, or 2) increased hydroxychloroquine, prednisone, and/or immunosuppressant dose(s) during the 4 weeks preceding day 0. The study was approved by the local ethics committee (no. 30052012), and informed consent was provided by all participants. The research was carried out in compliance with the Helsinki Declaration.

IFNα bioassay. Serum IFNα biologic activity (in IU/ml) was determined by assessing the protection afforded by each patient's serum to cultured Madin-Darby bovine kidney cells infected with vesicular stomatitis virus, as described previously [START_REF] Lebon | Early intrathecal synthesis of interferon in herpes encephalitis[END_REF][START_REF] Gresser | Pronounced antiviral activity of human interferon on bovine and porcine cells[END_REF][START_REF] Lebon | Production of human type I interferon by lymphocytes in contact with cells infected by herpes virus and fixed with glutaraldehyde[END_REF][START_REF] Batteux | FCγRII (CD32)dependent induction of interferon-α by serum from patients with lupus erythematosus[END_REF] (see Supplementary Methods, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/ art.40792/abstract). Bioassay sensitivity (the lower limit of detection) was 2 IU/ml. Serum IFNα activity in healthy individuals is undetectable at a level of <2 IU/ml [START_REF] Lebon | Intrathecal synthesis of interferonɑ in infants with progressive familial encephalopathy[END_REF][START_REF] Vezinet | Herpes encephalitis in adults and interferon[END_REF].

IFNα digital ELISA. Serum IFNα concentrations (in fg/ml) were determined with an IFNα digital ELISA technology reagent kit (Simoa; Quanterix), which is based on a 3-step protocol using an HD-1 Analyzer (Quanterix) [START_REF] Rissin | Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations[END_REF] (see Supplementary Methods, available at http://onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract).

Statistical analysis.

Qualitative variables are expressed as the number (percentage), and quantitative parameters as the mean ± SD or median (range), as appropriate. Differences between patient groups were tested with the Mann-Whitney U test or Student's t-test for continuous data, and Fisher's exact test or chi-square test for categorical data. Spearman's correlation coefficients were computed for quantitative values. The diagnostic performances of the Farr assay, IFNα digital ELISA, and the bioassay to detect SLE disease activity were investigated by analyzing receiver operating characteristic (ROC) curves, with the SELENA-SLEDAI-assessed clinical activity as the gold standard for those analyses. Because the SELENA-SLEDAI measure includes the Farr assay among the domains scored, we used for this analysis the clinical SELENA-SLEDAI that refers to symptoms, signs, and routine laboratory testing, disregarding the points that can be given for the presence of anti-DNA antibodies and/or low complement levels [START_REF] Van Vollenhoven | A framework for remission in SLE: consensus findings from a large international task force on definitions of remission in SLE (DORIS)[END_REF]. The areas under the ROC curves (AUCs) to differentiate active versus inactive SLE and an SLE flare versus no flare according to the digital ELISA, bioassay, and Farr assay were compared using a nonparametric approach [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF]. The optimal thresholds were determined using a compromise among the maximum correct classification rate, the minimum distance to the upper left corner of the ROC curve, the minimum sensitivity -specificity difference, and the Youden index. Sensitivities, specificities, likelihood ratios, positive predictive values, and negative predictive values were calculated. McNemar's test for paired proportions was used to compare sensitivities and specificities. No statistical analyses were performed on likelihood ratios and predictive values.

To identify SLE parameters independently associated with the absence of detectable serum IFNα in patients with active disease, variables significantly associated with the falsenegative rate in bivariable analyses were entered into a multivariable logistic regression model with stepwise selection of variables (P = 0.30 for entry and P = 0.10 for exit). Alternatively, to identify SLE parameters independently associated with bioassay-or digital ELISA-detected abnormal IFNα levels, univariable and multivariable logistic regression analyses using backward stepwise variable elimination were performed (with the variable exit threshold set at P > 0.10). All potential explanatory variables included in the multivariable analyses were subjected to collinearity analysis with a correlation matrix. None of these variables were associated with each other. Model goodnessof-fit was assessed with the determination coefficient (R 2 ). All tests were 2-sided, and P values less than 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism (version 5.0), IBM SPSS Statistics (version 22.0), and SAS 9.4 software.

RESULTS

Patient characteristics. The baseline characteristics of the patients are described in Table 1. Approximately half of the patients had SELENA-SLEDAI-defined active SLE (score ≥4) or a SELENA-defined flare. Immunosuppressant therapy was mycophenolate mofetil for 18 patients, methotrexate for 13 patients, and azathioprine for 6 patients.

Serum IFNα concentrations in SLE patients.

Digital ELISA-determined serum IFNα concentrations in patients with active SLE (median 1,396 fg/ml [range 0-53,000]) were significantly higher than those in patients with inactive SLE (median 14 fg/ml [range 0-4,328]) (P < 0.0001) and healthy controls (median 0 fg/ml [range 0-269]) (P < 0.0001). Concentrations also differed significantly between patients with inactive SLE and healthy controls (P < 0.0001) (Figure 1).

The IFNα digital ELISA positivity threshold was 136 fg/ml, which is 3 SD above the mean serum IFNα concentration calculated from the serum samples from the 68 healthy blood donors. Using that cutoff value, the digital ELISA was able to detect abnormal serum IFNα concentrations (>136 fg/ml) in 78 SLE patients (52%).

Bioassay-determined serum IFNα levels were significantly higher in patients with active SLE (median 5 IU/ml [range 0-200]) than those with inactive disease (median 0 IU/ml [range 0-12]) (P < 0.0001) (Figure 1). The bioassay was able to detect abnormal serum IFNα levels (≥2 IU/ml) in 56 SLE patients (37%).

Sensitivity of digital ELISA versus the bioassay to detect abnormal serum IFNα levels.

Although the digital ELISA-and bioassay-determined serum IFNα levels were significantly correlated (Spearman's rank r = 0.77 [95% confidence interval 0.69-0.83]) (P < 0.0001), the digital ELISA was able to identify 29 SLE patients with a serum IFNα concentration of >136 fg/ml (Figure 2) and negative biologic activity (data not shown), thereby indicating the digital ELISA's higher sensitivity than the bioassay to detect abnormal serum IFNα concentrations. We also compared digital ELISA IFNα measurements with the ISG score of samples from 22 SLE patients (see Supplementary Figure 2, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract). We used an ISG score, described previously by Rice et al, based on quantitative polymerase chain reaction assessment of 6 ISGs [START_REF] Rice | Assessment of interferon-related biomarkers in Aicardi-Goutieres syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study[END_REF][START_REF] Rice | Assessment of type I interferon signaling in pediatric inflammatory disease[END_REF]. In this test, the median fold change of the ISGs when compared with the median in healthy controls is used to create an IFN score. Serum IFNα levels were highly positively correlated with the ISG scores (r = 0.74, P < 10 ˗4 ). Only 2 patients showed a positive ISG score and a serum IFNα level below the positivity threshold for the digital ELISA (<136 fg/ml).

Digital ELISA and bioassay diagnostic performances to discriminate active SLE. The ROC AUC for the IFNα digital ELISA to differentiate between active and inactive SLE was 0.83, better than that of the Farr assay (0.69; P = 0.007) (Figure 3). The ROC AUC for the IFNα digital ELISA to differentiate between an SLE flare and no flare was 0.84, better than that of the Farr assay (0.70; P = 0.006). The ROC AUC for the IFNα bioassay was 0.79 to differentiate between active and inactive SLE (P = 0.04 versus the Far assay) and 0.78 to differentiate between an SLE flare and no flare (P = 0.09 versus the Farr assay).

The optimal thresholds to distinguish between patients with active SLE and those with inactive SLE or between patients with a flare and those without a flare were identical, equal to 266 fg/ml for the digital ELISA, 2 IU/ml for the bioassay, and 14.3 IU/ml for the Farr assay. Using ROC curve-defined thresholds, the IFNα digital ELISA and the IFNα bioassay had significantly better specificity than the Farr assay to differentiate between active and inactive SLE and between a flare and no flare (Table 2). The IFNα digital ELISA and the IFNα bioassay also had a better likelihood ratio for positive results and positive predictive value to identify active disease and flare than the Farr assay.

Sensitivity of IFNα digital ELISA versus IFNα bioassay to identify patients with active SLE.

The IFNα digital ELISA had significantly better sensitivity than the IFNα bioassay to differentiate between an SLE flare and no flare. Supplementary Table 2 (available on the Arthritis & Rheumatology web site at http:// onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract) reports positivity rates of the IFNα digital ELISA, bioassay, and Farr test according to SLE activity. The digital ELISA was able to identify a subset of 20 SLE patients with serum IFNα levels between 266 fg/ml (the cutoff associated with active SLE) and 1,130 fg/ml (the cutoff associated with the best agreement between the digital ELISA and the bioassay). In this subset of patients the median SLEDAI score was slightly, but significantly, higher than that in the subset of patients with serum IFNα levels below normal (4 versus 2; P = 0.01) (see Supplementary Figure 3, at http://onlinelibrary. wiley.com/doi/10.1002/art.40792/abstract). The vast majority of the subset of patients with serum IFNα levels of 266-1,130 fg/ ml (17 of 20) had negative results on the bioassay. Thus, the digital ELISA was more sensitive than the bioassay to detect low-abnormal serum IFNα concentrations and patients with low disease activity.

Characteristics of digital ELISA false-negatives.

Patients with a digital ELISA false-negative result (active SLE but with a serum IFNα concentration of <266 fg/ml), as compared to patients with a true-positive result (active SLE and a serum IFNα concentration of ≥266 fg/ml), were more likely to be treated with hydroxychloroquine, have more frequent arthritis without other organ involvement, lower anti-RNP antibody rates, and lower SELENA-SLEDAI activity scores (Table 3).

Patient characteristics associated with abnormal serum IFNα levels.

According to multivariable analyses, abnormal digital ELISA-determined IFNα concentrations were significantly associated with SLE-specific fever, active mucocutaneous lupus, active lupus nephritis, and anti-Sm antibodies, but no other anti-RNP antibodies (anti-Ro/SSA 52, anti-Ro/SSA 60, anti-La/ SSB, and anti-RNP) (see Supplementary Table 3, at http://onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract). Similar results were obtained with the bioassay except that abnormal IFNα levels were significantly associated with the presence of anti-RNP antibodies, while anti-Ro/SSA 52, anti-Ro/SSA 60, anti-La/SSB and anti-Sm antibodies were not (see Supplementary Table 4, at http://onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract).

DISCUSSION

The contribution of IFNα as a biomarker of SLE activity has been limited so far by the absence of a simple and standardized sensitive assay to quantify this cytokine routinely. The new ultrasensitive digital ELISA Simoa technology enabled direct IFNα quantification, with a 5,000-fold increased sensitivity over commercial ELISAs [START_REF] Rissin | Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations[END_REF][START_REF] Wilson | The simoa HD-1 analyzer: a novel fully automated digital immunoassay analyzer with single-molecule sensitivity and multiplexing[END_REF]. Recent findings described by Rodero et al suggested that this assay would enhance our understanding of IFNα biology and potentially improve the diagnosis and stratification of pathologies associated with IFNα dysregulation [START_REF] Rodero | Detection of interferon α protein reveals differential levels and cellular sources in disease[END_REF]. Based on a large series of SLE patients, we confirmed that routine, highly sensitive IFNα quantification is feasible and of interest for monitoring SLE activity. First, using serum samples from healthy blood donors, we calculated that the threshold for abnormal serum IFNα levels was 136 fg/ml. Next, by studying a series of patients with widely ranging SLE activity and organ involvement, we determined that the threshold associated with active disease in the patients was 266 fg/ml. Finally, according to ROC curve analysis, we demonstrated that the digital ELISAdetermined serum IFNα concentration was a better biomarker of SLE activity than concentrations measured with the Farr assay.

To date, anti-dsDNA antibody titration, better achieved with the Farr assay, has been used to monitor global disease activity in SLE (4-7). Indeed, anti-DNA antibody positivity is associated with overall SLE activity and can be useful in monitoring that activity. However, the sensitivity and specificity of that association are relatively low, both ~66% [START_REF] Kavanaugh | Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests[END_REF], which is in good accordance with our results. Clearly, some SLE populations have persistently elevated anti-DNA antibody titers but not active disease [START_REF] Gladman | Serologically active clinically quiescent systemic lupus erythematosus: a discordance between clinical and serologic features[END_REF][START_REF] Leblanc | Serologically active clinically quiescent systemic lupus erythematosus-predictors of clinical flares[END_REF]. The latter observation implies that positivity for anti-DNA antibodies will have limited impact on the pretest likelihood of active disease for a given SLE patient [START_REF] Kavanaugh | Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests[END_REF]. That discordance might be overcome by using the IFNα digital ELISA, which has better specificity, likelihood ratio for positive results, and positive predictive value to identify active disease and a flare than the Farr assay. Importantly, the bioassay-assessed serum IFNα level, already used routinely in our institution, was also a better biomarker of disease activity than the Farr assay. However, the ability of the digital ELISA to detect low-abnormal serum IFNα concentrations and low disease activity was more sensitive than the bioassay. Furthermore, it is important to note that bioassays based on IFNα antiviral properties are difficult to standardize, which could limit the generalization of the data presented in this study.

Considering the unprecedented number of new agents being developed to treat SLE via targeting of IFN signaling, having an ultrasensitive, valid, easy-to-use, and standardized assay to directly assess IFNα expression in SLE patients will certainly help guide physicians' treatment choices. Whether the direct IFNα determination can be used to predict an SLE flare in the ensuing weeks after assessment remains to be tested. IFN scores have been dis- ‡ Multivariable analysis (stepwise logistic regression model with P for entry = 0.3 and P for exit = 0.1). Using bivariable analysis, low C3 level, SELENA-SLEDAI score, SELENA-SLEDAI score of ≥8, and flare were associated with the detection of IFNɑ. These 4 items were clearly linked and interdependent. We therefore chose to include in the multivariable analysis only the items that seemed the most relevant and the most significant in bivariable analysis (i.e., SELENA-SLEDAI score ≥8). § Excluding antimalarials and prednisone. ¶ Value shown is the number of positive assay results/number of patients assessed (%). # Defined using the SELENA flare instrument.

appointing for that purpose, with several studies showing a lack of association between the IFN signature and longitudinal disease activity changes or risk of SLE flare [START_REF] Petri | Longitudinal expression of type I interferon responsive genes in systemic lupus erythematosus[END_REF][START_REF] Landolt-Marticorena | Lack of association between the interferon-ɑ signature and longitudinal changes in disease activity in systemic lupus erythematosus[END_REF]. Despite that lack of association, the possibility that IFNα-related biomarkers could predict future flares was highlighted by the monitoring of IFN-regulated chemokine levels in SLE patients [START_REF] Bauer | Interferon-regulated chemokines as biomarkers of systemic lupus erythematosus disease activity: a validation study[END_REF]. In that study, serum levels of CXCL10, CCL2, and CCL19 chemokines were linked with SLE activity and performed better as biomarkers than the currently available laboratory tests to predict a flare over the following year [START_REF] Bauer | Interferon-regulated chemokines as biomarkers of systemic lupus erythematosus disease activity: a validation study[END_REF]. However, monitoring of those 3 chemokines remains difficult in routine practice. We are currently studying the abilities of the IFNα digital ELISA and bioassay to predict a lupus flare.

Using an ultrasensitive assay may also contribute to improving our understanding of SLE pathogenesis. Indeed, with the digital ELISA, abnormal IFNα levels were significantly associated with the presence of anti-Sm antibodies, but not with other anti-RNP antibodies tested. That finding contrasts with the bioassay analysis and other previous studies that used different cytokine dosage methods and showed abnormal IFNα levels to be associated with other anti-RNP antibodies, such as anti-RNP or anti-Ro/SSA antibodies [START_REF] Feng | Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus[END_REF][START_REF] Kirou | Coordinate overexpression of interferon-α-induced genes in systemic lupus erythematosus[END_REF][START_REF] Kirou | Activation of the interferon-α pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease[END_REF][START_REF] Petri | Longitudinal expression of type I interferon responsive genes in systemic lupus erythematosus[END_REF][START_REF] Weckerle | Network analysis of associations between serum interferonɑ activity, autoantibodies, and clinical features in systemic lupus erythematosus[END_REF][START_REF] Ko | Genetic ancestry, serum interferonɑ activity, and autoantibodies in systemic lupus erythematosus[END_REF]. Whether this new information is important in SLE pathogenesis remains to be elucidated.

Our study has certain limitations. We compared the IFNα digital ELISA measurements with the ISG score and found a highly positive correlation between the 2 parameters, suggesting that the digital assay may be used in assignment of patients to anti-IFN drugs [START_REF] Khamashta | Sifalimumab, an anti-interferon-α monoclonal antibody, in moderate to severe systemic lupus erythematosus: a randomised, double-blind, placebo-controlled study[END_REF]. According to a recent study, for some patients an ISG score could be more sensitive than a digital assay to detect low IFNα concentrations (<5 fg/ml) [START_REF] Rodero | Detection of interferon α protein reveals differential levels and cellular sources in disease[END_REF]. However, because our calculated threshold associated with active disease was 266 fg/ ml, which is far above the lower limit of detection of the digital ELISA, a very low IFNα concentration might not contribute to identifying patients with clinically active SLE. Furthermore, unless the digital ELISA results are compared directly to ISG scores in a larger panel of patients, it will not be possible to determine their clinical value in patient assignment to anti-IFN treatment. Moreover, these results must be validated in other independent cohorts.

In conclusion, our data support the notion that direct serum IFNα determination with a highly sensitive, easy-to-standardize assay might be useful for clinical monitoring of SLE activity and the selection of the best candidates for anti-IFNα treatment.

1

  Alexis Mathian, MD, PhD, Fleur Cohen-Aubart, MD, PhD, Julien Haroche, MD, PhD, Zahir Amoura, MD, MSc: Sorbonne Université, Assistance Publique-Hôpitaux de Paris, Groupement Hospitalier Pitié-Salpêtrière, French National Referral Center for Systemic Lupus Erythematosus, Antiphospholipid Antibody Syndrome and Other Autoimmune Disorders, Service de Médecine Interne 2, Institut E3M, INSERM UMRS, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France; 2 Suzanne Mouries-Martin, MD, MSc: Centre Hospitalier Universitaire de Dijon, Hôpital François-Mitterrand, service de médecine interne et maladies systémiques (médecine interne 2), Dijon, France; 3 Karim Dorgham, PhD, Elyès Ben Salah, PharmD, Laura Garrido Castillo, MSc, Makoto Miyara, MD, PhD, Delphine Sterlin, PharmD, PhD, Guy Gorochov, MD, PhD: Sorbonne Université, INSERM UMRS, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Assistance Publique-Hôpitaux de Paris, Groupement Hospitalier Pitié-Salpêtrière, Paris, France; 4 Hervé Devilliers, MD, PhD: Centre Hospitalier Universitaire de Dijon, Hôpital François-Mitterrand, service de médecine interne et maladies systémiques (médecine interne 2) and Centre d'Investigation Clinique, INSERM CIC 1432, Dijon, France; 5 Laura Barnabei, MSc, Frédéric Rieux-Laucat, PhD: Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, INSERM UMR-Institut Imagine, Sorbonne Paris Cité, Paris, France; 6 Miguel Hie, MD, MSc, Marc Pineton de Chambrun, MD, MSc, Micheline Pha, MD, MSc, Du Lê Thi Huong, MD, PhD: Assistance Publique-Hôpitaux de Paris, Groupement Hospitalier Pitié-Salpêtrière, French National Referral Center

Figure 1 .

 1 Figure 1. Serum interferon-α (IFNα) concentrations according to systemic lupus erythematosus (SLE) activity assessed with digital enzyme-linked immunosorbent assay (ELISA) (left) or functional bioassay (right). Patients were divided into groups with inactive SLE (iSLE; Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] score of <4) or active SLE (aSLE; SLEDAI score of ≥4). The IFNα digital ELISA positivity threshold (solid lines) was 3 SD above the mean of the 68 serum samples from healthy control (HC) blood donors, i.e., 136 fg/ml. The lower limits (broken lines) and upper limits (dotted lines) of quantification were 5-52,200 fg/ml for the digital ELISA and 2-200 IU/ml for the functional bioassay. Symbols represent individual subjects; bars show the median. Statistical analyses were conducted using the Mann-Whitney U test.

Figure 2 .

 2 Figure 2. Correlation between the interferon-α (IFNα) digital enzymelinked immunosorbent assay (ELISA)-and bioassay-determined concentrations. The IFNα digital ELISA positivity threshold (solid line), the upper limit of quantification (dotted line), and the lower limit of detection (broken lines) are shown. The IFNα cutoff concentration of 1,130 fg/ml gave the best agreement between the digital ELISA and the bioassay (see Supplementary Figure 1 and Supplementary Table1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley. com/doi/10.1002/art.40792/abstract). Symbols represent individual patients. Statistical analyses were conducted using Spearman's rank correlation coefficient. 95% CI = 95% confidence interval.

1

 1 Figure 2. Correlation between the interferon-α (IFNα) digital enzymelinked immunosorbent assay (ELISA)-and bioassay-determined concentrations. The IFNα digital ELISA positivity threshold (solid line), the upper limit of quantification (dotted line), and the lower limit of detection (broken lines) are shown. The IFNα cutoff concentration of 1,130 fg/ml gave the best agreement between the digital ELISA and the bioassay (see Supplementary Figure 1 and Supplementary Table1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley. com/doi/10.1002/art.40792/abstract). Symbols represent individual patients. Statistical analyses were conducted using Spearman's rank correlation coefficient. 95% CI = 95% confidence interval.

  Figure 2. Correlation between the interferon-α (IFNα) digital enzymelinked immunosorbent assay (ELISA)-and bioassay-determined concentrations. The IFNα digital ELISA positivity threshold (solid line), the upper limit of quantification (dotted line), and the lower limit of detection (broken lines) are shown. The IFNα cutoff concentration of 1,130 fg/ml gave the best agreement between the digital ELISA and the bioassay (see Supplementary Figure 1 and Supplementary Table1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley. com/doi/10.1002/art.40792/abstract). Symbols represent individual patients. Statistical analyses were conducted using Spearman's rank correlation coefficient. 95% CI = 95% confidence interval.

Figure 3 .

 3 Figure 3. Receiver operating characteristic (ROC) curves of interferon-α (IFNα) concentrations to discern active systemic lupus erythematosus (SLE). A-C, The diagnostic performances of the serum IFNα digital enzyme-linked immunosorbent assay (ELISA) (A), bioassay (B), and Farr assay (C) to detect SLE activity (active versus inactive) were investigated by analyzing ROC curves, with the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-assessed clinical activity score serving as the gold standard. D, The area under each ROC curve (AUC) is given for the digital ELISA (blue), the Farr test (green), and the bioassay (red). The optimal cutoff point represented for each ROC curve was determined using the maximum correct classification rate, the minimum distance to the upper left corner of the curve, the minimum sensitivity -specificity difference, and Youden's index score. In A-D (left), patients were divided into groups with inactive (clinical SLEDAI score of 0) or active SLE (clinical SLEDAI score of >0). In A-D (right), using the SELENA flare identifier, patients were divided into groups with no flare or experiencing a flare.

Table 1 .

 1 Baseline characteristics and disease parameters in SLE patients (n = 150)*

	Women	139 (92.7)
	Age, mean ± SD years	36.2 ± 12.5
	Disease duration, mean ± SD years	9.8 ± 9.1
	SELENA-SLEDAI score, median (range)	4 (0-36)
	SELENA-SLEDAI score ≥4	82 (54.7)
	Mild/moderate flare †	21 (14)
	Severe flare †	53 (35.3)
	Fever	31 (20.7)
	Weight loss or anorexia	18 (12)
	Lymphadenopathy	21 (14)
	Active cutaneous lupus	37 (24.7)
	Active lupus serositis	17 (11.3)
	Active lupus arthritis	44 (29.3)
	Active lupus nephropathy	20 (13.3)
	Active neuropsychiatric lupus	6 (4)
	Hydroxychloroquine use	124 (82.7)
	Prednisone use	90 (60)
	Prednisone ≥10 mg/day	38 (25.3)
	Immunosuppressive agent use ‡	37 (24.7)
	Positive Farr assay	87 (58)

Positive for anti-RNP antibodies 53 (35.3) Positive for anti-Sm antibodies 28 (18.7) Positive for anti-Ro/SSA 52 antibodies 48 (32) Positive for anti-Ro/SSA 60 antibodies 64 (42.7) Positive for anti-La/SSB antibodies 20 (13.3) Low C3 level 54/147 (36.7) § * Except where indicated otherwise, values are the number (%). SLE = systemic lupus erythematosus; SELENA-SLEDAI = Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index. † Defined using the SELENA flare instrument. ‡ Excluding antimalarials and prednisone. § Value shown is the number of positive assay results/number of patients assessed (%).

Table 2 .

 2 Sensitivity and specificity of the IFNɑ digital ELISA, bioassay, and Farr assays to detect active SLE or flare* No statistical analyses were performed on likelihood ratios and predictive values. IFNɑ = interferon-ɑ; ELISA = enzyme-linked immunosorbent assay; 95% CI = 95% confidence interval; PLR = positive likelihood ratio; NLR = negative likelihood ratio; PPV = positive predictive value; NPV = negative predictive value. † Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-defined groups were classified as having inactive systemic lupus erythematosus (SLE; clinical SLEDAI score of 0) or active SLE (clinical SLEDAI score of >0).‡ The chosen positivity threshold for this assay was 266 fg/ml. § P < 0.05 versus Farr assay. ¶ The chosen positivity threshold for this assay was 2 IU/ml. # P < 0.001 versus Farr assay. ** The chosen positivity threshold for this assay was 14.3 IU/ml. † † Defined using the SELENA flare instrument. ‡ ‡ P < 0.05 versus bioassay. § § P < 0.01 versus Farr assay.

		Sensitivity, %	Specificity, %
		(95% CI)	(95% CI)	PLR	NLR	PPV, %	NPV, %
	Active SLE †
	Digital ELISA ‡	68.7 (57.6-78.4)	85.1 (74.3-92.6) §	4.6	0.37	85.1	68.7
	Bioassay ¶	62.7 (51.3-73.0)	94.0 (85.4-98.4)#	10.5	0.40	92.9	67.0
	Farr assay**	63.9 (52.6-74.1)	67.2 (54.6-78.2)	1.9	0.54	70.7	60.0
	SLE flare † †
	Digital ELISA ‡	73.0 (61.4-82.7) ‡ ‡	82.9 (72.5-90.6) §	4.3	0.32	80.6	75.9
	Bioassay ¶	63.5 (51.5-74.4)	88.2 (78.7-94.4) § §	5.4	0.41	83.9	71.3
	Farr assay**	67.6 (55.7-78.0)	67.1 (55.4-77.5)	2.0	0.48	66.7	68.0

*

Table 3 .

 3 Baseline characteristics of and SLE features in patients with active disease according to detection of IFNɑ with digital ELISA*

	IFNα	IFNα not
	detectable,	detectable,
	true-positive	false-negative
	(n = 57)	(n = 25)	P †	P ‡	OR (95% CI) ‡

* Except where indicated otherwise, values are the number (%). Systemic lupus erythematosus (SLE) was defined as active based on a Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score of ≥4. ELISA = enzyme-linked immunosorbent assay; OR = odds ratio; 95% CI = 95% confidence interval; NS = not significant; ND = not done. † Bivariable analysis (Mann-Whitney U test for continuous variables and chi-square test for categorical variables).

ACKNOWLEDGMENTS

We thank the patients, the healthy donors, the nurses, and the Department of Internal Medicine 2 staff who participated in this study.

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final ver-sion to be submitted for publication. Dr. Mathian had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Mathian, Mouries-Martin, Dorgham, Barnabei, Rozenberg, Gorochov, Amoura. Acquisition of data. Mathian, Mouries-Martin, Dorgham, Barnabei, Ben Salah, Cohen-Aubart, Garrido Castillo, Haroche, Hie, Pineton de Chambrun, Miyara, Sterlin, Pha, Lê Thi Huong, Rieux-Laucat, Rozenberg. Analysis and interpretation of data. Mathian, Mouries-Martin, Dorgham, Devilliers, Barnabei, Pineton de Chambrun, Rozenberg, Gorochov, Amoura.

AUTHOR CONTRIBUTIONS


Sayena Molaie Lidilem

Françoise Raby

Laura M Hartwell

A multiple-intelligence approach to creativity in ESP courses

Introduction

The manner through which a student is taught in a classroom is an influential factor on the student's achievement. Furthermore, the presence of creativity within that teaching context is of great importance. Creativity is linked to different levels of attainment in second language learning [START_REF] Maley | Creativity[END_REF].

The human capability to act effectively in a novel or difficult situation constitutes the modern definition of intelligence (UNESCO, 2002) and precisely Gardner's theory of multiple intelligences (MIT). According to Gardner (1983: 2) there is more than only one intelligence an individual may rely upon when facing a novel or a difficult situation. In parallel, one of the first requirements for constructing products is creativity [START_REF] Naiman | Orchestrating Collaboration at Work: Using Music, Improv, Storytelling and other Arts to Improve Teamwork[END_REF]. In order to implement a creative approach in a language classroom, we must first agree on a definition of creativity.

As language teachers and researchers, the kind of creativity that we look for in the classroom consists of the production of something new or of presenting already known material in a new fashion [START_REF] Richards | Creativity in Language Teaching[END_REF]). An essential characteristic of creativity is the process of becoming sensitive to problems, identifying problems, searching for solutions, formulating a hypothesis and testing it, and finally communicating this procedure to others involved in the same situation (DeHaan, 2009: 172-181). This is also the common ground with the definition of modern intelligence, in particular the definition of multiple intelligences (MI). Becoming attuned to a learner's ability to think creatively provides clues about different preferences for ways of learning, the personal preferences throughout learning and the individual strategies learners employ when dealing with a learning difficulty.

According to numerous studies such as those of [START_REF] Tilman | The ecological consequences of changes in biodiversity: a search for general principles[END_REF]Tilman ( : 1455Tilman ( -1477)), [START_REF] Elton | The Ecology of Invasions by Animals and Plants[END_REF] and [START_REF] Odum | Fundamentals of Ecology[END_REF], increasing diversity appears to increase creativity. In a language class, curriculum variety maximizes chances for learners to become more creative [START_REF] Pashler | Learning styles: concepts and evidence[END_REF]. When learners are exposed to a set of facts several times, in various forms and in creative contexts, their knowledge shifts from a purely theoretical construct to a more practical one. According to [START_REF] Gardner | Multiple Intelligences[END_REF], not all students learn the same way and, therefore, they should not be taught in one similar manner. Taking into account learners' different learning styles in a language classroom provides students with the freedom to express what they know in different creative ways. It also allows them to come up with creative strategies when facing a learning difficulty.

In this article, we will describe Gardner's theory of multiple intelligences (MIT) and its nine types of intelligences. We will also explore how becoming familiar with learners' dominant type(s) of intelligence and allowing them to experience the given topic using specific tasks might nurture creativity in the classroom. Next, we will focus on the creative characteristics of a multiple intelligence classroom. Then, we will show examples of re-designing the already existing classroom tasks and activities in a way that is both creative and MIT-based. Finally, we will discuss the current results of the pre-experimentation part of a PhD thesis in progress on the theory of multiple intelligences.

Theoretical framework

Defining creativity

There are many different scientific definitions of creativity. Some definitions are formulated in terms of a product, i.e. when the outcome is an invention or a discovery. Other definitions are expressed in terms of a process, a kind of person or a set of conditions. However, the production of something new to a person or to a culture is included in almost all definitions [START_REF] Cutraro | How Creativity Powers Science[END_REF]. There are also some traits that stand true in all the definitions of creativity, such as the fact that creative contributions must be true, generalizable, and surprising in view of what existed at the time of the discovery [START_REF] Torrance | Scientific views of creativity and factors affecting its growth[END_REF]. It is an interesting fact that the definition above cannot guarantee that whoever displays these features will consistently act creatively but, if one lacks these features, the chances of acting creatively decrease significantly. Creativity is a natural process that occurs when there is a problem. When tension arises and the already-existing conditions and solutions do not suffice, one attempts to come up with new ideas and that can be the start of creativity (UNESCO, 2002).

If we agree on these characteristics of creativity, we can see that the rise of an issue or a problem is the starting point of creativity. This means that one must be sensitive to problems and deficiencies and find a different way to approach and solve a potential problem [START_REF] Raby | La classe de langue. Cours d'ergonomie de la formation langagière[END_REF]. Being sensitive to existing problems and using different strategies to solve or overcome the issues at hand is also one of the definitions of intelligence or, more precisely the notion of multiple intelligences.

The multiple intelligences theory and foreign language teaching and learning

The first intelligence test was invented by the French psychologist, Binet, in 1904. This test represented intelligence with a single number, which is known as the "intelligence Quotient" (IQ). The IQ score is based on a test of mental intelligence, divided by age and multiplied by one hundred. Although Binet himself did not mention that intelligence is a fixed trait, expressing the level of intelligence with a single number often leads people to think that way and, unfortunately, gives certain educators an excuse to ignore the students who did poorly on the IQ test [START_REF] Perkins | Outsmarting IQ: The Emerging Science of Learnable Intelligence[END_REF]. For decades, IQ tests were embedded in examinations in different countries (such as Japan, the United States, Australia, Iran, etc.) to underpin school selection procedures. Despite continued use, the prevalence of IQ tests has faded with the rise of the modern view towards intelligence (Bartholomew, 2004: 279-293).

The modern view towards intelligence holds that intelligence is learnable. A growing number of educators and psychologists believe that intelligence is carried by genes along with innumerable experiences that shape human beings [START_REF] Perkins | Outsmarting IQ: The Emerging Science of Learnable Intelligence[END_REF]. As a result of this view, the tests of intelligence now measure different types of intelligence and no longer operate on the basis that intelligence is fixed. Educators can therefore use this information to create curricula that fit learners' needs, and help them improve their weaknesses [START_REF] Shearer | MIDAS: A Professional Manual[END_REF]. We can conclude that the modern views towards intelligence hold two major beliefs: a) intelligence is not a single trait, b) intelligence is not a fixed trait.

MIT has led to the application of eight frames in the contexts of language teaching and learning. Various empirical studies (Dörnyei, 1998: 117-135;[START_REF] Skehan | A Cognitive Approach to Language Learning[END_REF] have investigated the relationship between Binet's IQ scores and the capacity for learning foreign languages but have found only a moderate level of correlation. Therefore, Binet's traditional static concept of intelligence cannot be used as a predictor of successful language learning. Arnold & Fonseca (2004: 119-136) studied the relation of cognitive aptitudes with success in foreign language learning of over 5,000 students at American military academies and concluded that future research should involve experimentation with alternative models not narrowly dependent on a general aptitude test results. [START_REF] Skehan | A Cognitive Approach to Language Learning[END_REF] emphasizes that in classroom language learning, MIT can provide a basis for developing more creative descriptors in the target language by helping learners to connect with the learning activities and to activate linguistic information stored in memory. However, it is not a question of addressing all the individual MI profiles of each learner in every language class but of offering a balanced approach where different windows on the same concept are incorporated [START_REF] Gardner | Multiple Intelligences: The Theory in Practice[END_REF].

What are the different types of intelligences?

In 1983, Howard Gardner posited that there were at least seven different types of intelligences. Following further research, he later defined an eighth type of intelligence [START_REF] Gardner | Intelligence Reframed[END_REF]. More recently (2006), he also mentioned that there is a possibility for the existence of another type of intelligence, which is different from all the rest. Below is a brief description of these nine intelligences by Howard [START_REF] Gardner | Frames of Mind: The Theory of Multiple Intelligences[END_REF][START_REF] Gardner | Multiple Intelligences[END_REF].

1. Musical intelligence refers to the ability to perform, compose, but also appreciate music, ranging from the ability to keep in tune while humming or singing a song to more professionally playing a musical instrument. 2. Bodily/kinesthetic intelligence refers to the movement and ability of muscles in different body parts, ranging from being good with one's hands, fixing and repairing things, to being a good athlete. 3. Logical/mathematical intelligence refers to the ability to work and reason with numbers and figures, ranging from calculating sums in one's head to a liking for mathematics and algebra. 4. Visual/spatial intelligence refers to the ability to imagine the three dimensions of things, ranging from finding one's direction in a new location to being a good sculptor or a painter. 5. Linguistic intelligence refers to the ability to use words, ranging from having a convincing way of speaking to note-taking and being a good public speaker. 6. Interpersonal intelligence refers to one's ability to maintain and use social skills, ranging from having successful long-lasting friendships to understanding other peoples' moods and needs and behaviors. 7. Intrapersonal intelligence refers to one's ability to understand oneself, ranging from being productive when working alone to making appropriate decisions for oneself and being able to see it through. 8. Naturalistic intelligence refers to the ability to relate to nature and the natural environment in general, ranging from taking care of a pet to being able to categorize different breeds of animals, flowers or plants [START_REF] Gardner | Intelligence Reframed[END_REF]. 9. Existential intelligence is defined as the ability to be sensitive to, or have the capacity for, conceptualizing or tackling fundamental questions about human existence; questions that revolve around the meaning of life, why we are born, why we die, how we got here [START_REF] Gardner | Multiple Intelligences[END_REF]. Most of the credibility of Gardner's work rests on neurological evidence of specific locations within the brain; however, the exact location of existential intelligence in the human brain has not been pinpointed [START_REF] Sisk | Spritual Intelligence : Developing Higher Conscienceness[END_REF]. Accordingly, in the present study, the first eight intelligences defined by Gardner are taken into account.

Language learning can be supported by bringing into play not only the linguistic but also the musical, visual-spatial, bodily-kinesthetic, interpersonal, intrapersonal, mathematical and naturalistic abilities as they constitute distinct frames for working on the same linguistic content [START_REF] Molaie | Language sustainability and the theory of multiple intelligences[END_REF]. Not only does this variety allow students to learn in their own best ways, it also helps to reduce boredom as language learning requires frequent circling back over the same material, and thereby being more creative (Schumann et al., 1998). In this context, the research question explored in this article is: does creative use of a multiple-intelligence approach to language teaching boost English performance among second-year undergraduate (L2) science students?

Methodology

During two semesters (2014 and 2015), the researcher/teacher randomly selected one group as the experimental group (total of 30 students) and one as the control group (total of 30 students). MIT-based activities were implemented by adapting activities found in the program's coursebook Minimum Competence for Scientific English (Blattes, Vans & Upjohn, 2013) in the two experimental classes. In class, the control group followed only the activities of the coursebook. The activities in the control group were not MIT-based. However, all students also had the requirement of completing common multimedia tasks during sessions in the language laboratory (see Hartwell, 2010a;Hartwell 2010b).

The participants in this study were all second-year science students at the University Joseph Fourier, now part of the Université Grenoble Alpes. They were all in a three-year undergraduate degree program or Licence, taking classes at the Service des Langues. As the field of study of all the students was science-based, and as the name of their coursebook suggests, the English course was for specific purposes (ESP), in this case, English for the sciences.

In order to answer the research question, the common work of the teacher and students in each English semester-long course was divided into five categories, and the findings on these categories will be reported accordingly. In a creative MIT-based approach to language teaching, we looked at:

-teaching materials and classroom activities, -students' learning styles and activities, -homework, -testing tools, -scoring criteria.

Creativity in teaching material and classroom activities

An important stage of MIT application to teaching is to choose and design appropriate MIT-based material to use for classroom tasks and activities. There are many different activities and tasks that are considered MIT-based, some of which many language teachers use in their classrooms without necessarily being aware of MI theory. Table 1 below shows examples of MIT activities, materials and instructional strategies, adapted and modified from [START_REF] Armstrong | You're Smarter than You Think: A Kid's Guide to Multiple Intelligences[END_REF]. Most classroom tasks and activities used by the teacher/researcher for the experimental group were an adaptation of the activities in Table 1 As can be seen in Table 1, there are recommended activities that can be considered creative, such as journal writing, number games, visualization, drama, cooperative learning, peer tutoring, etc. General observations of teaching at the Service des Langues suggests that MIT-based activities are encouraged, although not always labeled as such. For example, L2 students are asked to work in pairs on a poster activity called an innovation project, in which they choose an already existing machine and think of ways to improve it. During this task, students are asked to work with each other (interpersonal activity), choose a machine and make a list of its weak points (logical activity), imagine ways to improve the machine (logical and spatial activities) and, finally, make a poster expressing their ideas (spatial and linguistic activities).

An important point here is that MIT-based activities are not necessarily eccentric or completely new, but that they are creative and various. Consequently, if any course is designed in a fashion that contains a high level of variety and creativity, MIT-based activities are found among its activities. For instance, if designing posters or completing multimedia exercises are common activities that all L2 students are required to do, it means that even those students randomly chosen to serve as the control group participate in some MI activities.

Logical intelligence class activity

In the grammar and vocabulary descriptions of Unit 1 of the coursebook Minimum Competence in Scientific English, the focus is on using measurement vocabulary, and making grammatically correct sentences, while describing measurements in English.

The students in the control group did not work with real tools. The teacher wrote the grammar lesson on the board, and then asked the students to look up words such as height, width, depth, diameter, etc. in the dictionary. Then, they were asked to do another coursebook exercise for which they were asked to provide synonyms and antonyms for the measurement words. Once they had finished answering, students checked their answers in pairs. The teacher was there to answer questions about the exercise, and when there was none, the class moved on to the next exercise in the coursebook.

On the contrary, in the experimental group, the teacher provided students with various objects in different forms and shapes and a number of calipers (instruments for measuring thicknesses and internal or external diameters inaccessible to a scale), measuring tapes and a scale to calculate their weight, diameter, length, thickness, etc. This exercise triggers spatial and logicalmathematical intelligences. Next, students were asked to create charts using information on the objects and their measures. This exercise triggers spatial intelligence. The chart in Figure 1 below is one example.

Measurements→ Objects ↓ Height Width Length Depth Weight Diameter Span

A carton box An encyclopedia A test tube A laboratory flask A beaker A crucible A funnel An evaporator dish Figure 1  A chart activity for practising measurement vocabulary

As can be seen above, the objects chosen for this activity mostly belong to biology and chemistry labs. This material is familiar to science students as they are used in their field of academic and professional lives; therefore, learning their names in English and using them for a classroom task can be productive while working with real tools is a creative way for learners to connect their professional field to their knowledge of English. Then, students were asked to work in groups and write sentences comparing different objects and their measures. They also asked each other questions with "how' about their charts, such as "how wide" or "how deep". This exercise triggers linguistic intelligence.

Interpersonal, logical and linguistic intelligences class activity

All lessons in the Minimum Competence for Scientific English (MCSE) course book start with a self-evaluation entry test. The questions on these tests evaluate students' knowledge on lesson content before they have studied it. The aim of these entry tests is, first, to see whether students already know about the vocabulary they are going to study in the lesson and, secondly, to arouse their curiosity about the topic. After the students were asked to answer the questions on the self-evaluation entry test, the researcher/the teacher asked them to announce how many questions they were able to answer. This gives a general idea about the extent of student knowledge of content before they study each unit. Consistently, the number of questions students could answer before studying the grammar and vocabulary of each unit was very low (one or two students out of eighteen were able to successfully complete the test, and most could complete approximately two questions out of ten). The exercises at the beginning of each unit of MCSE is an example of a selfevaluation test. The students were asked to answer the test questions on their own and without referring to the lesson or the answer key at the end of the book. In this exercise, the teacher asks the students to do the test on their own in the classroom.

In the experimental group, after the students answered as many questions as they could in the entry test, they formed pairs to participate in a peer teaching activity. In each unit of the coursebook, the entry test is followed by a grammar lesson. The teacher divided this grammar lesson into two equal parts. Each individual in a pair is responsible for studying and then teaching his/her partner. In this way, all pairs cover the whole grammar lesson. The teacher helps them in all the steps of this peer teaching activity. This exercise is designed to trigger interpersonal, logical and linguistic intelligences.

In the control group, after the students answer as many questions as they can in the entry test, the teacher teaches the grammar lesson that follows. Learners are then asked to go back to the entry test to see if they are able to answer the questions they could not before the lesson. This activity ends with students checking their answers together.

Students' learning styles and activities

In an exercise in the Minimum Competence for Scientific English coursebook (page 43), students are asked to follow a certain grammatical pattern to compare and contrast two objects. The coursebook provides them with a number of suggestions such as a rat, a diamond, a village, and Mars. As an example, Figure 2 shows an actual classroom activity that occurred in one of the classes. Most of the students came up with factual and true statements. However, a student whom we will call student C, came up with a more creative example.

Definitions -defining by comparison

Use the following pattern: "X is similar to Y but much … + er." Example: "A tiger is similar to a cat but much larger." Define the following words: a rat, diamond, a village, Mars Student A: Mars is similar to Earth, but much smaller. Student B: Mars is similar to Earth, but much dryer. Student C: Mars is similar to Snickers, but much more delicious. As can be seen in this real classroom situation in Figure 2, student C compared the famous Mars candy bar to another famous candy bar, Snickers. The vocabulary and the grammar student C used fulfilled the goal of this exercise, although what was originally requested by this exercise was to compare planet Mars to another planet. As far as the goal is achieved, in this case using the correct grammatical formula for comparison in English, more freedom can be given to students to come up with their own strategies for learning and production and therefore, open up to their creative potential. In this regard, MIT is a method that welcomes and appreciates learners' individuality and creative ways of production.

Creativity in homework

Learning is not limited only to the classroom. Each individual is influenced by an immediate and a proximal environment. That proximal environment includes outside-of-the classroom environments such as the students' homes. Consequently, learning is extended to students' homes and homework can play a key role in learning (Carbone, 2009: 1). In the semesters through which MIT was integrated into teaching English to science students, we tried to increase the level of creative homework as much as possible.

As homework, learners were assigned to work on a three-minute presentation on the thirtieth week of pregnancy and on the main causes of death between the ages fifteen and thirty. The students in the experimental group were asked to be creative with their visual supplies, whether displayed in a chart, a drawing or a graph. For example, a student presented his own drawing of the embryo in the thirtieth week of pregnancy and the changes it goes through. Another student had prepared an illustration of the main causes of death in the age range of fifteen to thirty, namely road accidents and suicides. It was followed by a quotation from Woody Allen that says "I'm not afraid of death, I just don't want to be there when it happens." This exercise can trigger student's logical, linguistic and spatial intelligences.

Another task the experimental students were assigned to do for homework entailed practicing the grammar elements of Unit 5 in their coursebook which focuses on linking words. Students were asked to find English songs with the linking words besides, moreover, as, since, in other words, for instance, in fact and actually present in the lyrics, then to make a selection of those parts of the songs with these linking words, and finally mix and record them on their cellphones to be played during the next classroom session. This was a creative listening exercise designed to aid students to become familiar with the usage of these linking words in contexts outside their coursebook. In the next session, students played their playlist in the classroom and their peers were asked to record as many of the phrases or sentences they heard on a piece of paper. They compared their answer sheets in a group including the student who created the playlist. Finally, they were asked to use the same linguistic units and phrases to link two scientific ideas, beliefs, or concepts in order to use the creative exercise and reproduce scientific ideas. This exercise can trigger students' logical, musical and interpersonal intelligences.

Creativity in testing tools

Midas test

Another stage of integrating MI into teaching is to determine the strongest type of intelligence of each individual. In order to do this, the standardized test of intelligence named Multiple intelligences developmental assessment scales (Midas), designed by Dr. Branton Shearer in 1996, and modified by the teacher/researcher was used. Midas represents the first effort to measure the multiple intelligences and has been developed according to standard psychometric procedures [START_REF] Gardner | Multiple Intelligences[END_REF]. There are approximately twelve questions for each type of intelligence and a total of 100 questions in the Midas test (See Appendix 1 for sample questions). There are five options for each question enabling test takers to choose the option best fitting their personal experiences. For example, the first question in the test is: "As a child, did you have a strong liking for music or take music lessons?" and is followed by six options: a) Always or almost always, b) Often c) Sometimes, d) A few times, e) Almost never, f) I do not know/I do not remember. The options have the following scores: a = 5, b = 4, c = 3, d = 2, e = 1 and f = 0. For each type of intelligence, the scores are totalled and then multiplied by 100. By comparing the percentages accumulated for different types of intelligences for each test taker, the strongest type of intelligence is identified. Figure 3 below shows results of one of the students who took the Midas test. After a thorough analysis of their answers is completed, a detailed report is also given to them (cf. Appendix 2).

Continuous assessment

In the testing system used at the Service des Langues, continuous assessment marks are valued and compose 60% of a student's final mark. The continuous assessment mark consists of classroom activities, homework, and oral presentations. These marks are notified to the English learners gradually over the course by their English teacher. Consequently, continuous assessment can also be conceived as a type of evaluation that allows the teacher to be more creative and gives learners more freedom to express themselves in creative fashions.

One of the testing ways encouraged by MIT is peer teaching and peer testing. During one session, students were divided into two different groups, and a "teacher" was chosen randomly for each group. While the students were busy answering the questions on the self-evaluation entry test that begins each unit of the coursebook and checking their answers together, the student who was chosen to be the teacher had time to study two pages of grammar and prepare a lesson plan to teach his/her fellow students. Then, each "teacher" was provided with a few markers and a board, and students were asked to move the chairs and sit in front of the board. The session was very successful as the students helped each other in all the steps of this teaching-learning process. Also, there were many other sessions through which students were asked to reflect on each other's answers and even give scores to one another. Other criteria for continuous assessment are those shared with other colleagues and teachers, such as the use of correct grammatical forms in speaking, use of lexis, avoiding certain frequent mistakes, level of cooperation in pair and group work, avoiding unjustified absences, and completing lab work.

Observation and process folios

According to [START_REF] Gardner | Multiple Intelligences[END_REF], fill-in-the-blank tasks have a limited ability to encompass the wide range of learners' abilities and potentials. To the contrary, MIT-based teaching requires gradual and thorough observation and taking continuous notes of likes, dislikes, preferences, strategies and weak and strong traits of the learners. In order to achieve that goal, teachers are encouraged to keep a process-folio of learners. "Process-folio", a term coined by Gardner, refers to the need of learners to know continuously about their progress. Keeping a process-folio can help learners remain positive about the process of learning and at the same time help them to be better aware of the areas in which progress and improvement is needed. A sample process-folio is demonstrated in Figure 4. Students were asked to have the process-folios with them in all sessions. In Figure 4, the first report starts from week three of the course, when sufficient time has passed from the start, course participants are fixed, learners have had enough time to become acquainted with the pace of the course and they are ready to display their work. The process-folios are updated every two weeks so that students regularly know how they have been doing.

There are four main categories in the process-folios: classroom activities, writing activities, homework, and speaking and oral presentations. Detailed marks are allocated to each of the categories based on regular observation. Progress and improvement is taken into account. For each category, a certain mark is given for creativity, originality of the ideas, and novelty. A benefit of using process-folios worth mentioning is that students will be aware of their end of the course continuous assessment mark evaluated by the teacher.

Another testing tool used in all courses is the final written exam which is common to all L2 students, irrespective of the many teachers participating in L2 teaching.

Pre-experimental procedure

In order to answer the research question "Does creative use of a multipleintelligence approach to language teaching boost performance among L2 English learners?", we analyzed the marks for both continuous assessment (Figure 5) and final exams (Figure 6), both of which were scored between 0 and 20. We found a correlation between creative class tasks based on an MI approach and performance as expressed through marks.

First, we examined all the individual student scores concerning creativity of the experimental group, notably the continuous assessment marks, the process-folio marks, and the MIDAS scores pertaining to creativity. Each student often had similar marks for the continuous assessment and process folio marks. Students with high MIDAS creativity scores tended to also have higher continuous assessment and process-folio marks. Inversely, those who scored low on the MIDAS creativity questions, also had lower continuous assessment and process-folio marks.

Secondly, we looked at the possible differences between the continuous assessment marks of the experimental group and the continuous assessment marks of the control group. Figure 5 shows that the experimental group had a higher overall mean (13.783) and standard deviation (1.617) than the control group (mean 12.633, SD 1.351). A t-test produced a P-value of less than 0.0001, indicating that the difference is not simply produced by chance, but by a difference of marks between the two groups. However, because there were different activities for the experimental and control group, we then examined final exam results. Both the experimental and the control groups took a final exam that was prepared by the English Department for all of the students in the given program. This means that the results are highly objective and were not designed to evaluate the impact of MIT-teaching. Figure 6 shows that the experimental group also had a higher mean for the final exam (13.733) than the control Group (12.266) as well as higher means of standard deviation (2.756 compared to 2.016). As for the continuous assessment scores shown in Figure 5, we used a paired t-test to compare the final exam marks of the experimental group with those of the control group. The two-tailed P-value was 0.0042. Once again, this value would indicate that the higher final exam means for the experimental group is not due to chance, but to an actual significant difference of results between the two groups. To summarize, the statistical tests performed to evaluate the differences between the experimental and the control group results showed an apparent statistically significant difference between the groups.

Groups

Analysis of the results

Statistical procedures in this study showed that teaching English for the sciences based on a creative use of the theory of multiple intelligences has a significant effect on the performance level of students during the course. Students who showed a higher level of creativity on the Midas test and participated in classroom tasks based on MIT, demonstrated a higher level of creativity in their continuous assessment scores, and also exhibited a higher level of creativity during each semester through observation. These students also performed better on the standard L2 final exam which is shared by all other L2 classes taught by other teachers. It is of importance to mention that the final exam papers were anonymous and were randomly corrected by all L2 teachers. As a result, the teacher/researcher had no access or influence on the final exam marks of the students.

Based on this data and statistics, results suggest that teaching English for sciences based on a creative use of the theory of multiple intelligences can boost performance level in learners.

Conclusion

Based on the procedures undertaken in this research, it seems that using an MIT-based approach to teaching English can boost creativity among science students in their leaning styles, how they do their homework, how they perform their oral presentations and writing exercises. Also, learners' scores on creativity had a high correlation with their scores of continuous assessment as well as a high correlation with their scores on process-folios and observations. Using regular and planned observation can be a useful tool to measure learners' creative potential in a more detailed and fair manner.

Furthermore, using a creative MIT-based approach to teaching ESP proved to have a positive effect on the learners' performance on the standard L2 final examinations. Learners in the experimental group who participated in creative MIT-based activities and were assigned more creative homework, performed better on the standard course final exam common with other L2 students.

Another interesting point is that creativity does not only blossom in arts, music and literature, but it can also manifest itself in science. In fact, observations in this study showed that there are many ways to be creative in teaching and learning English for the sciences, and that science students are able to manifest creativity in different tasks and activities during the course of an English semester.

One of the characteristics mentioned in the adopted definition of creativity was an element of novelty and surprise when the tested-and-true does not seem to work anymore. To be fully capable of investigating the relationship between creativity and MI, the testing tools should also be MIT-based. This does not mean that the only tool for measuring MIT-based experiments is MIT-based assessment. However, in order to be able to measure the true results of teaching based on MIT, we need a testing tool that allows learners to manifest their knowledge in different ways. As such testing tools do not fit into the limited time of many courses, the use of processfolios throughout the semester might be of great help for a more detailed and fair judgement of learners' progress. Further research will include analyzing process testing methods, targeting specific intelligences or the impact on different populations from other disciplines and a survey of the evolution of students' motivation.

Figure 2 

 2 Figure 2  Students' answers for an activity from the coursebook Minimum Competence in Scientific English (p. 43)

Figure 3 

 3 Figure 3  Sample Midas result of a random student

Figure 4 

 4 Figure 4  A sample process-folio of the student's progress

Table 1  MIT activities for a classroom, adapted from Armstrong, 2000

 1 and the original activities in the book Minimum Competence in Scientific English.

	INTELLIGENCE Teaching activities Teaching materials	Instructional strategies
	Linguistic	lectures, discussions, word games, journal writing	books, podcasts	read, write, or discuss the topic/subject
	Logical-mathematical	problem-solving, science experiments, mental calculation, number games	calculators, science equipment, math games	quantify, think critically, define a logical framework, or experiment the topic/subject
	Spatial	making charts and diagrams, estimating distances	maps, charts, videos, tools for measurements	see, draw, or visualize the topic/subject
	Bodily-kinesthetic	drama, dance, sports	building tools, sports equipment	build, act out, or touch the topic/subject
		songs that can be
	Musical	sung or listened to in order to practice and better understand a given lexical or	tape recorder, tape collection, musical instruments	sing, rap, listen to the topic/subject
		grammatical point
	Interpersonal	cooperative learning, peer tutoring, social gatherings	board games, props for role plays	teach, collaborate on, interact with respect to the topic/subject
	Intrapersonal	independent study, options in course of study, self-esteem building	self-checking materials, journals, materials for projects	connect the topic/subject to your personal life or make choices with regard it
				connect the
	Naturalist	nature study, care for animals	plants, animals, naturalists' tools	topic/subject to living things and
				natural phenomena

of continuous assessment mean and standard deviation between the experimental and control groups

	Groups	Experimental	Control
	Continuous assessment mean	13.783	12.633
	Continuous assessment standard	1.617	1.351
	deviation
	Number of students	30	30
	Figure 5  Comparison

of final exam score mean and standard deviation between the experimental and control groups

		Experimental	Control
	Final exam mean	13.733	12.266
	Final exam standard deviation	2.756	2.016
	Number of students	30	30
	Figure 6  Comparison

Appendix 1  Sample questions in each type of intelligence on the Midas test

Musical: Do you ever make up songs or write music? Kinesthetic: Do you enjoy working with your hands on projects such as mechanics, building things, preparing fancy food or sculpture? Logical: How are you at inventing "systems" for solving long or complicated problems? Spatial: Are you creative and like to invent projects, or often draw or sketch to explain an idea? Linguistic: Have you ever written a story, poetry, or words to songs? Interpersonal: Are you able to come up with unique or imaginative ways to solve problems between people or settle arguments? Intrapersonal: Have you ever been able to find unique or unusual ways to solve personal problems or achieve your goals? Natural: Have you taken photographs of nature or written stories or done artwork with/about nature?

Appendix 2  Sample report on a random student's Midas results "As you see, mathematical/logical intelligence is your strongest trait. Generally, it means that you are good at solving problems, finding solutions, and that you are good with figures and numbers. In an English classroom, that can mean that activities which involve numbers, or tasks that require you to experiment or think critically about a topic can aid your learning.

According to Howard Gardner, the scientist who proposed the theory of multiple intelligences, we are not born with a fixed degree of intelligence and die with it, i.e. intelligences can improve! We can all become smarter or more intelligent through the right practices and being exposed to the right kind of material. Also, all of us are intelligent in many different fields and domains, as you can see yourself in your results above.

Please note that no test of intelligence has yet been designed that can really estimate one's true intelligence. Therefore, this is mainly to help you know your own strengths, know yourself better, and be able to pick the best strategies for your own learning in different subjects and in different fields.

There is also a link provided on MIT in case you are interested to know more, or another link of a brief interview with Howard