Noemie Jourde-Chiche

Elizabeth Whalen

Bertrand Gondouin

Cate Speake

Vivian Gersuk

Bertrand Dussol

Stéphane Burtey

Virginia Pascual

Damien Chaussabel

Laurent Chiche

Noe ´mie Jourde-Chiche

Stephane Burtey

Noe ´ Mie Jourde-Chiche

Modular transcriptional repertoire analyses identify a blood neutrophil signature

Keywords: lupus nephritis, neutrophil, gene expression, systemic lupus erythematosus, biomarkers

Introduction

LN is a severe and frequent complication of SLE, affecting 2030% of patients in Europe [START_REF] Cervera | Systemic lupus erythematosus in Europe at the change of the millennium: lessons from the ''Euro-Lupus Project[END_REF], and with rates as high as 70% in other ethnicities [START_REF] Ferna ´ndez | A multiethnic, multicenter cohort of patients with systemic lupus erythematosus (SLE) as a model for the study of ethnic disparities in SLE[END_REF]. LN affects patients' survival [START_REF] Cervera | Morbidity and mortality in systemic lupus erythematosus during a 10year period: a comparison of early and late manifestations in a cohort of 1,000 patients[END_REF][START_REF] Thomas | Mortality associated with systemic lupus erythematosus in France assessed by multiple-cause-of-death analysis[END_REF], and severe forms lead to end-stage renal disease (ESRD) in 510% of patients after 10 years [START_REF] Houssiau | The 10-year follow-up data of the Euro-Lupus Nephritis Trial comparing low-dose and high-dose intravenous cyclophosphamide[END_REF][START_REF] Go ´mez-Puerta | Racial and ethnic differences in mortality and cardiovascular events among patients with end-stage renal disease due to lupus nephritis[END_REF]. LN prognosis is improved by early diagnosis and treatment [START_REF] Esdaile | The benefit of early treatment with immunosuppressive agents in lupus nephritis[END_REF], whereas diagnostic delay is associated with increased risk of ESRD [START_REF] Faurschou | Prognostic factors in lupus nephritis: diagnostic and therapeutic delay increases the risk of terminal renal failure[END_REF]. A kidney biopsy is the gold standard for assessing LN severity and guiding treatment [START_REF] Bertsias | Joint European League Against Rheumatism and European Renal AssociationEuropean Dialysis and Transplant Association (EULAR/ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis[END_REF][START_REF] Hahn | American College of Rheumatology guidelines for screening, treatment, and management of lupus nephritis[END_REF].

Classification of LN according to the current International Society of Nephrology/Renal Pathology Society (ISN/RPS) [START_REF] Weening | The classification of glomerulonephritis in systemic lupus erythematosus revisited[END_REF] allows stratification of patients between those with proliferative LN, who require immunosuppressive therapy [START_REF] Bertsias | Joint European League Against Rheumatism and European Renal AssociationEuropean Dialysis and Transplant Association (EULAR/ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis[END_REF][START_REF] Hahn | American College of Rheumatology guidelines for screening, treatment, and management of lupus nephritis[END_REF], and those with non-proliferative or chronic LN lesions. However, a kidney biopsy remains an invasive procedure [START_REF] Bataille | Comparative safety and efficiency of five percutaneous kidney biopsy approaches of native kidneys: a multicenter study[END_REF][START_REF] Dhaun | Utility of renal biopsy in the clinical management of renal disease[END_REF] that cannot be easily performed repeatedly [START_REF] Pagni | The value of repeat biopsy in the management of lupus nephritis: an international multicentre study in a large cohort of patients[END_REF], even less pre-emptively, although SLE immune-mediated renal injury can precede the detection of proteinuria [START_REF] Zabaleta-Lanz | Silent nephritis in systemic lupus erythematosus[END_REF]. Moreover, the response to treatment is unpredictable, and clinical remission is not always associated with resolution of histological activity in LN [START_REF] Malvar | Histologic versus clinical remission in proliferative lupus nephritis[END_REF]. Non-invasive biomarkers are therefore needed for identifying patients prone to LN, and for determining the severity and a prognosis for renal flares when they occur. To date, although some serological or urinary markers have been associated with LN occurrence or severity, none is sufficiently reliable for replacing a kidney biopsy, guiding treatment and predicting LN outcomes [1721].

Over the last decade, omics-based techniques have been used successfully to discover biomarkers in renal diseases [START_REF] He | Systems biology of kidney diseases[END_REF] and in SLE [START_REF] Chaussabel | A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus[END_REF][START_REF] Bennett | Interferon and granulopoiesis signatures in systemic lupus erythematosus blood[END_REF]. Modular transcriptional repertoire analysis [START_REF] Chiche | Current perspectives on systems immunology approaches to rheumatic diseases[END_REF][START_REF] Chaussabel | Democratizing systems immunology with modular transcriptional repertoire analyses[END_REF] of whole-blood samples, in particular, has revealed new aspects of the SLE IFN signature [START_REF] Chiche | Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures[END_REF]. The aim of this study was to use modular repertoire analyses to analyse the links between blood transcriptional signatures and LN.

Methods

Selection of patients and characterization

The study comprised 143 patients and controls overall. The 62 consecutive patients with SLE who fulfilled the 1997 ACR criteria were enrolled and followed-up prospectively at a French reference centre for autoimmune disease (Ho ˆpital de la Conception, Marseille, France). Blood was collected by peripheral venipuncture using Tempus tubes (3 ml) at inclusion and longitudinally at each follow-up visit, and distant from any infectious event. Complete clinical and biological evaluation of disease activity was performed, as well as pathological analysis of renal biopsies (see supplementary Methods, pathological analysis of renal biopsies section, available at Rheumatology Online).

Healthy controls (n = 21) matched for age, gender and ethnicity, were sampled once. Pathological controls (n = 40) comprised patients with crescentic GN caused by ANCA-associated vasculitis (AAV) (n = 10); patients with primitive glomerular diseases (n = 15) matched for renal parameters with patients with LN, and who were sampled once at the time of a renal biopsy; and patients with bacterial pneumonia who were sampled once at the time of infection (n = 15). Healthy controls of these pathological controls (n = 20) were also sampled once.

Blood samples from SLE patients were split into three groups, according to disease activity and renal involvement: biopsy-proven LN comprised samples collected at the time of a biopsy-proven LN flare with active lesions, whether they were proliferative (class III or IV ± V) or not (class II or V of the ISN/RPS 2003 classification). Patients showing only chronic lesions (class III-C or IV-C with no activity) were not included in this group. Extra-renal SLE flare comprised samples collected at the time of an extrarenal flare, with no sign of active LN. Quiescent lupus comprised samples from SLE patients at their first clinically quiescent visit, defined by the absence of flare or treatment modification in the 60 days prior to the visit and a SLEDAI of 44 (immunological activity authorized). In each group, the existence of a past history of LN was recorded and the patients were considered as ever renal if they had a past and/or a current history of LN and/or had developed LN during the follow-up.

RNA preparation and microarray hybridization RNA was processed as described elsewhere [START_REF] Chiche | Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures[END_REF] using Illumina beadchips (see supplementary Methods, RNA preparation and microarray hybridization section, available at Rheumatology Online). Data are deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO Series accession number GSE49454). PCR analyses were performed on the same samples using a Fluidigm Real-Time PCR platform (see supplementary Methods, PCR analyses section, available at Rheumatology Online). Publicly available blood and tissue gene-expression profiles were also used (see supplementary Methods, public domain datasets section, available at Rheumatology Online).

Modular transcriptional repertoire analyses

Analyses were performed using the second generation of a modular framework as previously described [START_REF] Chaussabel | A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus[END_REF][START_REF] Chaussabel | Democratizing systems immunology with modular transcriptional repertoire analyses[END_REF][START_REF] Chiche | Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures[END_REF] (see supplementary Methods, modular transcriptional repertoire analyses section, available at Rheumatology Online).

The level of regulation of each module was calculated as the per cent difference: % upregulated probes % downregulated probes. A module was considered active (upregulated) if the percentage difference was 520% and silenced (downregulated) if the percentage difference was À20% or less.

Statistical analyses

Numerical data were processed and analysed using R statistical software. For continuous data, comparisons between groups were conducted using analysis of variance (ANOVA) (assuming normality was appropriate) or the non-parametric KruskalWallis test. Student's t-test or Wilcoxon's test was conducted if further testing was needed to determine which group was different. Linear models were used to test for trend. For categorical variables, Fisher's exact test was used to determine differences in contingency tables, and the Chi-squared test to determine trends in proportions if the categorical variable was ordinal. Correlations were assessed by Pearson's (assuming normality was appropriate) or Spearman's correlation test.

For longitudinal analyses, a mixed model was used to determine the relationship between module activity variation and response to treatment in patients with proliferative LN who had at least three blood samples. This model comprised fixed effects of the group (responder vs nonresponder), time (as a continuous variable), and an interaction term between time and group, with a random effect for individual. Response was defined by complete or partial remission at M6 [START_REF] Gordon | European consensus statement on the terminology used in the management of lupus glomerulonephritis[END_REF].

Values of P < 0.05 were considered to indicate statistical significance, with adjustment by multiple-testing correction when needed (BenjaminiHochberg procedure).

Ethics and informed consent

Patients were included in the LUPUCE study: Estimate of the Activity and the Forecast of the Lupus Disease of the Adult by a Transcriptomic Score (NCT00920114): the design details are reported in [START_REF] Chiche | Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures[END_REF]. This study was conducted according to the principles expressed in the Declaration of Helsinki. The LUPUCE study and this study were approved in France by the Comite ´de Protection des Personnes Sud Me ´diterrane ´e 1 (IDRCB 2009-A00257-50) and in the USA by the Institutional Review Boards of the Baylor Institute of Immunology Research (IRB 011-173) and the Benaroya Research Institute (IRB 12085). Informed written consent was obtained from each patient and enrolled control prior to any study-related procedure.

Results

Characteristics of the SLE patients

The characteristics of the 62 enrolled SLE patients are detailed in supplementary Table S1, available at Rheumatology Online. The median age was 38 years, 85% of patients were women, and 89% were White. The median duration of SLE was 7.8 years. Data on 157 visits were collected. Twenty-four patients were sampled at the time of a renal biopsy and showed active LN that was either proliferative [n = 14, class III or IV-(A) or (A/C) ± class V] or non-proliferative (n = 10, class II, class V or interstitial nephritis). It was the first LN flare event for 13 and a relapse for 11 patients. Nephrotic syndrome was present in 14 patients and 9 had acute kidney injury. During a median follow-up of 42 months (range: 655), one patient died (sudden death) after the initiation of dialysis, two other patients reached ESRD, and the median serum creatinine at the last visit was 68 mmol/l (range: 431178). Detailed renal parameters of patients with active LN are provided in supplementary Table S1, available at Rheumatology Online. Eleven patients were sampled at the time of an extra-renal (cutaneous, articular and/or haematological) flare (supplementary Table S1, available at Rheumatology Online). Thirty-four samples were collected from SLE patients at the time of their first quiescent visit. Among them, 22 patients had a past history of LN (supplementary Table S1, available at Rheumatology Online).

Module repertoire analysis reveals a strong neutrophil signature in SLE At the group level, in addition to the previously reported IFN signature (upregulation of the three IFN-related modules M1.2, M3.4 and M5.12) [START_REF] Chiche | Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures[END_REF], modular repertoire analysis revealed strong upregulation of module M5.15 (Fig. 1). A similar upregulation pattern was also observed in three other SLE datasets generated in children and adults from various ethnicities (supplementary Fig. S1, available at Rheumatology Online). Module M5.15, annotated neutrophil, comprises 24 probes corresponding to 22 genes (supplementary Table S2, available at Rheumatology Online) strongly related to neutrophil functions (supplementary Fig. S2, available at Rheumatology Online) [29]. In this cohort of SLE patients, 20/24 (83%) probes belonging to M5.15 were upregulated, whereas no probe was downregulated compared with matched healthy controls (Fig. 1). At the individual level, M5.15 was active (520% difference) in 92 (59%) SLE samples, and 40/62 patients (65%) showed this modular neutrophil signature at least once during the follow-up (characteristics according to the presence of a modular neutrophil signature are detailed in supplementary Tables S3 andS4, available at Rheumatology Online, respectively).

Modular neutrophil signature and clinical or biological activity parameters of SLE

There was no significant correlation between IFN modules activity, either individually or as a combined modular IFN score, and M5.15 activity (Fig. 2). At the individual level, M5.15 activity was not correlated to age or ethnicity, nor with SLEDAI or the titre of anti-dsDNA (Table 1). M5.15 was associated with renal flares, independently of CS dose (Table 1). Samples with M5.15 activity were less likely to be from patients experiencing an isolated cutaneous or articular flare. Importantly, among the 62 modules tested, M5.15 was the module exhibiting the strongest association with active LN (adjusted P-values = 0.0014 after BenjaminiHochberg multiple-testing correction), whereas none of the three IFN modules (M1.2, M3.4, M5.12) showed a significant association (only M5.12 had a P-values of 0.007 before multiple-testing correction). M5.15 was moderately correlated with daily CS dose (r = 0.43, P < 0.001), independently of neutrophil count.

Modular neutrophil signature and severity of LN M5.15 activity was stronger in patients with active LN than in those with extra-renal flares (88% vs 17% probes upregulated, with no probe downregulated, respectively).

The activation of M5.15 was associated with some indicators of LN severity, such as serum-creatinine level, 24-h proteinuria and lower serum albumin (supplementary Table S3, available at Rheumatology Online). M5.15 activity was associated with the presence of acute kidney injury (P = 0.03) and inversely correlated with serum albumin (r = À0.30, P = 0.01), but not with the activity of urinary sediment. M5.15 at inclusion was correlated with serum creatinine at the last follow-up visit (r = 0.28, P < 0.001).

Among patients with active LN, median M5.15 activity was higher in those with proliferative than in those with non-proliferative LN (respectively, 66.7 vs 18.8%, P = 0.04). Yet, there was no correlation between M5.15 and pathological parameters such as the National Institutes of Health (NIH) activity and chronicity indices or tubulo-interstitial score, or with the percentage of activity or chronicity according to the ISN/RPS classification.

After the initiation of immunosuppressive therapy in patients with proliferative LN and 53 longitudinal samples (n = 10), a significant association was observed between the decrease in M5.15 activity and remission at M6 (P = 0.046) (Fig. 3). Conversely, there was no relation between IFN signature and response to treatment in these patients (data not shown).

The analysis of published transcriptomic data [START_REF] Berthier | Cross-species transcriptional network analysis defines shared inflammatory responses in murine and human lupus nephritis[END_REF], generated from glomeruli and tubulo-interstitium of micro-dissected kidneys from SLE patients with active LN, revealed that the expression of the genes LTF (lactotransferrin) and FIG. 1 Modular repertoire analyses at the group level in SLE patients Four modules were strongly upregulated in SLE patients (vs healthy controls): three INF-related modules (M1.2: 100% probes upregulated; M3.4: 90% probes upregulated; M5.12: 79% probes upregulated, with 0% probes downregulated) and M5.15 (83% probes upregulated, 0% downregulated). Other active modules (per cent difference 520%) were those related to inflammatory response (M3.2, M4.2, M4.6), cell cycle (M3.3), cell death (M6.13) and apoptosis/survival (M6.6). In addition, silencing of modules related to B cells (M4.10), T cells (M4.1 and M4.15), cytotoxicity/NK cells and protein synthesis (M4.3) was observed. MPO (myeloperoxidase), belonging to M5.15, was significantly upregulated compared with healthy renal tissue (supplementary Table S5, available at Rheumatology Online).

Modular neutrophil signature and potential confounding factors SLE patients with active LN, who displayed a strong neutrophil signature, also received higher doses of CSs and had worse renal function than others (supplementary Table S1, available at Rheumatology Online). We thus conducted additional analyses to confirm the link observed between the modular neutrophil signature and LN.

M5.15 activity was correlated with blood neutrophil count (r = 0.38, P < 0.001), but this correlation was not as strong as for different types of lymphocytes with their corresponding modules (supplementary Fig. S3, available at Rheumatology Online). M.15 activity could be high in some quiescent patients receiving low doses of CSs, and low in some patients receiving high doses of CSs (Fig. 4), which mirrors what was observed in a paediatric cohort (supplementary Fig. S4, available at Rheumatology Online). Among quiescent SLE patients with low and stable doses of CSs, M5.15 activity was higher in those with a past history of LN (50% vs 8% in those without, respectively) (Fig. 5A).

Modular repertoire analyses of SLE patients with active LN were compared with those of control patients from this study or from publicly available datasets (supplementary Table S6, available at Rheumatology Online). The modular neutrophil signature was shared by patients with bacterial sepsis and patients with AAV (Fig. 5B). Conversely, patients with primary glomerular diseases, matched for renal FIG. 2 Neutrophil signature is not correlated to IFN signature in SLE patients M5.15 activity was not correlated to (A) modular IFN signature (absent: 0, mild: 1, moderate: 2, strong: 3 active IFN modules) (r = 0.1, P = 0.2) or (B) activity of each IFN module individually (r = 0.06, P = 0.4 for M1.2; r = 0.15, P = 0.06 for M3.4 and r = 0.13, P = 0.09 for M5.12). Thirteen samples (from seven patients) had a strong modular neutrophil signature (M5.15 >50%) and an absent/mild IFN signature. function and levels of proteinuria, displayed no activation of M5.15. Of note, active LN and active tuberculosis both displayed an IFN signature (Fig. 5B) and the activation of modules related to inflammation, cell death and apoptosis. Silencing of modules related to B-cells, T cells and cytotoxicity/NK cells were common to patients with SLE, tuberculosis, AAV or bacterial sepsis.

Additionally, we tested the association of smoking status with M5.15 activity. There was no correlation between M5.15 activity and smoking in SLE patients, and Among the nine other patients, only two eventually developed LN (class IV, after 12 and 33 months). In quiescent patients, 10/16 with a modular neutrophil signature had a past history of LN, and 4/16 subsequently developed a LN flare (class IV after 18, 21 and 36 months; class V after 13 months). Overall, the presence of a modular neutrophil signature at least once during the follow-up was associated with past, current or future LN (82.5% of ever renal patients vs 54.5% of patients who never presented LN, P = 0.039) (supplementary Table S4, available at Rheumatology Online).

PCR validation and neutrophil score

To validate the microarray data, we confirmed the overexpression of four genes (DEFA4, ELANE, CEACAM6, CEACAM8) belonging to M5.15, using whole-blood RT-PCR in the same SLE samples. There were excellent correlations between microarray and TaqMan assays across the samples (supplementary Fig. S5, available at Rheumatology Online). A PCR neutrophil score was calculated, corresponding to the mean of the log2 (fold change) values of SLE samples compared with matched healthy controls for these four genes. This score correlated well with global M5.15 activity, with a 93% accuracy in predicting the presence of a modular neutrophil signature (supplementary Fig. S6, available at Rheumatology Online).

Discussion

Over the last decade, omics-based techniques (e.g. genomics, transcriptomics and proteomics) have extended our understanding of the molecular basis of SLE and provided candidate biomarkers for disease prognosis and response to treatment [2325]. In this study, conducted in an adult European cohort of SLE patients recruited in Nephrology and Internal Medicine, two-thirds of patients displayed a modular neutrophil signature at least once during the study period. Importantly, a comparable signature was observed in independent cohorts of paediatric patients or adult patients with different ethnic backgrounds. More importantly, we have shown, for the first time, a strong association between this modular FIG. 4 M5.15 activity according to CS dose in SLE patients (n = 157 visits) neutrophil signature (i.e. module M5.15 activity) and the occurrence, severity and prognosis of LN. Strikingly, the modular neutrophil signature was not correlated with markers for global disease activity, such as SLEDAI or anti-dsDNA antibody titres, or to the IFN signature of SLE. However, the association between M15.5 and LN was reinforced by further analyses. First, the proportion of patients with M5.15 activation was higher in patients with LN than in those with extra-renal SLE flares. Second, among patients with biopsy-proven active LN, M5.15 activity was stronger among those with proliferative (vs non-proliferative) nephritis, and M5.15 was associated with various indicators of LN severity, including serum-creatinine level (at diagnosis and at last follow-up), 24-h proteinuria, serum albumin and acute kidney injury. Third, an association was observed between the decrease in M5.15 activity and remission at M6 after the initiation of immunosuppressive therapy in patients with proliferative LN and who were followed longitudinally.

Finally, some of the neutrophil-specific transcripts belonging to the M5.15 module were also identified as overexpressed in the kidney tissues of SLE patients [START_REF] Berthier | Cross-species transcriptional network analysis defines shared inflammatory responses in murine and human lupus nephritis[END_REF]. These observations are in accordance with a previous report by Bennett et al. [START_REF] Bennett | Interferon and granulopoiesis signatures in systemic lupus erythematosus blood[END_REF], where a neutrophil signature had been initially reported in 25/30 (83%) children, of which 18 (60%) had LN. Altogether, these results suggest FIG. 5 Modular repertoire analysis in various groups of SLE patients and in control patients (A) Modular repertoire analysis of SLE patients, stratified according to renal activity/involvement. In contrast to the IFN signature, which was present in all SLE groups, a modular neutrophil signature was present in patients with active LN (1) and in quiescent patients (2), particularly those with a past history of LN (3). (B) Modular repertoire analysis of patients with LN and other conditions, with or without renal involvement. Active LN (1) and tuberculosis (2) shared an IFN signature and clustered together (hierarchical clustering), ANCA-associated vasculitis (3) and bacterial sepsis (4) shared the neutrophil signature of SLE and clustered together, while other renal diseases (5) displayed no particular modular signature.

that the modular neutrophil signature could be associated with severe manifestations of SLE (i.e. LN) as well as with the severity of such manifestations (i.e. proliferative LN and/or renal severity markers), providing a non-invasive marker for monitoring patients with SLE.

As patients with LN had both increased doses of CSs and worse renal function, complementary analyses were performed to investigate these potential confounding factors. Indeed, although M5.15 was associated with LN independently of CS dose, CSs are known to promote demargination of circulating neutrophils [START_REF] Nakagawa | Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes[END_REF], and could increase neutrophil gene expression in whole blood because of mere neutrophilia. Yet, conversely to some cell counts that were strongly correlated to corresponding modules, M5.15 was only moderately correlated with neutrophil count. This suggests that M5.15 reflects a functional activation of neutrophils. Of note, such a neutrophil signature was first reported in SLE and AAV in studies using peripheral blood mononuclear cells, and was attributed to low-density granulocytes, a distinct class of neutrophils migrating with the peripheral blood mononuclear cell fraction [START_REF] Bennett | Interferon and granulopoiesis signatures in systemic lupus erythematosus blood[END_REF][START_REF] Nakagawa | Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes[END_REF][START_REF] Grayson | Rituximab in ANCA-Associated Vasculitis-Immune Tolerance Network Research Group et al. Neutrophil-related gene expression and low-density granulocytes associated with disease activity and response to treatment in antineutrophil cytoplasmic antibody-associated vasculitis[END_REF]. Also, some SLE patients receiving no or low-dose steroids had high M5.15 activity, and others receiving high-dose steroids displayed no neutrophil signature. This supports M5.15 activation being a reflection of an inflammatory response in some SLE patients with an aggressive disease. We did not observe a modular neutrophil signature in patients with various glomerular diseases that were matched for renal function and proteinuria; this excludes the possibility that M5.15 activation could be a consequence of acute kidney injury or nephrotic syndrome. Additionally, although cigarette smoking can induce neutrophilia and neutrophil activation in lungs [START_REF] Alexander | Inflammatory diseases of the lung induced by conventional cigarette smoke: a review[END_REF], there was no association of blood M5.15 activity with smoking status in patients with SLE.

Interestingly, we also observed a modular neutrophil signature in quiescent SLE patients, including patients with absent or low modular IFN signatures. In addition, most quiescent patients displaying this signature had a past history of LN or had developed LN during the follow-up period. Overall, this suggests that M5.15 activity could allow the identification/stratification of SLE patients who are at risk for LN. Interestingly, an enrichment for a granulocyte signature in patients developing LN was also observed in a large longitudinal cohort of paediatric patients with SLE [START_REF] Banchereau | Personalized immunomonitoring uncovers molecular networks that stratify lupus patients[END_REF]. This finding is of the utmost importance, as LN does not occur in all SLE patients and, although genetic, hormonal and ethnic backgrounds are risk factors for LN, no clinical or biological parameter can currently identify patients at high risk of developing LN [START_REF] Ntatsaki | Risk factors for renal disease in systemic lupus erythematosus and their clinical implications[END_REF]. Finally, these observations further support a pathogenic role for neutrophils in the severity of immune dysregulation observed in adult SLE patients, in particular those with LN.

The understanding of the potential role of neutrophils in SLE and LN has expanded with the discovery of neutrophil extracellular traps (NETs) [START_REF] Brinkmann | Neutrophil extracellular traps kill bacteria[END_REF]. NETs are fibrous networks of DNA and antimicrobial factors that are released by dying neutrophils to trap and kill pathogens, but can also lead to tissue damage [START_REF] Villanueva | Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus[END_REF]. NETs are also involved in the pathogenesis of many autoimmune diseases, including SLE and AAV [3744], and the impaired degradation of NETs is associated with LN in SLE patients [START_REF] Hakkim | Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis[END_REF]. In our study, we observed a similar modular neutrophil signature in SLE patients with active LN and in patients with AAV or bacterial sepsis. Indeed, 8 of the 11 genes identified in a recent publication as correlated with AAV disease activity are included in M5.15 [START_REF] Grayson | Rituximab in ANCA-Associated Vasculitis-Immune Tolerance Network Research Group et al. Neutrophil-related gene expression and low-density granulocytes associated with disease activity and response to treatment in antineutrophil cytoplasmic antibody-associated vasculitis[END_REF]. Genes belonging to M5.15 include several genes implicated in early granulopoiesis, including MPO, neutrophil elastase (ELANE), cathepsin G (CTSG), defensin A4 (DEFA4) and LTF. Most of these genes have established roles in neutrophil maturation and NETs formation (including DEFA4, CTSG and ELANE). In particular, DEFA4 participates in immune-mediated tissue damage consecutive to autoantibody deposition in SLE [START_REF] Villanueva | Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus[END_REF]. Furthermore, both DEFA4 and CTSG (a serine protease that is also released by neutrophils during NETs formation) are expressed at higher levels in SLE patients with an active disease [START_REF] Sthoeger | High alpha-defensin levels in patients with systemic lupus erythematosus[END_REF].

This study has several strengths. Namely, samples were collected together with careful recording of clinical and laboratory data. In addition, active LN was systematically documented by a renal biopsy and classified by an expert pathologist according to the ISN/RPS [START_REF] Weening | The classification of glomerulonephritis in systemic lupus erythematosus revisited[END_REF]. Access to various control conditions allowed us to validate our findings across independent datasets.

This study also has limitations. First, the absence of correlation between M5.15 activity and pathological markers of activity implies that blood modular analysis is not meant to replace renal biopsy, and M5.15 activity is to be considered as a risk marker of LN, but not a diagnostic pathological tool. Then, the use of whole-blood samples prevented investigation of the exact cellular source of the neutrophil signature observed in SLE patients, and no functional assay on neutrophil functions was conducted. However, this study was mainly dedicated to biomarker discovery. PCR validation of the microarray results, which showed very good correlation with the four-gene PCR neutrophil score, suggests a possible translation into clinical practice. Finally, the limited number of longitudinal samples warrants further studies for confirming the prognostic value of the modular neutrophil signature in the progression of quiescent patients to LN and in the monitoring of responses to treatment in patients with LN.

After the discovery of an IFN signature in SLE a decade ago, immune dysregulation of SLE has often been compared with a chronic virus-like response, and is also quite close to the IFN signature observed during infection with intracellular bacteria, such as tuberculosis [START_REF] Berry | An interferoninducible neutrophil-driven blood transcriptional signature in human tuberculosis[END_REF]. Alternatively, the strong neutrophil signature observed in SLE patients with LN, as in patients with AAV but also in those with severe bacterial sepsis, demonstrates that a bacterial-like immune response may also be involved in SLE pathogenesis, especially in patients with severe organ involvement. A practical consequence of these observations is that these various biomarker signatures are not specific to SLE and should be assessed at a distance from infectious events.

Collectively, our results suggest that the presence of a modular neutrophil signature is associated with a more aggressive course of SLE with the occurrence of LN, and could be associated with LN severity and prognosis. Interestingly, simultaneous assessment of both modular IFN and neutrophil signatures could allow a molecular stratification of patients with various inflammatory conditions and identify pathways to be targeted for treatment.

FIG. 3

 3 FIG. 3 M5.15 activity over time in patients with proliferative LN

TABLE 1

 1 Correlation of M5.15 activity with SLE patients' characteristics and disease-activity parameters

		Correlation		p-values adjusted
		coefficient	p-values	for neutrophil count
	Characteristics	(Spearman)	(Spearman)	and steroid use
	Age	À0.037	0.647	0.314
	SELENA-SLEDAI	0.136	0.089	0.862
	Anti-dsDNA titre	À0.054	0.514	0.346
	Neutrophil count	0.376	2.27 Â 10 À6	8.01 Â 10 À5
	Daily CS dose	0.427	2.38 Â 10 À8	0.0042
			Wilcox test	p-values adjusted for neutrophil
			p-values	count and steroid use
	Gender, male		4.16 Â 10 À5	8.88 Â 10 À5
	Ethnicity		0.866	0.914
	Cutaneous flare		0.029	0.0015
	Articular flare		0.0081	0.0007
	Haematological flare		0.897	0.992
	Renal flare		2.33 Â 10 À5	0.026

SELENA: Safety of Estrogens in Lupus Erythematosis National Assessment trial. Values highlighted in bold are for adjusted p < 0.05. smoking status was not significantly different between SLE groups. Modular neutrophil signature and prognosis of LN IFN signature could not differentiate between patients with active LN and those with extra-renal flares or quiescent SLE. Conversely, M5.15 activation (M5.15 520%) was observed, respectively, in 16/24 (67%), 2/11 (18%) and 16/34 (47%) within these three groups (Fisher's exact test: P = 0.027). Interestingly, of the two patients with an extra-renal SLE flare and a modular neutrophil signature, one had a past history of LN and the other subsequently developed LN (class III, after 24 months).

Acknowledgements

We thank Scott Presnell and Peter Lindsay (Systems Immunology Department, BRI) for their help in this study.

Funding

: Appel d'Offres de Recherche CliniqueAssistance Publique Ho ˆpitaux de Marseille; Centre de Recherche en Ne ´phrologie (La Conception, Marseille). Disclosure statement: The authors have declared no conflicts of interest.

Supplementary data

Supplementary data are available at Rheumatology Online.


Lazare S Sidjui

Kenneth O Eyong

Kenneth G Hull

Gabriel N Folefoc

Valérie Leddet

Gaëtan Herbette

Evelyne Ollivier

Joseph J Taube

Kevin Klausmeyer

Daniel Romo

Valerie M Leddet

 Gaёtanherbette

∥ Evelyneollivier

Bioactive Seco-Lanostane-Type Triterpenoids from the Roots of Leplaea mayombensis

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

However, recent morphological studies show that the group is distinctfrom the South-American members of Guarea and deserves generic status. Therefore, the genus Leplaeahas been reinstated to accommodate five of the species formerly placed in Guarea.These species are:Leplaealaurentii(De Wild.) E. J. M. Koenen& J.J. de Wilde, Leplaeathompsonii(Sprague & Hutch.) E. J. M. Koenen& J.J. de Wilde, LeplaeaadenopunctataE. J. M. Koenen& J.J. de Wilde, Leplaeacedrata(A. Chev.) E. J. M. Koenen& J.J. de Wilde,andLeplaeamayombensis(Pellegr.)

Staner. 1 Leplaeamayombensis is distributed in Gabon, West Cameroon, Congo, D.R. Congo (Zaire) and was first identified in 2010. 2 This plant is used in African folk medicine forthe treatment of cancer and microbial infections. Phytochemical studies on this speciesreveal that triterpenoids are its main constituents. 3 Triterpenoids are the C-30 class of natural products derived from squalene. The biosynthetic origin of most triterpenoids lies in cascade cyclizations and rearrangements of the acyclic precursors squalene (S) and 2,3-oxidosqualene (OS), leading to thetetra-and pentacyclictriterpenoidskeletons. 4,5,6,7 Lanostanes aretetracyclic triterpenoids and are the principal constituents in some genera such as the genus KadsuraJuss., e.gK. coccinea(Lem.) A.C. Sm.

andgenus AbiesL. such asA. balsamea(L.) Mill.Several of these lanostanetriterpenoids have potential anti-HIV, anticancer, and cholesterol biosynthesis inhibitory activities. 8 For example, schisanlactone E and changnanic acid isolated from K. longipedunculatashowedantiproliferative effects against murine leukemia P388 cells, 9 and ananosic acids B and C isolated from K. ananosmaexhibited cytotoxicity against human CCRF-CEM cells and HeLa cells. 10 In the present study,the isolation and structure elucidation of three new lanostane-type triterpenoids (1-3, Figure 1) from the air dried roots of L. mayombensisare reported.Derivatives, 4, 11,and 12 were prepared by esterification and amidation of compound 1for SAR studies.

RESULTS AND DISCUSSION

The CH 2 Cl 2 -MeOH (1:1) extract of the air-dried roots of L. mayombensis was partitioned with hexanes, EtOAc, and n-BuOH successively. Three new lanostane-type triterpenoids1-3 and six known compounds, 5-10(Figure 4) were obtained from the EtOAc fraction through a series of column chromatographic separations.Esterification and amidationof compound 1 afforded its dimethyl ester4, monoamide11, and diamide12 for structure-activity relationship (SAR) studies (Scheme 1). The lupeol-type structure was however discarded based on the indices of hydrogen deficiency. The 1 H and 13 C NMR data were assigned using 1 H- ( 1.80). Collectively, the NMR data supported the assigned structure of compound 1 and the connectivity and relative configuration were unambiguously confirmed using single crystal Xray diffraction 12 (Figure 2).Compound 1 was thus defined as (5, 9, 10, 13, 14, 17, 20)-3,4-secolanostan-4(28),7,24-triene-3,21-dioic acid, a new seco-lanostane-typetriterpenoidand named, leplaeric acid A.In an attempt to unambiguously assign the absolute configuration of leplaeric acid(1)by X-ray analysis,p-bromobenzamides11 and 12possessing a heavy atomwere prepared (Scheme 1). However, none of the crystals obtained of these derivativeswere of X-ray quality. ) and an ester (1739cm -1 )group.

The 1 H NMR (600 MHz, CDCl 3 ) and 13 C NMR (150MHz, CDCl 3 ) spectra were similar to compound 1 except for the presence of a three-proton singlet at  H 3.60 attributed to an estergroup. The HMBC spectrum shows correlations between H 2 -2 protons ( 2.39, td, J=6.0,12.0Hz and 2.19) andMeO-31 at 51.5 indicating that the methoxy group is at C-3.

Theshielding of H 2 -2 as compared to compound 1 (2.62 and 2.42) indicatesa difference in its chemical environment.The 13 C NMR spectrum of compound 2 indicates a 3-methoxycarbonyl (174.4)as compared to a 3-hydroxycarbonyl function( 181.3) in compound 1. On the basis of these 1D and 2D spectroscopicvalues (Table 1), the structure of compound 2, leplaeric acid B, was assigned as the 3-methyl ester ofleplaeric acid A (Figure 1). ( H 1.11, s) and Me-29 (1.04, s) respectively.Compound 3 also exhibited differences from compound 1 in the 13 CNMR spectrum with the presence of a carbonyl group ( C 216.9)indicative of a ketone and the absence of the terminal olefinic moiety 4(28) ( C 147.5, 114.1). In the HMBCspectrum,correlations of H 2 -2 (2.76,2.32), H 2 -1 (1.97 and 1.45) with the C-3 ( 216.9) carbonyl groupwereobserved. AditionalHMBC correlations were also observed between the methyl protons H 3 -28 ( 1.11) and H 3 -29 (1.04)with C-3( 216.9), confirming the position of the carbonyl group of leplaeric acid C(Figures1 and 3).It is likely that leplaeric acidsA (1) and C (3) are derived biosynthetically from the same lanosterol precursor. Oxidation of the C-3 hydroxy group of the lanosterol would give rise to the C-3 carbonyl in ketone3, while an oxidosqualenecyclase-catalyzed Grob fragmentation of the A-ring 13 of the sterol precursor yields an intermediate that would provideleplaeric acid A (1) after additional oxidation and olefin isomerization reactions. To obtain derivatives for structure-activity relationship (SAR) analysis, ester derivatives2 and 4 wereprepared fromleplaeric acid A(1) using diazomethane generated in situ from 1methyl-3-nitro-1-nitrosoguanidine (MNG) and amidation of1with 4-bromobenzylamine provided amide derivatives11 and 12(Scheme 1).

Scheme 1. Diazomethane esterification and amidationof compound 1.

Treatment of compound 1with 2 equivalents of diazomethane 14 afforded compound 2 in a biomimetic-type transformation. When excess diazomethane(7 equiv.)was used, the dimethyl diester4was produced. Synthesized compound 2 was identical withthe natural productby comparing their TLC R f values and NMR spectroscopic data.

Compound 4 was purified as a white amorphous compound from a silica gel column eluting with 95% hexanes-EtOAc from the diazomethane reaction of compound 1. Its IR spectrum shows an intense peak for an ester group at 1733 cm -1 . Its 1 H NMR (600 MHz, CDCl 3 ) and 13 C NMR (150MHz, CDCl 3 ) spectra were similar to compound 1 except for the presence of two 11 methoxyproton resonances at 3.60(s, OMe) and 3.59(s, OMe)in the 1 H NMR spectrum and at 51.1(OMe),174.0 and176.5(two -COOMe groups) in the 13 C NMR spectrum (Table 1). These results were supported by the HRESIMS (m/z 499.3782 [M+H] + ) for C 32 H 50 O 4 .Based on spectroscopic analysis, compound 4 was determined to be3, 21-dimethyl-leplaeric ester.Compound 1 was treated with thionyl chloride followed by 2 equiv. of 4bromobenzylamine in a two pot reaction to afford compounds11 and 12 in a 1:3 ratio that was purified on a silica gel column eluting with 30% (12) and 45% (11) hexanes-EtOAc respectively.

In addition to the lanostane derivatives, five known compounds were also isolated and included: andirobin 15 (5), 1,2-dihydroandirobin 15 (6), -sitosterol 16 (7), stigmasterol 16 (8), αspinosterol 17 (9),and sigmoidin B 18 (10) (Figure 4). viability of the MDAMB231 breast cancer cell line exposed to triterpenoids at multiple concentrations was assayed. For this, MDAMB231 cells were plated in a 96 well format. The indicated compounds, dissolved in DMSO were serially diluted in culture media prior to addition to the culture. After a 3-day incubation time, relative viability was determined indirectly through the measurement of total ATP in each well using CellTiter-Glo (Promega, Figure 5). Following this assay, the IC 50 value was determined with 95% confidence interval (Table 2).The methyl esters 2 and 4 were more active than the diacidagainst the MDA MB 231 breast cancer cell line which may be due to greater cell permeability. The methylesters 2 and 4 were equally more active than the amides 11 and 12. Additionally, a significant loss in activity of the diamide12 compared to diacid1 was observed. For the determination of cytotoxicity, cells were plated in 96-well plates (Nest Scientific) at a density of 2000 cells per well in 100 μL of media per well. On the following day, the media was aspirated, and 100 μL of media in which a serial dilution of DMSO-dissolved compound (or DMSO as a control) was added to wells in triplicate. Three days later, the relative number of metabolically active cells was determined by addition of the CellTiter-Glo reagent (Promega)

and measurement of chemiluminesence according to the manufacturer's protocolusing a Fluoroskan Ascent FL (Thermo Scientific). To calculate the percent viability, the signal from background wells (media only) was subtracted from each well and then the remaining chemiluminesence value was normalized to the signal from wells with the equivalent amounts of DMSO. Graphing was done using GraphPad Prism 6 (GraphPad Software). IC 50 values were calculated by fitting a non-linear curve using the "log-inhibitor vs normalized response" function given by the equation Y=100/{1+10 [(X-LogIC50)] }.

Leplaeric acidA(1): White micro-crystallinepowder obtained from 70% hexanes-EtOAc.

Compounds 1 , 2 , 4 ,

 124 and 11 possess modest cytotoxicity against MDA MB 231 breast cancer cell lines.

Figure 1 .

 1 Figure 1. Lanostane-typetriterpenoids fromL. mayombensis.

Figure 2 .

 2 Figure 2. ORTEP drawing (left) and main HMBC correlations of 1 (right).

  Compound 3was obtained as a white powder from a silica gel column chromatography eluting with 82% hexanes-EtOAc. Its IR spectrum indicated the presence of two carbonyl groups(1704 and 1654 cm -1 ) and it has a melting point range of 274-276 o C.The HRESIMSshowed (m/z 455.3427 [M+H] + ) and a molecular formula of C 30 H 46 O 3 . It is soluble in CH 2 Cl 2 and stains on TLC with iodine or H 2 SO 4. The 1 H NMR (600 MHz, CDCl 3 ) spectrumof compound 3was similar to compound 1 except for the absence of the terminal double bond protons H 2 -28 ( H 4.83), thevinylic Me-29(1.80), and the presence of two methyl groupsMe-28

Figure 3 .

 3 Figure 3. Main HMBC correlations of ring A in 3.

Figure 4 :

 4 Figure 4: Known compounds from the EtOAc extract of Leplaeamayombensis.

Figure 5 .

 5 Figure 5. Triterpenoids1, 2 and 4 have modest cytotoxic activity against MDAMB231 breast

  (1): R 1 = R 2 = H Leplaeric acid B (2): R 1 = Me, R 2 = H H

  Compound 1 was obtained as a white powder from silica gel column chromatography eluting with 30% hexanes-EtOAc. It is soluble in CH 2 Cl 2 , stains on TLC with iodine or H 2 SO 4 , melts at 160

o Cand has an

[]

Table 1 . 1 H and 13 C NMR Data (600 and 150 MHz) of Compounds 1-4, 11-12 ( H , CDCl 3 , J in Hz)

 1

	1		2		3			4		11	12
	1 H	13 C	1 H	13 C	1 H	13 C	1 H		13 C	1 H	1 H
	1 2.11(1H, m)	27.8 2.00(1H, m)	27.3	1.97(IH, m)	38.7	2.00(1H, m)		26.1	2.11(1H, m)	2.11(1H, m)
	1.33(1H,m)	1.33 (1H,m)		1.45(1H, m)		1.33(1H,m)			1.33(1H,m)	1.33(1H,m)
	2 2.62 (1H, m)	27.6 2.39(1H,td, J=12.0,	28.1	2.76(1H, m)	35.0	2.37 (1H, m)		27.1	2.48 (1H, m)	2.48 (1H, m)
	2.42(1,H, m)	6.0Hz) 2.25(1H, m)		2.32(1H, m)		2.11(1,H, m)			2.42(1,H, m)	2.42(1,H, m)
	3	181.3		174.4		216.9			174.0
	4	147.5		147.3		35.2			147.3
	5 2.00(1H, m) 50.0 2.09 (1H, m)	49.9	1.72 (1H, t, J=6Hz) 52.5	2.00 (1H, m)		49.9	2.00(1H, m)	2.00(1H, m)
	6 2.25(1H, m)	30.2 2.23(1H, m)	30.2	2.10(2H, m)	24.6	2.12(1H, m)		30.2	2.25(1H, m)	2.25(1H, m)
	2.02 (1H, m)	2.05(1H, m)				1.99(1H, m)			2.02 (1H, m)	2.02 (1H, m)
	7 5.26(1H, m) 117.9 5.28 (1H, m)	118.3 5.31 (1H, m)	118.4 5.18(1H,t, J=6Hz) 118.2	5.26(1H, m)	5.26(1H, m)
	8	145.7		145.8		145.6			145.9
	9 2.60(1H, m) 40.9 2.48(1H, m)	40.6	2.04 (1H, t, J=6Hz) 50.0	2.45(1H,m)		40.6	2.48(1H, m)	2.48(1H, m)
		43.3		43.3		43.5			43.1
	1.55(1H, m)	18.1 1.55(1H, m)	18.1	1.53 (2H, m)	18.0	1.53 (2H,m)		17.9	1.55(1H, m)	1.55(1H, m)
	1.35(1H, m)	1.45(1H, m)							1.35(1H, m)	1.35(1H, m)
	1.52(2H, m) 33.5 1.55(2H, m)	33.8	1.55(2H, m)	33.7	1.57(2H,m)		33.8	1.52(2H, m)	1.52(2H, m)
		36.4		36.9		35.0			36.8
		51.4		51.3		51.1			51.6
	1.50(2H, m) 32.3 1.56(2H, m)	32.2	1.56(2H, m)	32.5	1.57 (2H,m)		32.4	1.50(2H, m)	1.50(2H, m)
	1.45 (2H, m) 30.5 1.75(1H, m)	30.0	1.75(1H, m)	30.2	1.46 (2H,m)		30.1	1.45 (2H, m)	1.45 (2H, m)
		1.55(1H, m)		1.45(1H, m)
	2.27(1H, m) 48.8 2.33 (1H,td,J=12.0,	47.3	2.29 (IH, m)	48.3	2.25 (1H,td,		47.3	2.27(1H, m)	2.27(1H, m)
		6.0Hz)					J=6..0;12.0Hz)
	0.88(3H, s)	16.2 0.84(3H, s)	15.8	0.95(3H, s)	12.9	0.75(3H,s)		15.9	0.88(3H, s)	0.88(3H, s)
	1.01 (3H, s)	27.0 1.01(3H, s)	27.4	1.01(3H, s)	27.5	0.91(3H,s)		27.4	1.01 (3H, s)	1.01 (3H, s)
	2.48(1H, m) 50.4 2.44(1H, m)	49.3	2.32 (1H, m)	47.3	2.35(1H, m)		49.4	2.41(1H, m)	2.41(1H, m)
		183.4		182.4		181.4			176.5
	1.60(2H, m) 32.0 1.62(2H, m)	31.9	1.94 (1H, m)	27.3	1.57(1H, m)		31.8	1.60(2H, m)	1.60(2H, m)
					1.34 (1H, m)		1.23(1H,m)
	2.09(1H,m)	26.0 2.00(2H, m)	26.0	1.99(2H, m)	26.2	1.82(2H,m)		26.1	2.09(1H,m)	2.09(1H,m)
	1.99(1H, m)									1.99(1H, m)	1.99(1H, m)
	5.12 (1H, m) 123.4 5.12 (1H, m)	123.5 5.10 (1H, m)	123.7 5.01(1H, t)		123.9	5.07 (1H, m)	5.07 (1H, m)
		132.4		132.3		132.5			132.1
	1.68(3H, d,	25.7 1.70(3H, d, J=1.3Hz ) 25.7	1.68(3H, s)	25.8	1.50(3H,s)		25.7	1.68(3H, s)	1.68(3H, s)
	J=1.3Hz )
	1.61(3H, d,	17.7 1.60(3H, d, J=1.3Hz ) 17.6	1.58 (3H, s)	17.8	1.61(3H,s)		17.6	1.61(3H, s)	1.61(3H, s)
	J=1.3Hz )
	4.83(2H, m) 114.1 4.86 (1H, m)	113.9 1.11 (3H, s)	21.7	4.76	(1H,d,	113.7	4.83(2H, d, J=12.0	4.83(2H, d, J=12.0
		4.81 (1H, d, J=2.1Hz)				J=6Hz)			Hz)	Hz)
							4.72	(1H,d,
							J=6Hz)
	1.80 (3H, m) 21.7 1.80 (3H, m)	22.5	1.04 (3H, s)	24.7	1.71(3H,s)		22.3	1.80 (3H, m)	1.80 (3H, m)
	0.92(3H, s)	21.4 0.95(3H, s)	21.8	0.89(3H, s)	21.8	0.82(3H, s)		21.9	0.92(3H, s)	0.92(3H, s)
		3.61(3H, s)	51.5			3.60(s, OMe)	51.1	7.48(2H, t, J=6Hz ),	7.49(2H, t, J=6Hz ),
										7.25(2H, d, J=12Hz),	7.25(2H, d,J=12Hz),
										5.71(NH, t, J=6Hz),	5.71(NH,t, J=6Hz),
										4.58 (1H, dd, J= 12.0	4.58 (1H, dd, J=
										6.0Hz) 4.29 (1H, dd,	12.0 6.0Hz) 4.29
										J= 12.0 6.0Hz) for 4-	(1H, dd, J=12.0
										bromobenzylamide	6.0Hz) for 4-
											bromobenzylamide
							3.59(s, OMe)	51.1		7.49(2H, t, J=6Hz ),
											7.19(2H, d,J=12Hz),

Table 2 . IC 50 Values Calculated from Scheme 1 and Figure 4for the Indicated Compounds Toward MDAMB231 Breast Cancer Cells after 3 Day Exposure.

 2

		1	2	4	8	11
	IC 50 (μM)	127	55.0	63.5	564	140
	95% Confidence Intervals (μM)	102 to 158	47.5-63.8 51.5-78.3	409-779	107-183
	EXPERIMENTAL SECTION
	General Experimental Procedures.Melting points were measured on a MEL-TEMP R
	apparatus. Optical rotations were determined using a Rudolph Autopol IV polarimeter. IR
	spectra were recorded on a BrukerTensor 27 Spectrometer. 1D ( 1 H, 13 C and DEPT) and 2D ( 1 H-
	1 H COSY, HSQC, HSQC-TOCSY, HMBC, TOCSY, NOESY, and ROESY) NMR spectra were
	recorded on a Bruker Ascend 600 ( 1 H 600 MHz and 13 C 150 MHz) NMR spectrometer equipped
	with a 5 mm cryoprobe. 1 H NMR chemical shifts are reported as δ values in ppm relative to
	CDCl 3 (7.26 ppm,), coupling constants (J) are reported in Hertz (Hz), and multipilicity follows
	convention. Unless indicated otherwise, CDCl 3 served as an internal standard (77.2 ppm) for all
	13 C spectra. The chemical shifts are quoted relative to TMS. X-ray data were recorded on a
	Bruker400 Machine.HRESIMS data were aquired on a Thermo LTQ Orbitrap mass spectrometer

  Soluble in CH 2 Cl 2 , mp 160 o C, IR  max 2953, 1702, 1449 cm -1 , and has an [] 20 D -3.4 (c 2.0, CHCl 3 ). 1 H and 13 C NMR data, see Table 1; HRESI (m/z 471.3468 [M+H] + ) for C 30 H 46 O 4 . Leplaeric acid B(2): white micro-crystalline powder from 88% hexanes-AcOEt. Soluble in CH 2 Cl 2 , mp 174 o C, IR  max 1700, 1739 cm -1 , and has an [] 20 D +21.7 (c 0.35, CHCl 3 ). 1 H and 13 C NMR data, see Table 1; HRESIMS (m/z 485.3627 [M+H] + ) for C 31 H 48 O 4 .

Table of Contents/Abstract Graphic:

 of

			R 2 O 2 C
	R 1 O 2 C	2	1
			H
			1

ACKNOWLEDGEMENTS

The authors would like to acknowledge the Council for International Exchange of Scholars (CIES) for a J. William Fulbright Visiting Scholar award to Dr. Kenneth Eyong. The Molecular Biosciences Center, the CPRIT Synthesis and Drug Lead Discovery Laboratory, (supported by the Cancer Prevention and Research Institue of TexasR1309 and Baylor University, College of Arts and Sciences), the Department of Chemistry and Biochemistry, and the Department of Biology atBaylor University and Baylor University, Waco-Texas for infrastructure and financial support.The University of Yaounde I and the Government of Cameroon are acknowledged for financial support through the Fondsd'Appuis a la Recherche.

with ESI direct infusion and a Bruker Amazon SL for LRMS. Flash column chromatography was performed using 60Å Silica Gel (200-300 mesh). Sample Collection.L. mayombensis fruits and roots were harvested in Yaoundé-Cameroon, (October, 2014) and identified by Mr. Victor Nana (Plant taxonomist) of the Cameroon National Herbarium (HNC), where voucher specimens were deposited (46220/HNC). The root was collected, cut into small pieces, dried at room temperature and powdered.

Extraction and Isolation. Powdered roots (1.5 kilograms) were extracted by maceration in MeOH:CH 2 Cl 2 (1:1 v/v) at room temperature. The filtrates were concentrated ona rotary evaporator under reduce pressure to yield 250 g of crude extract. The crude extract (240 g) was subjected to fractionation using hexanes, EtOAc and n-BuOH. The EtOAc rich fraction (70 g) was subjected to silica gel 60 (0.063 -0.200 mm) column chromatography using hexanes, hexanes-EtOAc gradient and EtOAc. Fractions (150) of 150 mL each were collected and concentrated under vacuum. Eight compounds namely, leplaericacid A (1, 200mg), leplaeric acid B(2, 30 mg), leplaeric acid C (3, 15 mg),andirobin (5, 20mg), 1,2-dihydroandirobin (6, 15mg), βsitosterol (7, 13 mg), stigmasterol (8, 20mg), and α-spinosterol (9, 15 mg), were obtained.

Preparation of Compound 4.

To a stirred solution of 50% KOH (10 mL) under diethyl ether (5.0 mL) at 0 °C, MNG (11.25 mg, 0.08mmol) was added gently. The resulting yellow coloured organic layer was separated, dried over KOH pellets, and added to compound 1 (18 mg, 0.04mmol) in Et 2 O (2 mL), the reaction mixture was stirred for 2 h and the solvent was removed under reduced pressure. Column chromatographic purification of the crude material over silica gel using 5% EtOAc in hexanes as solvent afforded pure compound 4 (70% yield). X-ray Crystallographic Analysis. White crystals of compound 1 were obtained from CHCl 3 -hexanes. Data for complex1were collected at 150 K on a Bruker D8 Quest with IµS microfocus source using Mo Kα radiation (λ = 0.71073 Å). The structure was solved by direct methods and refined by full-matrix least-squares refinement on F 2 after multiscan absorption correction of the data using SADABS. [START_REF]BRUKER APEX2 (Version 1.0-28) and SAINT-PLUS(Version 6.25[END_REF][START_REF] Sheldrick | SHELXS97 and SHELXL97[END_REF] Data was processed using the Bruker AXS SHELXTL software, version 6.14. [START_REF] Sheldrick | Version 6.14[END_REF] 5983 unique (Rint = 0.0810), which were used for all calculations; the final refinement produced R1 = 0.0607 (>2(I)) and wR2 = 0.1249 (all data); and Flack parameter = -0.3 (10), which does not allow for absolute stereostructure determination by X-ray. All non-hydrogen atoms were refined anisotropically, all hydrogen atoms attached to carbon were placed in calculated positions and the carboxylate hydrogens were located in the difference map and their positions refined. A disordered water molecule was removed from the structure (8 e-) using the SQUEEZE procedure implemented in PLATON. [START_REF] Spek | PLATON-90[END_REF]

Preparation of

Supporting Information

NMR data for lanostane-type terpenoids (1-4) and (11-12). This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Tel (Kenneth Eyong):+237-677-951126. E-mail: eyongkk@gmail.com

Notes

The authors declare no competing financial interests.


Marie-Laure Mille

Pascal Vallée

Romain Tisserand

Richard C Fitzpatrick

Jean-Louis Vercher

Experimental vs. model based determination of

Background and aim.

Stepping is a common protective strategy for dynamic balance recovery following external perturbations of stance. The present study investigated the threshold for triggering a step during force-controlled forward perturbations of different durations delivered at waist level, and confronted the experimental results with a simple biomechanical model that could predict when a subject had to step.

Methods. Twenty-two healthy young adults (5 women; 19-37 years old) were asked to try not to step in response to 86 different force/time combinations of forward waist-pulls (Fig A ). Each trial perturbation was characterized by its force (Fn -normalized according to the subject body weight), its duration (T), and its consequence (step or no-step). The probability to step as a function of perturbation characteristics was then calculated for the entire group and the force at which 50% of the subjects stepped (F50) were identified for each tested perturbation duration (Fig B). Experimental results were compared to a numerical criterion used to estimate if a recovery step was necessary for a given square force perturbation. It was obtained from the dynamics of a linear inverted pendulum + foot model, considering that the maximal balance recovery reactions. These were an instantaneous shift of the center of pressure at the edge of the functional base of support (CoPmax=15,3 cm) that arises after the perturbation (delay representing the reaction time RT = 116 ms). Values of CoPmax and RT were obtained from the experimental measures. Conclusions. The stepping boundary describes the maximal force that has to be applied for a specific time to trigger a step. Experimentally, this boundary corresponded to a constant impulse (i.e. constant perturbation force and duration product), which could be easily applied in a clinical environment. When compared to the biomechanical model, the stepping boundary was mainly explained by the RT and the CoP displacement within the functional BoS (CoPmax). Future work could investigate clinical population to further test validity of both stepping boundary methods and their predictive capabilities.

Results

  Experimental vs. model-based comparison of stepping threshold in response to external force-controlled perturbation Marie-Laure Mille, Pascal Vallee, Romain Tisserand, Jean-Louis Vercher, Richard C. Fitzpatrick, Thomas Robert To cite this version: Marie-Laure Mille, Pascal Vallee, Romain Tisserand, Jean-Louis Vercher, Richard C. Fitzpatrick, et al.. Experimental vs. model-based comparison of stepping threshold in response to external forcecontrolled perturbation. 2017 ISPGR World Congress, Jun 2017, FORT LAUDERDALE, United States. 2 p. hal-01791265

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  The experimental stepping boundary was well described by a simple hyperbolic function with a positive horizontal asymptote: F50 = a/T + C, with a the constant defining the radius of curvature of the function and C the horizontal asymptote describing the smallest force necessary to trigger a step -any force smaller than C could be sustained indefinitely without a step. The results of this fitting (Gauss-Newton, nonlinear, least-mean-squares) is represented on Fig C(red line) and the mean squared error was very small (MSE 0.12). The values of Fmax computed using the biomechanical model (Fig C solid blue line) correctly predicted the pulling force threshold.