Julien Bonnet
email: julien.bonnet@unilim.fr

Camille Garcia
email: camille.garcia@ijm.fr

Thibaut Leger
email: thibaut.leger@ijm.fr

Marie-Pauline Couquet
email: mariepauline.couquet@gmail.com

Philippe Vignoles
email: philippe.vignoles@unilim.fr

Vatunga Gedeao
email: vmlgedeao@live.com

Joseph Ndung
email: joseph.ndungu@finddx.org

Clotilde Boudot
email: boudot.clotilde@gmail.com

Sylvie Bisser
email: sbisser@pasteur-cayenne.fr

Bertrand Courtioux
email: bertrand.courtioux@unilim.fr

Proteome characterization in various biological fluids of Trypanosoma brucei gambiense-infected subjects

Keywords: Sleeping sickness, biological fluids, proteomic

Human African trypanosomiasis (HAT) is a neglected tropical disease that is endemic in sub-Saharan Africa. Control of the disease has been recently improved by better screening and treatment strategies, and the disease is on the WHO list of possible elimination. However, some physiopathological aspects of the disease transmission and progression remain unclear. We propose a new proteomic approach to identify new targets and thus possible new biomarkers of the disease. We also focused our attention on fluids classically associated with HAT (serum and cerebrospinal fluid (CSF)) and on the more easily accessible biological fluids urine and saliva.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) established the proteomic profile of patients with early and late stage disease. The serum, CSF, urine and saliva of 3 uninfected controls, 3 early stage patients and 4 late stage patients were analyzed.

Among proteins identified, in CSF, urine and saliva, respectively, 37, 8 and 24 proteins were differentially expressed and showed particular interest with regards to their function. The most promising proteins (Neogenin, Neuroserpin, secretogranin 2 in CSF; moesin in urine and intelectin 2 in saliva) were quantified by enzyme-linked immunosorbent assay in a confirmatory cohort of 14 uninfected controls, 23 patients with early stage disease and 43 patients with late stage disease. The potential of two proteins, neuroserpin and moesin, with the latter present in urine, were further characterized.

Our results showed the potential of proteomic analysis to discover new biomarkers and provide the basis of the establishment of a new proteomic catalogue applied to HAT-infected subjects and controls.

Introduction

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic disease, caused by subspecies of Trypanosoma brucei, that mainly affects poor and rural communities in more than 20 sub-Saharan African countries [START_REF] Büscher | Human African trypanosomiasis[END_REF]. HAT is one of 20 neglected tropical diseases (NTDs) recognized by the World Health Organization (WHO) [START_REF]Strategic and Technical Advisory Group on NTDs (STAG).10th meeting of the Strategic and Technical Advisory Group for Neglected Tropical Diseases[END_REF], and it is estimated that 55 million people live at risk of contracting HAT infection [START_REF] Who | Report of the second WHO stakeholders meeting on gambiense human African trypanosomiasis elimination[END_REF]. In the human population, T. b. gambiense is responsible for more than 98% of HAT cases in central and western Africa, T. b. rhodesiense is responsible of the remaining 2% of cases, which is prevalent in eastern and southern Africa. However, since 2009, fewer than 10,000 cases have been reported each year [START_REF] Who | Report of the second WHO stakeholders meeting on gambiense human African trypanosomiasis elimination[END_REF]- [START_REF] Franco | Epidemiology of human African trypanosomiasis[END_REF].

Trypanosomes are inoculated into human hosts by tsetse fly vectors; the trypanosomes then multiply and spread into the blood and lymphatic system. T. b. gambiense causes a chronic form of disease that develops over months to years in two stages. The first stage (S1), or hemolymphatic stage, is generally characterized by intermittent fever, lymphadenopathy, hepatosplenomegaly and headaches [START_REF] Franco | Epidemiology of human African trypanosomiasis[END_REF]. These symptoms are discrete and nonspecific for HAT. After a variable duration of time, trypanosomes cross the blood-brain barrier and reach the central nervous system (CNS); this stage is called the second stage (S2) or nervous stage. S2 is characterized by neurological signs and symptoms and usually leads to death if untreated [START_REF] Brun | Human African trypanosomiasis[END_REF], [START_REF] Mogk | Cyclical Appearance of African Trypanosomes in the Cerebrospinal Fluid: New Insights in How Trypanosomes Enter the CNS[END_REF].

Currently, several tests adapted to the field are used to diagnose HAT, the card agglutination test for trypanosomiasis (CATT) is still the most widely used screening test although single-format rapid diagnostic tests have been recently validated [START_REF] Bisser | Sensitivity and Specificity of a Prototype Rapid Diagnostic Test for the Detection of Trypanosoma brucei gambiense Infection: A Multi-centric Prospective Study[END_REF]. When the screening test is positive, the disease must be confirmed by demonstrating the presence of the parasite by microscopy. The tests used include blood concentration techniques such as the capillary tube centrifugation (CTC) and mini anion exchange centrifugation technique (mAECT) [START_REF] Lutumba | Validity, cost and feasibility of the mAECT and CTC confirmation tests after diagnosis of African of sleeping sickness[END_REF], [START_REF] Büscher | Improved Models of Mini Anion Exchange Centrifugation Technique (mAECT) and Modified Single Centrifugation (MSC) for Sleeping Sickness Diagnosis and Staging[END_REF] or examination of lymph node aspirates when lymph nodes are swollen. Once diagnosis of the disease is established by one or more of these tests, it is important to stage the disease to guide treatment regimen. The staging criteria defined by the WHO are the absence of trypanosomes in the cerebrospinal fluid (CSF), and fewer than 5 white blood cells (WBC)/µL CSF for patients with S1 disease, and the presence of trypanosomes in the CSF and/or more than 6 WBC/µL CSF for patients with S2 disease. These staging criteria are not always easy to use in settings where the disease occurs; microscopy is not a sensitive enough technique to reliably detect the parasite in the CSF, and the CSF WBC count is not specific for HAT [START_REF] Ngoyi | Stage determination in sleeping sickness: comparison of two cell counting and two parasite detection techniques[END_REF]. Without the right diagnosis, S1 disease may progress to S2 disease, and patients may not get the correct treatment. Patients with S1 T. b. gambiense HAT are treated with pentamidine. S2 patients require treatments that can cross the blood-brain barrier. Second stage Trypanosoma b. gambiense S2 infection can be treated with a nifurtimox-eflornithine combination therapy (NECT), but this treatment is given by infusion which is invasive and difficult to perform in some areas [START_REF] Fairlamb | Chemotherapy of human African trypanosomiasis: current and future prospects[END_REF]. For T. b. rhodesiense, melarsoprol is the only treatment available [START_REF] Who | Report of the second WHO stakeholders meeting on gambiense human African trypanosomiasis elimination[END_REF]. A new easy-to-use treatment, named Fexinidazole, is expected to be available in 2018. However, this treatment, even if it appears to be active in both stages of the disease, is proposed as an alternative to NECT, therefore for the treatment of S2. The use of pentamidine to treat S1 remains the current course of action [START_REF] Mesu | Oral fexinidazole for late-stage African Trypanosoma brucei gambiense trypanosomiasis: a pivotal multicentre, randomised, non-inferiority trial[END_REF]. Even if we can hope for the arrival of a molecule that will allow us to treat both stages of the disease, greatly facilitating the diagnostic and therapeutic plan of patients, the determination of the stage of the disease is still necessary at this time.

The composition of the proteome of different biological fluids change with disease progression and may lead to the generation of biomarkers that could be used in diagnosing and staging of the disease [START_REF] Meng | Proteomic analysis of serum, plasma, and lymph for the identification of biomarkers[END_REF]. Currently, proteome mapping is experiencing substantial growth owing to very high-resolution techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry analysis allows a large proteome screen compared with more specific techniques such as enzyme-linked immunosorbent assay (ELISA), immunoblotting techniques and others that focus only on a single protein or small part of the proteome. The data obtained with this technique, compared with the current human protein database, allow identification and categorization of the proteins present in a biological sample with extensive proteome coverage. Studies using LC-MS/MS have already discovered new biomarkers in many diseases, such as Duchenne muscular dystrophy, amyotrophic lateral sclerosis, oral cavity squamous cell carcinoma and breast cancer [START_REF] Rouillon | Proteomics profiling of urine reveals specific titin fragments as biomarkers of Duchenne muscular dystrophy[END_REF]- [START_REF] Collins | Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis[END_REF]. To enable the discovery of new biomarkers from the proteome of biological fluids, it is important to characterize the entire proteomes of both healthy and infected individuals. In this study, the serum, CSF, saliva and urine proteomes obtained in patients with S1 and S2 T. b. gambiense HAT were compared with uninfected controls. Using a high-resolution mass spectrometer coupled to a nano-ultraperformance liquid chromatography (UPLC) system, several proteins with variable abundance levels between the different groups were revealed by label-free protein quantification analysis.

The objective of this study was to characterize the proteome of serum, CSF, urine and saliva from patients with T. b. gambiense HAT and to identify biological markers that could be used in disease diagnosis and staging.

Material and Methods

Subjects and sample collection

The samples used in this study were collected during a prospective cohort study carried out rating scale [START_REF] Hamilton | A rate scale for depression[END_REF], and the sleep and psychiatric disturbance using the Mini-International neuropsychiatric interview [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF]. Main co-infections were excluded in the field by microscopy for common parasitic co-infections (blood smear for malaria, CTC for detection of filariasis, examination of a urine sediment for schistosomiasis when urine was positive for blood).

Retrospectively, samples were tested for HIV and syphilis, and all positive samples were excluded from the study.

For both controls and patients, samples of 5 mL of serum, 4 mL of CSF, 2 mL of urine and 2 mL of saliva were stored in liquid nitrogen, transported to Limoges (France), in dry tanks and further stored at -80°C. Samples used for this study were carefully selected among the cohort and common co-infections were excluded.

Ethics statement

Ethical clearance was obtained from the Direccao National de Saude Publica, Ministerio da Saude, Angola. Written informed consent was obtained from all participants and, for participants below 18 years of age, from their parents or guardians, prior to enrolment. Any individual who declined to participate was managed according to the standard procedures of the national HAT control program.

Study design

Potential biomarkers were first screened in serum, CSF, urine and saliva on a small population of controls and patients by mass spectrometry and, second, the most promising biomarkers were then quantified on a larger cohort.

The screening cohort (n = 10)

LC-MS/MS analysis was done on uninfected controls (n = 3), S1 patients (n = 3) and S2 patients (n = 4). Only adult males were included to avoid variations of the proteome linked to the expression of hormonal proteins.

Quantitative analysis of the validation cohort by ELISA (n = 80)

Protein quantification was done on samples collected after inclusion for controls (n = 14), S1 patients (n = 23) and S2 patients (n = 43). All samples used for the LC-MS/MS analysis were included for protein quantification.

Two analyses were done: the first to identify among the detected proteins those that differentiated controls from patients with HAT (n = 66); the second to verify whether these proteins could discriminate S1 from S2 patients. The most promising proteins that could differentiate S1 and S2 patients were also quantified at 12 months follow-up to verify their potential to be a marker of cure.

LC-MS/MS analysis

Sample preparation

Serum, CSF, saliva and urine samples were used in this analysis. A preliminary step of depletion has been carried out to reduce the quantity of predominant proteins (α1-Acid Glycoprotein, α1-Antitrypsin, alpha 2-Macroglobulin, Albumin, Apolipoprotein A-I, Apolipoprotein A-II, Fibrinogen, Haptoglobin, IgA, IgG, IgM, Transferrin) in these biological fluids. The dynamic range of protein concentrations in different human fluids can be large. Without this step, the high abundance of these predominant proteins would overwhelm the data obtained for the analysis of low abundance proteins. Samples containing 10 µg of protein, after depletion (Proteome purify 12 Human, R&D Systems®) and concentration (AMICON ULTRA, Merck Millipore®), were adjusted to 120 µL of the reaction mixture containing 4 M urea, 1.5 M thiourea and 50 mM tris-HCl pH 8.3. Proteins were reduced with 10 mM dithiothreitol for 30 min and then alkylated with 55 mM iodoacetamide for 20 min. Alkylated proteins were first digested with 500 ng of endopeptidase lys-C (Wako®) for 3 h at room temperature (RT). Then the mixture was diluted with 3 volumes of MilliQ-water and treated with 500 ng of trypsin (Sequence Grade Trypsin, Promega®) for 16 h at RT. Enzymatic activity was stopped by addition of formic acid to a final concentration of 3%. Samples were stored at -20 °C until use. In the case of saliva, a preliminary acetone (100%) precipitation step at -20 °C was carried out. The saliva and urine samples were not depleted to avoid a loss of proteins as the sample volume was low (1.5mL).

Sample analysis

Peptide mixtures (10 corresponding to patients) were analyzed by a Q-Exactive Plus coupled to a Nano-LC Proxeon 1000 equipped with an easy spray ion source (all from Thermo Scientific).

Peptides were separated by chromatography with the following parameters: Acclaim PepMap100 C18 pre-column (2 cm, 75 μm i.d., 3 μm, 100 Å), Pepmap-RSLC Proxeon C18 column (50 cm, 75 μm i.d., 2 μm, 100 Å), 300 nL/min flow rate, gradient from 95 % solvent A (water, 0.1% formic acid) to 35% solvent B (100 % acetonitrile, 0.1% formic acid) over a period of 98 minutes, followed by a column regeneration for 23 min, giving a total run time of 2 hours.

Peptides were analyzed in the Orbitrap cell, in full ion scan mode, at a resolution of 70,000 (at m/z 200), with a mass range of m/z 375-1500 and an AGC target of 3 x10 6 . Fragments were obtained by high collision-induced dissociation (HCD) activation with a collisional energy of 30%, and a quadrupole isolation window of 1.4 Da. MS/MS data were acquired in the Orbitrap cell in a Top20 mode, at a resolution of 17,500, with an AGC target of 2x10 5 , with a dynamic exclusion of 30 seconds. The most intense precursor ions were acquired first by MS/MS. Peptides with unassigned charge states or monocharged were excluded from the MS/MS acquisition. The maximum ion accumulation time was set to 50 ms for MS acquisition and 45 ms for MS/MS acquisition.

Peptide and protein identification

For the peptide and protein identification step, all MS and MS/MS data were processed with Proteome Discoverer software (Thermo Scientific, version 2.1) and with Mascot search engine (Matrix Science, version 5.1). The mass tolerance was set to 6 ppm for precursor ions and 0.02 Da for fragments. The maximum number of missed cleavages was limited to two for the trypsin protease. The SwissProt database with the Homo sapiens and the T. b. gambiense taxonomies was used for the MS/MS identification step. The following variable modifications (2 maximum per peptide) were allowed: oxidation (M) and carbamidomethylation (C). Peptide identification in all fluids analyzed were validated using a 1% FDR (false discovery rate) threshold calculated with the Percolator algorithm [START_REF] Brosch | Accurate and sensitive peptide identification with Mascot Percolator[END_REF].

Peptide and protein abundance quantification and statistical validation

Progenesis QI for Proteomics software (version 4.0, Waters) was used for the relative quantification of the protein abundances by using co-detection to eliminate missing values. No filters based on peptide by protein occurrences were applied for the protein quantification. The relative quantitation of proteins according to the three groups (Control, S1 and S2 patients) was performed using a between subject analysis and a Hi-3 method for which the three most abundant peptides were used for protein quantification. Abundance variations of proteins with an ANOVA p-value under 0.05 were further considered. The protein fold change makes it possible to show a difference of this protein abundance according to the groups when it is greater than 2. The potential biological role of each selected protein was verified and completed by a complete bibliographic review to confirm its interest.

Validation of protein abundance variations by ELISA

Selection of ELISA assays

Human neuroserpin ELISA, BioVendor® (LOD-Limit of Detection: 0.03 ng/mL), human NEO1 ELISA Kit, Mybiosource® (LOD: 5.00 pg/mL) and human SCG2 ELISA Kit, Elabscience Biotechnology® (LOD: 0.75 ng/mL) were selected to detect levels of neuroserpin, neogenin and secretogranin 2 in CSF, respectively. The level of neuroserpin detection on diagnosis samples allowed further detection at 12 months follow-up. Human moesin ELISA kit, Abbexa Ltd® (LOD: 0.054 ng/mL) and human ITLN2 ELISA kit, Elabscience® (LOD: 3.75 ng/mL) were used to detect levels of moesin in urine and levels of ITNL2 in saliva, respectively. For each ELISA test, the protocol provided by the manufacturer was followed. Optical densities (OD) were measured with a Multiskan go (Thermo Scientific®) spectrophotometer.

Statistical analysis

Using the data obtained by ELISA, cut offs and receiver operating characteristic (ROC) curves were made to determine the best sensitivity/specificity ratio for each marker. The significance has been evaluated using Kruskal-Wallis test and Fisher's exact test for testing the null of independence. The level of agreement with cut offs found in the literature was estimated using Cohen's kappa coefficient and generalized linear models were used to make logistic regressions.

Tests were performed using R Core Team [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF]. The following packages were used: ROCR for drawing curves [START_REF] Sing | ROCR: Visualizing the Performance of Scoring Classifiers[END_REF] and PMCMR for pairwise comparisons of Kruskal-Wallis test [START_REF] Pohlert | PMCMR: Calculate Pairwise Multiple Comparisons of Mean Rank Sums[END_REF].

.

Results

Population characteristics

Demographic, clinical and biological data are summarized in Table 1. All screening cohort selected patients and controls were male. Biological, neurological and parasitological examination showed that no controls suffered from sleep or neurological disorders, no trypanosomes were found in their blood or CSF, and their average WBC count was 4.61 WBC/µL CSF. S1 patients had similar clinical characteristics to the control group but with presence of trypanosomes in blood and an average of 1.33 WBC/µL CSF. S2 patients had confirmed neurological signs and sleep disorders; their average WBC count was 195.79 WBC/µL CSF.

The protein quantification cohort included 14 controls (sex ratio: 0.56), 23 S1 patients (sex ratio: 0.92) and 43 S2 patients (sex ratio: 0.54). Biological, neurological and parasitological examination showed that no controls had trypanosomes in the blood or CSF. The CSF examination of one of these controls revealed the presence of 8 WBC/µL, but no co-infection was detected for this patient, and the neurological examination did not reveal any disorders. Among the controls only 2 patients had sleep disorders. All S1 patients had trypanosomes in the blood and a WBC count between 0 and 4 WBC/µL CSF. None of the S1 patients had trypanosomes in the CSF, and the neurological examination showed that 6 patients had neurological disorders, and 10 had sleep disorders. All S2 patients had trypanosomes in the blood, and 23 out of 43 patients had trypanosomes in the CSF. The WBC count of these patients ranged from 6 to 597 WBC/µL CSF. Neurological examinations showed that 70% (30/43) of these patients had neurological disorders, and 67% (29/43) had sleep disorders.

For the quantification of proteins in urine and saliva, samples were not available for all controls and patients. For urine samples, the analysis was done on 13 controls, 21 S1 patients and 38 S2 patients. For saliva samples, the analysis was done on 13 controls, 22 S1 patients and 38 S2 patients.

Global LC-MS/MS analysis

Searching the LC-MS/MS data for semi-tryptic peptides in the protein database allows the identification of any possible protein degradation of the samples caused by the method and duration of freezing. There were no major differences between the samples of occurrences of peptides derived from non-tryptic endogenous proteases. This analysis was carried out on the screening cohort samples and showed that the biological samples of these patients had not been degraded.

The samples were tested for the presence of peptides described in the T. b. gambiense protein database; however, although parasite proteins were identified in each of the 4 biological fluids tested, they were not studied further owing to a lack of validation of protein identification. However, they are present in the database published in supplementary files.

Analysis of the total number of different proteins identified in serum, CSF, saliva and urine for each group of individuals (controls, S1 patients and S2 patients) (Figure 1) showed that S2 patients had a decrease in the number of different proteins expressed in the CSF compared with control and S1 patients. For saliva, the mean total number of different proteins identified in S2 patients (472 different proteins) was less than the total number of different proteins identified in S1 patients (594 different proteins) and by the control group (627 different proteins). The total number of proteins expressed in sera was similar in the different groups. For urine, the lowest total number of proteins identified was found in the controls.

LC-MS/MS analysis and protein quantification in sera

The proteomic analysis of the 10 screening cohort serum samples showed the presence of 269 proteins. The 10 best-scored proteins are presented in table 2. These data are heavily skewed owing to the very high levels of some serum proteins, such as albumin and immunoglobulins, despite the use of depletion protocols. This is highlighted by the presence of proteins with high identification scores, which are normally depleted, in table 2. As such, any changes in protein levels specific to HAT are not statistically pertinent.

LC-MS/MS analysis and protein quantification in CSF

The proteomic analysis of the screening cohort CSF samples showed the presence of 491 proteins. The relative abundances of these proteins were estimated by label-free quantification with Progenesis QI software. Among these proteins, 159 were found differentially abundant (ANOVA p-value lower than 0.05 and Fold Change higher than 2) between controls, S1 or S2 patients. After analysis of these 159 proteins on the basis of their statistical data (ANOVA pvalue and fold change), their differences attributable to disease stage, and their biological role, 37 proteins presented potential interest in HAT characterization (Table 3). Neogenin, secretogranin 2 and neuroserpin were chosen for protein quantification in CSF samples as their results showed the best combination of our defined criteria. Briefly, the ANOVA p-value calculated for neogenin is 3.82x10 -5 , and the fold change is close to infinity; the average normalized abundance (ANA) calculations showed an expression of this protein only in controls and S1 patients (2.72x10 4 and 2.17x10 4 , respectively) (Table 3). The ANOVA p-value calculated for secretogranin 2 is 5.45x10 - 5 and the fold change is 11.27 (Table 3). More secretogranin 2 is found in the CSF of healthy controls (ANA: 2.49x10 6 ) and S1 patients (ANA: 2.01x10 6 ) than in S2 patients (ANA: 2.21x10 5 ).

The most significant ANOVA result was obtained for neuroserpin at 2.61x10 -8 , and the Fold Change was close to infinity (Table 3). This protein is present in the CSF of healthy controls (ANA: 2.5x10 4 ) and S1 patients (ANA: 2.32x10 4 ) but not in S2 patients.

Protein quantification by ELISA was performed for the 3 proteins meaningful results were not obtained for neogenin and secretogranin 2. For neogenin, the amount was too low to be detected by ELISA, and for secretogranin 2, similar concentrations were found for controls, S1 and S2

patients. Mean values obtained were 0.27 ng/mL, with a standard deviation of 0.003. It was thus not possible to use secretogranin 2 as a possible marker of differentiation for patients or controls. Protein quantification by ELISA was performed for the 3 proteins, but for neogenin an secretogranin 2, the amount was too low to be detected by ELISA. It was therefore not possible to use them as a possible marker of differentiation for patients or controls. Only the neuroserpin ELISA results showed significant differences (Figure 2) and allowed statistical tests to be performed for further analysis. There was no difference in neuroserpin CSF concentration between the control and patients at inclusion when analyzed by the Kruskal-Wallis test; however, neuroserpin levels were significantly different (p < 0.05) between S1 and S2 patients. ROC curve analysis was used to determine the threshold level of neuroserpin for discriminating S2 patients from S1 patients (area under the curve (AUC): 0.72). It was determined that a level of CSF neuroserpin below 4.99 ng/mL was indicative of a patient having S2 disease (sensitivity: 0.94 and specificity: 0.58). Logistic regression analysis showed that age and sex of individuals have no effect on CSF neuroserpin concentration; however, there was a correlation between neuroserpin concentration and presence of sleep disorders (odds ratio: 2.1x10 -02 (CI: 1.0x10 -3 ; 1.47x10 -1 ), p < 0.01) and neurological disorders (odds ratio: 6.3x10 -2 (CI: 0.9x10 -2 ; 2.32x10 -1 ), p < 0.001).

Similarly, a link could be made between the presence of trypanosomes in the CSF and the concentration of neuroserpin (odds ratio: 5.09x10 -4 (CI: 1.93x10 -4 ; 4.22x10 -2 ), p < 0.001). Neuroserpin levels were then tested for association with sleep and neurological disorders and the presence of trypanosomes in the CSF. The Kruskal-Wallis test revealed that CSF neuroserpin could act as a marker for these disease-associated pathologies (p < 0.05). A ROC curve was used to determine the threshold of 5.09 ng/mL, below which patients had sleep disorders (AUC: 0.79; sensitivity: 0.72 and specificity: 0.67). The ROC curve analysis also allowed us to determine a threshold of 4.97 ng/mL of CSF neuroserpin (AUC: 0.77), below which the patients had neurological disorders (sensitivity: 0.9 and specificity: 0.59). The threshold of CSF neuroserpin determined by ROC curve analysis for discriminating the presence of trypanosomes in the CSF (AUC = 0.95) is 4.83 ng/mL, below which is indicative of CSF invasion by trypanosomes (sensitivity: 0.98 and specificity: 0.91). The discriminating power of CSF neuroserpin was tested against the parasitological examination of CSF (gold standard) with an agreement of 0.88 (Cohen's kappa coefficient), which is an excellent concordance.

At follow-up, at 12 months following treatment, the neuroserpin concentration was quantified in the CSF in 5/14 of the controls, 16/23 of the S1 patients and 28/43 of the S2 patients. Kruskal-Wallis analysis showed that there was no difference in neuroserpin levels between the 3 groups after 12 months post-treatment, and the concentrations at 12 months were close to those found in the control and S1 patients at inclusion. The mean neuroserpin concentration in the CSF in all the groups is 5.13 ng/mL (Figure 2).

LC-MS/MS analysis and protein quantification in urine

The proteome analysis of urine samples showed the presence of 664 proteins, and 32 proteins were differentially abundant (ANOVA p-value lower than 0.05 and Fold Change higher than 2) between the control and the S1 or the S2 patients. From these 32 proteins, 8 were chosen as interesting (Table 4). Moesin appeared to be the most promising protein for quantification by ELISA at inclusion. The ANOVA p-value for moesin analysis was 9.38x10 -3 and its fold change was 47.11 (Table 4). Moesin is increased in S1 patients (ANA: 7.12x10 4 ) and S2 patients (ANA:

1.57x10 4 ) compared with the controls (ANA: 1511.77). Quantification of moesin at 12 months follow-up was not possible owing to the low number of urine samples available.

The moesin ELISA was performed on samples from 59 patients from both S1 and S2 patients and 13 uninfected controls. The Kruskal-Wallis test revealed a significant difference in moesin levels (p < 0.05) between controls and patients with HAT. The threshold for discriminating controls from patients was determined with ROC curve analysis (AUC = 0.70). A level of moesin above 0.448 ng/mL (sensitivity: 0.64 and specificity: 0.61) in urine is indicative of HAT disease.

Further characterization of moesin levels between S1 (n = 21) and S2 patients (n = 38) compared with uninfected controls (n=13) was performed. No statistical difference in moesin abundance levels was observed between uninfected controls and S1 patients and no significant difference was observed between S1 and S2 patients. However, when comparing the uninfected controls and S2 patients, a significant difference in moesin levels was found (p < 0.05). ROC curve analysis (AUC: 0.72) was used to determine a urine moesin threshold of 0.60 ng/mL (sensitivity: 0.63 and specificity: 0.65) above which patients had S2 HAT. Using logistic regression, it was shown that the moesin concentrations were not influenced by sex, age, presence of trypanosomes in CSF, or sleep and neurological disorders.

LC-MS/MS analysis and protein quantification in saliva

The screening was carried out on the screening cohort but with only 2 controls (owing to an error in tube labelling). The proteome screening of saliva allows the identification of 954 proteins. Among these 954 proteins, 137 were found to be differentially abundant (ANOVA p-value lower than 0.05 and fold change higher than 2) between the control and the S1 or the S2 patients, and 20 24 were chosen for their particular interest (Table 5). Among them intelectin 2 (ITLN2) was chosen for quantification by ELISA. ITLN2 had an ANOVA of 1.23x10 -4 and a Fold Change of 120.23 as determined by quantitative proteomics; ANA, determined by Progenesis QI software analysis, showed lower ITLN2 concentration in the saliva of patients in both stages (S1 ANA: 258.69; S2 ANA: 350.60) than controls (ANA: 3.11x10 4 ). Saliva ITLN2 concentrations measured by ELISA of controls (n = 13) and those of patients (n = 60) were compared with the Kruskal-Wallis test. There was no significant difference between the two populations (p = 0.40). Saliva ITLN2 concentrations were then compared between S1 patients (n = 22), the S2 patients (n = 38) and uninfected controls (n = 13) using the Kruskal-Wallis test. There was no significant difference in ITLN2 levels between controls, S1 and S2 patients (p = 0.06). Therefore, it was not possible to determine a threshold for this protein (AUC = 0.59).

Discussion

This is, to our knowledge, the first simultaneous proteomic characterization of 4 biological fluids (serum, CSF, saliva and urine) from the same HAT patients. The proteome screening analysis by LC-MS/MS has revealed 69 proteins (37 proteins in CSF, 8 proteins in saliva and 24 proteins in urine) that could be interesting for diagnosis or staging of the disease. The potential of 5 proteins (3 proteins in CSF, 1 protein in urine and 1 protein in saliva) were further verified as biomarkers in diagnosis or staging. These assays are a preliminary step in the identification of the entire proteome of HAT patients.

The semi-tryptic peptide analysis of the database showed that the method and the freezing duration of our samples did not alter the proteome quality. However, very few proteins or degraded fragments from the parasite were identified owing to poor Mascot scores. This may be due to the low quantity of parasitic proteins relative to the amount of human proteins and/or to limitations or absence of T. b. gambiense proteins in the existing database. This needs to be further investigated, which is why our study was focused on the human proteome and the detection of biomarkers for S2 patients and not on parasite proteins.

In sera, the label-free quantification did not identify any protein of notable interest (ANOVA: < 0.05 and Fold Change: > 2). The quantification by LC-MS/MS of an exhaustive set of proteins in serum is difficult; this is partly due to the complexity of the serum, which contains a large number of different proteins with an extremely large range of expression [START_REF] Anderson | Candidate-based proteomics in the search for biomarkers of cardiovascular disease[END_REF], [START_REF] Hu | Human body fluid proteome analysis[END_REF]. In this case, despite the depletion step carried out, the presence of a large quantity of proteins (mainly albumins and globulins) impedes quantification of other proteins as shown in other studies [START_REF] Anderson | The Human Plasma Proteome History, Character, and Diagnostic Prospects[END_REF].

In the CSF, the protein composition showed a clear decrease in the diversity and number of identified proteins in patients with S2 disease. This result is surprising but can be explained by two physiopathological mechanisms that may act together. The first is trypanosome-induced immunosuppression [START_REF] Low | Report of the Sleeping Sickness Commission[END_REF]- [START_REF] Baral | Immunobiology of African trypanosomes: need of alternative interventions[END_REF], which is a marked weakening of the humoral response owing to massive B cell death [START_REF] Radwanska | Trypanosomiasis-Induced B Cell Apoptosis Results in Loss of Protective Anti-Parasite Antibody Responses and Abolishment of Vaccine-Induced Memory Responses[END_REF], [START_REF] Bockstal | brucei Infection Reduces B Lymphopoiesis in Bone Marrow and Truncates Compensatory Splenic Lymphopoiesis through Transitional B-Cell Apoptosis[END_REF]. Furthermore, it has been described that the release of trypanosome suppression immunomodulating factor by the parasite triggers the development of suppressive M1 macrophages [START_REF] Gómez-Rodríguez | Identification of a parasitic immunomodulatory protein triggering the development of suppressive M1 macrophages during African trypanosomiasis[END_REF] and blocks T cell proliferation [START_REF] Stijlemans | Immune Evasion Strategies of Trypanosoma brucei within the Mammalian Host: Progression to Pathogenicity[END_REF]. This immunosuppression could explain the decrease in the number of proteins found in the CSF of the patients with S2 disease because the disappearance of the immune cells implies a decrease of the proteins related to them. The second explanation is a leakage or resorption of nervous system proteins owing to the permeabilization of the blood-brain barrier [START_REF] Grab | African trypanosome interactions with an in vitro model of the human blood-brain barrier[END_REF], [START_REF] Grab | Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells[END_REF]. The loss of nervous system proteins and increase in plasma-associated protein, such as complement system proteins, albumin, transthyretin and apolipoproteins has been described by others in CNS infections altering the blood-brain barrier [START_REF] Jurado | Cerebrospinal Fluid[END_REF]- [START_REF] Blyth | Validation of Serum Markers for Blood-Brain Barrier Disruption in Traumatic Brain Injury[END_REF] and was corroborated in the CSF analysis of our study.

In CSF, the analysis allowed the identification of 491 proteins, of which many had been previously associated with HAT, such as vascular cell adhesion protein 1 (VCAM1) [START_REF] Mulenga | Trypanosoma brucei brucei crosses the blood-brain barrier while tight junction proteins are preserved in a rat chronic disease model[END_REF], [START_REF] Hainard | Matrix metalloproteinase-9 and intercellular adhesion molecule 1 are powerful staging markers for human African trypanosomiasis[END_REF] and osteopontin (SPP1) [START_REF] Tiberti | Discovery and verification of osteopontin and Beta-2-microglobulin as promising markers for staging human African trypanosomiasis[END_REF]. The study focused on 37 proteins that were differentially expressed between the uninfected controls, S1 and S2 patients, and 3 proteins -namely neogenin, secretogranin 2 and neuroserpin -were chosen for further analysis by ELISA [START_REF] Fischer-Colbrie | Secretogranin II Is Synthesized and Secreted in Astrocyte Cultures[END_REF]- [START_REF] Fujita | The roles of RGMa-neogenin signaling in inflammation and angiogenesis[END_REF].

Neogenin is a protein associated with the nervous system, and it has already been proposed as a biomarker in other pathologies [START_REF] Mattsson | Cerebrospinal fluid concentrations of peptides derived from chromogranin B and secretogranin II are decreased in multiple sclerosis[END_REF], [START_REF] Fujita | The roles of RGMa-neogenin signaling in inflammation and angiogenesis[END_REF]. In the brain, neogenin is a receptor for many proteins, including repulsive guidance molecule (RGM) family members, which is involved in axon guidance, apoptosis and neuronal differentiation [START_REF] Wilson | Neogenin: one receptor, many functions[END_REF], [START_REF] Fujita | The roles of RGMa-neogenin signaling in inflammation and angiogenesis[END_REF]. The use of the RGMa-neogenin signaling pathway as a therapeutic target to overcome inflammatory and neurodegenerative diseases has already been proposed [START_REF] Fujita | The roles of RGMa-neogenin signaling in inflammation and angiogenesis[END_REF]. Secretogranin 2 is a soluble member of the granin protein family, which are widely distributed in neuroendocrine and nervous system tissues.

Granins are stored in large dense-core vesicles within neurons; secretogranin 2 can also be detected in glial cells [START_REF] Fischer-Colbrie | Secretogranin II Is Synthesized and Secreted in Astrocyte Cultures[END_REF]. Secretogranin 2 has numerous functions, including neurite outgrowth and chemoattractive effects on monocytes, eosinophils and endothelial cells [START_REF] Fischer-Colbrie | Secretogranin II Is Synthesized and Secreted in Astrocyte Cultures[END_REF]. In 2013, Jakobsson et al proposed secretogranin 2 as a CSF marker for severe forms of bipolar disorder.

Patients with bipolar disorder have defects in the regulatory secretory pathway; one of the altered proteins is secretogranin 2 [START_REF] Jakobsson | Decreased cerebrospinal fluid secretogranin II concentrations in severe forms of bipolar disorder[END_REF], and reduction is linked to severe forms. Reduced levels of secretogranin 2 and secretogranin 3 were observed in serum and CSF samples of patients with multiple sclerosis [START_REF] Mattsson | Cerebrospinal fluid concentrations of peptides derived from chromogranin B and secretogranin II are decreased in multiple sclerosis[END_REF], [START_REF] Li | Dysregulated expression of secretogranin III is involved in neurotoxin-induced dopaminergic neuron apoptosis[END_REF], [START_REF] Begcevic | Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas[END_REF]. In our HAT sample collection, neogenin and secretogranin 2 were highlighted by LC-MS/MS analysis; however, the ELISA assay used seemed unable to detect these proteins, which may be due to the detection limit of the commercial ELISA test being too high for the samples in this study. In our HAT sample collection, neogenin and secretogranin 2 were highlighted by LC-MS/MS analysis; however, our patients had neogenin and secretogranin 2 concentrations under the ELISA assay limit of detection.

Finally, among the tested proteins, only neuroserpin allowed the discrimination of disease stage with a decrease in neuroserpin concentrations with disease progression. This is the first study showing a link between neuroserpin and HAT. Neuroserpin is a protein belonging to the serine protease inhibitor superfamily, which is expressed throughout the nervous system but more particularly at the latter stages of the development of neuronal cells [START_REF] Hastings | Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival[END_REF], [START_REF] Krueger | Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse[END_REF], [START_REF] Lee | Physiological and pathological functions of neuroserpin: Regulation of cellular responses through multiple mechanisms[END_REF]. Neuroserpin inhibits the activation of tissue plasminogen and has an essential role in axogenesis, synaptic plasticity [START_REF] Caccia | Molecular bases of neuroserpin function and pathology[END_REF], memory and brain development [START_REF] Osterwalder | Neuroserpin, an axonally secreted serine protease inhibitor[END_REF], [START_REF] Lee | Physiological and pathological roles of tissue plasminogen activator and its inhibitor neuroserpin in the nervous system[END_REF]. Neuroserpin is linked to familial encephalopathy with neuroserpin inclusion bodies (FENIB) [START_REF] Davis | Familial dementia caused by polymerization of mutant neuroserpin[END_REF]. This dementia is due to point mutations favoring the polymerization of neuroserpin and its retention in endoplasmic reticulum of neurons [START_REF] Davis | Familial dementia caused by polymerization of mutant neuroserpin[END_REF]- [START_REF] Noto | The Tempered Polymerization of Human Neuroserpin[END_REF]. This protein is also associated with Alzheimer disease, but its role is not yet clearly identified [START_REF] Kinghorn | Neuroserpin binds Abeta and is a neuroprotective component of amyloid plaques in Alzheimer disease[END_REF]- [START_REF] Subhadra | Neuroserpin up-regulation in the Alzheimer's disease brain is associated with elevated thyroid hormone receptor-β1 and HuD expression[END_REF]. To date, we have no knowledge of work studying the relationship between neuroserpin and HAT. The decrease in late stage disease could be due to the fact that neuroserpin is predominantly expressed in the neurons of the hippocampus, caudate and cerebral cortex [START_REF] Teesalu | Tissue plasminogen activator and neuroserpin are widely expressed in the human central nervous system[END_REF], [START_REF] Uhlén | Proteomics. Tissue-based map of the human proteome[END_REF]. The hippocampus and the caudate are close to the choroid plexus, which is described in the literature as the first route of entry of the parasite into the brain owing to the absence of the blood-brain barrier at this location [START_REF] Schultzberg | Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia[END_REF]- [START_REF] Nikolskaia | Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease[END_REF]. It is probable that these regions are the first to be affected by the presence of the trypanosome. ELISA quantification allowed us to determine a cut-off point for neuroserpin, below which HAT patients may be considered to be in S2 (4.99 ng/mL; sensitivity: 0.94 and specificity: 0.58) at diagnosis. Patients with a neuroserpin concentration greater than 4.99 ng/mL are considered in S1 of the disease. Levels had returned to normal at 12 months follow up, suggesting that the decrease is directly linked to the presence of the parasite or the elicited immune reaction. It was also possible to determine that the onset of sleep disorders was associated with a CSF neuroserpin concentration of less than 5.09 ng/mL. This last cut-off could explain how some patients with S1 disease present symptoms of sleep disorders [START_REF] Bertrand | Symptomatologie générale de la trypanosomiase humaine africaine au moment du dépistage[END_REF], [START_REF] Urech | Sleeping Sickness in Travelers -Do They Really Sleep?[END_REF]. Similarly, the presence of trypanosome in the CSF was correlated with a CSF neuroserpin concentration of less than 4.84 ng/mL. Each of these cut-offs correlate with classical stage markers, indicating that neuroserpin may be considered as a good additional or unique marker if its concentration is easy to quantify.

The identification of biomarkers in alternative fluids such as urine and saliva is extremely interesting for field screenings or use in resource-limited countries. Access to these fluids is noninvasive, and collection can be done by non-qualified people, or they can even can be used for self-diagnosis [START_REF] Malamud | Saliva as a diagnostic fluid[END_REF], [START_REF] Nunes | Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid:a systematic review[END_REF]. Currently, saliva is a source of biomarkers for neurological diseases [START_REF] Al-Nimer | Saliva α-Synuclein and A High Extinction Coefficient Protein: A Novel Approach in Assessment Biomarkers of Parkinson's Disease[END_REF], HIV [START_REF] Fransen | Using conventional HIV tests on oral fluid[END_REF], diabetes [START_REF] Yoon | Inflammatory biomarkers in saliva: assessing the strength of association of diabetes mellitus and periodontal status with the oral inflammatory burden[END_REF]- [START_REF] Zalewska-Szajda | Activity of N-acetyl-β-D-hexosaminidase in the saliva of children with type 1 diabetes[END_REF] and cancer [START_REF] Jenzano | Levels of glandular kallikrein in whole saliva obtained from patients with solid tumors remote from the oral cavity[END_REF], [START_REF] Streckfus | A preliminary study of CA15-3, c-erbB-2, epidermal growth factor receptor, cathepsin-D, and p53 in saliva among women with breast carcinoma[END_REF]; urine is used for the diagnosis of osteoclastogenesis [START_REF] Robins | Collagen crosslinks in metabolic bone disease[END_REF], urological cancer [START_REF] Mlcochova | Urine microRNAs as potential noninvasive biomarkers in urologic cancers[END_REF], and proteins in the urine have been identified as potential biomarkers for breast cancer [START_REF] Shichkin | TNF receptor p55 and IL-8(72) and IL-8(77) isoforms: blood and urine levels in breast cancer patients[END_REF], Alzheimer disease [START_REF] Ghanbari | Biochemical assay for AD7C-NTP in urine as an Alzheimer's disease marker[END_REF] and Parkinson disease [START_REF] Connolly | F2 isoprostane levels in plasma and urine do not support increased lipid peroxidation in cognitively impaired Parkinson disease patients[END_REF].

In urine, proteins linked to infectious processes have been highlighted. Moesin was the most interesting according to our identification criteria. This protein belongs to the family of ezrinradixin-moesin (ERM) proteins, and it is involved in angiogenesis. In fact, the phosphorylation of moesin allows the mediation of endothelial angiogenesis [START_REF] Wang | Role of Moesin in Advanced Glycation End Products-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells[END_REF]. This protein also participates in the connection between the cytoskeleton and the plasma membrane of human cells. This property allows this protein to limit infection with herpes simplex virus 1 [START_REF] Henning | PDZD8 is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type 1[END_REF]. It has been shown to have a role in invasion of Trypanosoma cruzi amastigotes into human cells [START_REF] Ferreira | ERM Proteins play distinct roles in cell invasion by extracellular amastigotes of Trypanosoma cruzi[END_REF]. Moesin is also involved in the regulation, proliferation and adhesion of human lymphoid cells [START_REF] Pore | Ezrin-Radixin-Moesin family proteins in the regulation of B cell immune response[END_REF]. A mutation of this protein leads to primary immunodeficiency [START_REF] Lagresle-Peyrou | Xlinked primary immunodeficiency associated with hemizygous mutations in the moesin (MSN) gene[END_REF]. The involvement of moesin in kidney lesions was established by Chen et al [START_REF] Chen | Role of Moesin in Renal Fibrosis[END_REF]; however, its role is not clear. The ERM complex intervenes in the cellular dynamics by taking part in the mobility of cancer cells described in oral cancers and carcinomas [START_REF] Kinoshita | Tumor suppressive microRNA-133a regulates novel targets: moesin contributes to cancer cell proliferation and invasion in head and neck squamous cell carcinoma[END_REF], [START_REF] Li | Moesin regulates the motility of oral cancer cells via MT1-MMP and E-cadherin/p120-catenin adhesion complex[END_REF]. In our study, moesin levels were elevated in urine samples from S1 and S2 patients compared with uninfected controls. This result was corroborated by the ELISA quantification. ELISA quantification shows that there is a significant difference between moesin concentrations in uninfected controls compared with patients. Furthermore, a difference in moesin concentration was observed between the uninfected controls and S2 patients, which could make it possible to discriminate stage when the presence of the parasite is established in the blood. It was subsequently impossible to carry out the analysis at 12-months follow-up as not enough samples were available.

In saliva, 24 potential interesting proteins were identified that are differentially expressed, and ITLN2 was chosen as a potential candidate biomarker owing to its physiopathological background. However, ITLN2 levels detected by ELISA could not discriminate controls from patients, nor S1 from S2 patients (p = 0.06). It does not appear aberrant to find this protein in an inflammatory pathology such as HAT [START_REF] Peebles | The intelectins: a new link between the immune response to parasitic infections and allergic inflammation?[END_REF], [START_REF] Wesener | Recognition of Microbial Glycans by Human Intelectin[END_REF]. The intelectin families, and particularly ITLN2, are proteins expressed principally in the small intestine. ITLN2 is involved in host-pathogen interactions [START_REF] Lee | Human homologs of the Xenopus oocyte cortical granule lectin XL35[END_REF] and iron metabolism [START_REF] Yan | Comparative genomic and phylogenetic analyses of the intelectin gene family: implications for their origin and evolution[END_REF] and allergic inflammation [START_REF] Peebles | The intelectins: a new link between the immune response to parasitic infections and allergic inflammation?[END_REF]. Studies are currently determining any potential antimicrobial and anti-parasitic activity of ITLN2 [START_REF] Peebles | The intelectins: a new link between the immune response to parasitic infections and allergic inflammation?[END_REF], [START_REF] Wesener | Recognition of Microbial Glycans by Human Intelectin[END_REF], [START_REF] Pemberton | Innate BALB/c enteric epithelial responses to Trichinella spiralis: inducible expression of a novel goblet cell lectin, intelectin-2, and its natural deletion in C57BL/10 mice[END_REF].

However, the presence of ITLN2 and its role in saliva remains unexplained. The quantification of this protein on a larger sample could make it possible to identify the pathophysiological role of this protein in HAT.

There can be some bias in saliva and urine analysis. In case of saliva, intra-and inter-individual composition variations can be influenced by many parameters, such as salivary flow, sex, age, the subject's emotional state, season, nycthemeral rhythm and medication [START_REF] Vitorino | Identification of human whole saliva protein components using proteomics[END_REF]. In the same way, external elements to the salivary system, such as nasal and bronchial secretions, the gingival fluid, epithelial cells, microbial flora and food debris, can modify its composition [START_REF] Kaufman | The diagnostic applications of saliva-a review[END_REF], [START_REF] Esser | Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid[END_REF]. It is recommended to perform the saliva collection before brushing the teeth and at least 30 minutes after any ingestion of food [START_REF] Gröschl | Données actuelles sur l'analyse hormonale salivaire[END_REF]. For urine, the time of sample collection was not calibrated (between 7 am and 1 pm) and thus may lead to variations in urine composition and concentration.

In this study, these variations could not all be controlled and may constitute a bias.

One weakness of this study is the number of patients that were selected for the screening by LC-MS/MS analysis. Indeed, for practical reasons 10 patients were selected among the 247 included in the cohort for the analysis of screening, for which there is homogeneous data (age, sex and no co-infection detected). Despite the low number of patient samples analyzed by LC-MS/MS, the patient sampling in each group (controls, S1 and S2) is statistically homogenous. Another possible criticism is the lack of uniformity in the sampling times, which is especially important in the case of urine and saliva.

Conclusion

In conclusion, our work allowed the characterization of a large number of proteins presenting an interest in the understanding of HAT pathophysiology and diagnosis. The high sensitivity of new technologies such as LC-MS/MS allow the discovery of new candidate disease marker and open the path to other more accessible biological fluids like urine or saliva. Our work identified two potential new markers, neuroserpin and moesin, the latter of which is found in urine, which could simplify diagnostic strategies in the field.

Graphical abstract

Human samples of sera, CSF, saliva and urine from screening cohort were prepared for LC-MS/MS analysis. Human African trypanosomiasis samples (sera, CSF, saliva and urine) from a screening cohort were prepared for characterization of disease biomarkers by LC-MS/MS analysis. Bottom-up identification of proteins allows to categorize 269 proteins in the serum, 491 proteins in the CSF, 954 proteins in the saliva and 664 proteins in the urine. Among these proteins identified, 37 proteins in the CSF, 24 proteins in the saliva and 8 proteins in the urine were differentially abundant between the control, the S1 or the S2 patients and were of interest for HAT diagnosis. Among these proteins, 5 were assayed by ELISA in the protein quantification cohort: neogenin, secretogranin 2 and neuroserpin in the CSF; intelectin 2 in the saliva and moesin in the urine. *For saliva screening cohort: controls (n = 2), S1 patients (n = 3) and S2 patients (n = 4). Neuroserpin and moesin are two potential new markers that could simplify diagnostics. CSF protein composition showed a clear decrease of identified proteins in S2 patients. Easily accessible biological fluids, like urine, could be used for HAT diagnosis.

Supplementary data

Ethics approval and consent to participate

Ethical clearance was obtained from the Direccao National de Saude Publica, Ministerio da Saude, Angola. The authorization for automated processing of personal data relating to the study was obtained from the Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé (CCTIRS) (N° 08.228bis)

Consent for publication

Written informed consent was obtained from all participants and, for participants below 18 years of age, from their parents or guardians, prior to enrolment. Any individual who declined to participate was managed according to the standard procedures of the national HAT control program.

Availability of data and material

  between 2009 and 2011 in three provinces in Angola: Bengo, Uíge and Kwanza Norte. The study enrolled 247 Angolan subjects: 67 were classified as S1 patients, 161 as S2 patients, and 19 as controls. All patients were designated HAT positive by CATT (with or without titration), and trypanosomes demonstration by microscopy in the blood or in lymph node aspiration. Patients in the cohort had T. b. gambiense HAT, and were staged before treatment. Patients with no trypanosomes in the CSF and normal WBC counts (≤ 5 WBC/µL CSF) were classified as S1 patients. Patients with a WBC counts of more than 5 WBC/µL CSF and/or presence of trypanosomes in the CSF were classified as S2 patients. Controls were subjects who had tested positive for HAT by CATT but without parasite confirmation in blood, lymph node or CSF and without development of the disease during the whole follow-up period. Every subject was examined clinically and submitted to a questionnaire to document clinical and neurological characteristics. Neurological data included an index of depression, measured with the Hamilton

Figure 1 :

 1 Figure 1: Distribution of the number of proteins identified in the serum, CSF, saliva and urine of the screening cohort with respect to disease stage.

Figure 2 :

 2 Figure 2: Changes in the neuroserpin concentration in CSF of controls and patients with stage 1 and stage 2 HAT at inclusion and after 12 months

  epidemiological and biological data of 3 controls, 3 patients with stage 1 (S1) disease and 4 patients with stage 2 (S2) disease who constituted the screening cohort, as well as the 14 controls, the 23 patients with S1 disease and the 43 patients with S2 disease enrolled in protein quantification cohort. For urine and saliva analysis, some samples were not present in the protein quantification cohort. *For urine (n = 72): 13 Controls, 21 S1 and 38 S2. For saliva (n = 73): 13 controls, 22 S1 and 38 S2.

  Table represents the average number of different proteins identified by LC-MS/MS analysis in serum, cerebrospinal fluid (CSF), urine and saliva. For serum, CSF and urine data there are 3 controls (C), 3 patients with stage 1 (S1) disease and 4 patients with Stage 2 (S2) disease. For saliva there are 2 controls, 3 patients with S1 disease and 4 patients with S2 disease. Highlights LC-MS/MS analysis allow the discovery of new candidate disease markers for HAT.

Table 1 :

 1 Characteristics of the screening and protein quantification cohort

	Screening cohort		Control	S1	S2
	n		3	3	4
	Sex-ratio (M/F)		3/0	3/0	4/0
	Age (SD)		36.67 (27.15)	43.33 (21.13)	31.25 (18.14)
	Neurological disorders	0	0	4
	Sleep disorders		0	0	4
	CSF WBC/µL (SD)		4.61 (3.05)	1.33 (0.58)	195.79 (147.44)
	Trypanosome per µL blood	0	3	4
	Trypanosome per µL CSF	0	0	4
	Protein quantification cohort	Control	S1	S2
	n *		14	23	43
	Sex ratio (M/F)		5/9	11/12	15/28
	Mean (SD)	38 (20.14)	39.09 (14.17)	34.55 (12.93)
	Age	Min	12	15	14
		Max	70	58	58
	Neurological disorders	0	6	30
	Sleep disorders		2	10	29
	CSF WBC/µL (SD)	2.64 (1.90)	1.34 (0.93)	137.82 (165.73)
	CSF WBC/µL	Min	0	0	6
		Max	8	4	597
	Trypanosome per µL blood		0	23	43
	Trypanosome per µL CSF		0	0	23
	This table represents the

Table 2 :

 2 Top-10 best scored proteins identified in serum 915

	Accession	Description	Score	ANOVA	Max Fold Change	Average normalized abundances Control S1 S2
	CO3_HUMAN	Complement C3	8016.88	0.82	1.31	1.91e+007 1.74e+007 1.46e+007
	A2MG_HUMAN	Alpha-2-macroglobulin	5874.13	0.94	1.37	1.52e+007 1.67e+007 1.22e+007
	VTDB_HUMAN	Vitamin D-binding protein	5127.27	0.87	1.11	9.21e+006 8.29e+006 8.67e+006
	APOB_HUMAN	Apolipoprotein B-100	4819.10	0.35	2.68	2.89e+006 1.08e+006 1.08e+006
	TRFE_HUMAN	Serotransferrin	4375.11	0.67	1.54	8.19e+006 1.26e+007 1.18e+007
	CERU_HUMAN	Ceruloplasmin	4249.73	0.18	1.47	3.09e+007 2.30e+007 2.10e+007
	HPT_HUMAN	Haptoglobin	3150.25	0.36	4.37	9.20e+006 1.34e+007 4.02e+007
	A1AT_HUMAN	Alpha-1-antitrypsin	3085.22	0.63	1.22	3.80e+007 4.45e+007 3.64e+007
	IGHM_HUMAN	Ig mu chain C region	2899.80	0.89	2.05	4.60e+007 3.58e+007 2.25e+007
	IGKC_HUMAN	Ig kappa chain C region	2880.99	0.63	2.20	8.53e+007 7.25e+007 3.88e+007
	IGHG1_HUMAN	Ig gamma-1 chain C region	2813.11	0.33	3.20	1.29e+007 4.14e+007 1.72e+007
	916
	917	S1: stage 1 disease; S2: stage 2 disease. Score: mascot protein score
	918

Table 3 :

 3 Most notable proteins in the CSF with altered abundance levels between control, S1 and S2 patients (classified by the increase in ANOVA p-value)

	All shown proteins passed thresholds of a fold change ≥ 2 and a p-value ≤ 0.05. Score: mascot
	protein score. * Protein quantified by ELISA. S1: stage 1 disease; S2: stage 2 disease.

Table 4 :

 4 Most notable proteins in the urine with altered levels between control, S1 and S2 patients (classified by increase in ANOVA p-value) All shown proteins passed thresholds of a fold change ≥ 2 and a p-value ≤ 0.05. Score: mascot protein score. * Protein quantified by ELISA. S1: stage 1 disease; S2: stage 2 disease.

		Description	Score	ANOVA	Fold Change	Average normalized abundances Control S1 S2
	P51688	N-sulphoglucosamine sulphohydrolase	89.58	1.51e-004	Infinity	0.00	5.23e+004	1856.39
	P16870	Carboxypeptidase E	23.90	1.61e-004	Infinity	0.00	2.06e+004	8488.37
	P25774	Cathepsin S	151.85	5.80e-003	371.97	37.12	1.38e+004	621.63
	P26038	Moesin *	319.43	9.38e-003	47.11	1511.77	7.12e+004	1.57e+004
	P59190	Ras-related protein Rab-15	47.97	0.02	Infinity	0.00	1.74e+005	2088.10
	P29622	Kallistatin	120.72	0.02	170.86	5936.72	4.28e+004	250.56
	Q9H8L6	Multimerin 2	52.32	0.05	Infinity	0.00	8618.93	4.37e+004
	P02766	Transthyretin	78.07	0.05	17.94	1.36e+004	1.42e+005	2.44e+005

Table 5 :

 5 Most notable proteins in the saliva with altered levels between control, S1 and S2 patients (classified by increase in ANOVA p-value) All shown proteins passed thresholds of a fold change ≥ 2 and a p-value ≤ 0.05. Score: mascot protein score. * Protein quantified by ELISA, S1: stage 1 disease; S2: stage 2 disease.

	Accession	Description	Score	ANOVA	Fold Change	Average normalized abundances Control S1 S2
	P19878	Neutrophil cytosol factor 2	26.41	1.21e-005	Infinity	0.00	1207.94	8375.66
	Q8WWU7	Intelectin 2 *	44.75	1.23e-004	120.23	3.11e+004	258.69	350.60
	Q15643	Thyroid receptor-interacting protein 11	25.42	3.24e-004	Infinity	0.00	0.00	1.06e+005
	P22392	Nucleoside diphosphate kinase B	36.48	1.87e-003	7.52	2.34e+004	4.61e+004	1.76e+005
	Q04446	1,4-alpha-glucan-branching enzyme	40.31	0.01	28.76	515.25	7843.70	1.48e+004
	P35241	Radixin	357.98	0.02	2.48	3.30e+005	3.79e+005	8.19e+005
	P27348	14-3-3 protein theta	106.60	0.02	11.21	2444.76	1.19e+004	2.74e+004
	Q04323	UBX domain-containing protein 1	101.23	0.02	408.77	27.88	5286.01	1.14e+004
	P49913	Cathelicidin antimicrobial peptide	93.06	0.02	13.27	1.99e+004	3.28e+004	2.65e+005
	O00602	Ficolin-1	84.63	0.02	43.65	620.60	1512.69	2.71e+004
	Q9BQI0	Allograft inflammatory factor 1-like	41.75	0.02	Infinity	0.00	914.27	7081.79
	P02753	Retinol-binding protein 4	24.27	0.02	3.59	2.92e+004	3.97e+004	1.05e+005
	O60437	Periplakin	353.95	0.02	16.22	6164.29	1.30e+004	1.00e+005
	Q9HC84	Mucin-5B	2457.44	0.03	11.30	2.98e+007	2.64e+006	5.98e+006
	P05120	Plasminogen activator inhibitor 2	255.32	0.03	25.13	2266.87	3.00e+004	5.70e+004
	P62136	Serine/threonine-protein phosphatase PP1-alpha catalytic subunit	130.27	0.03	23.02	1150.30	1.80e+004	2.65e+004
	Q15631	Translin	49.17	0.03	20.39	722.18	2713.99	1.47e+004
	P51888	Prolargin	22.23	0.03	17.73	3887.41	2.59e+004	6.89e+004
	Q13813	Spectrin alpha chain, non-erythrocytic 1	257.23	0.04	11.90	2035.25	4051.54	2.42e+004
	P99999	Cytochrome c	231.53	0.04	4.55	2.80e+004	4.21e+004	1.27e+005
	P04632	Calpain small subunit 1	220.74	0.04	5.69	9299.74	2.29e+004	5.29e+004
	P31997	Carcinoembryonic antigen-related cell adhesion molecule 8	60.26	0.04	26.63	3450.76	1.15e+004	9.19e+004
	P30153	Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform	52.04	0.04	6.29	1660.25	6513.70	1.04e+004
	Q15907	Ras-related protein Rab-11B	43.85	0.04	6.84	4755.71	2.47e+004	3.25e+004

This work was supported by the Foundation for Innovative and New Diagnostic (FIND 2008) (https://www.finddx.org) and Conseil Regional de la Nouvelle Aquitaine and INSERM.

The complete data sets are available in the PRIDE partner repository [START_REF] Vizcaíno | update of the PRIDE database and its related tools[END_REF] under the identification number : PXD007842 as .raw files, Proteome Discoverer 2.1 .pdResult file, associated pep.xml and xlsx files, and label-free report generated by Progenesis QI.

Username: reviewer10851@ebi.ac.uk

Password: NL6d28RK

Supporting Information Legends: Identification and relative quantification of all peptides/proteins from the proteomics experiments and according to the different experimental conditions are publicly available via ProteomeXchange and the PRIDE partner repository under the identification number: PXD007842 as.raw files, Proteome Discoverer 2.1. pdResult file, associated pep.xml and xlsx files, and label-free report generated by Progenesis QI.

Competing interests

The authors declare that they have no competing interests


Pan Dan Robin Augustine

Inbar Schlachet

Didier Rouxel

Patrick Menu

Alejandro Sosnik
email: sosnik@technion.ac.il

Dan Pan

Chitosan ascorbate hydrogel improves water uptake capacity and cell adhesion of electrospun poly(epsilon-caprolactone) membranes

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

Introduction

Impaired wound healing is a medical condition leading to non-healing chronic wounds and foot ulcers resulting in discomfort, increased health care costs and poor quality of life [START_REF] Guo | Factors affecting wound healing[END_REF]. The primary goal of a wound dressing is to prevent the access of pathogens to the wound bed and to maintain appropriate temperature and moisture to facilitate wound healing [START_REF] Boateng | Wound healing dressings and drug delivery systems: A review[END_REF]. Excess build-up of exudates in the wound bed is a leading cause of infection and pathogenesis. Wound dressings with good exudate uptake capacity helps to maintain a moist environment and promotes healing. The use of polymeric membranes that facilitate cell adhesion, migration and proliferation can further enhance the healing rate and improve the quality of the regenerated tissue [START_REF] Augustine | Electrospun polycaprolactone membranes incorporated with ZnO nanoparticles as skin substitutes with enhanced fibroblast proliferation and wound healing[END_REF].

Synthetic biodegradable polymers are robust biomaterials for wound healing applications due to the ability to tailor their bioactivity, mechanical properties and biodegradability [START_REF] Lutolf | Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering[END_REF]. However, their capability to support cell adhesion under in vitro conditions is not always high [START_REF] Gautam | Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method[END_REF]. Strategies such as functionalization with oligopeptides (e.g., arginyl glycyl aspartic acid, RGD), blending with natural proteins that contain this sequence (e.g., collagen) and interact with cellular integrins or incorporation of other agents such as growth factors is often required [START_REF] Liu | Functionalized Synthetic Biodegradable Polymer Scaffolds for Tissue Engineering[END_REF]. Natural polymers are more favourable for promoting cell adhesion and proliferation [START_REF] Malafaya | Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications[END_REF], though they display lower and less tuneable stability under physiological conditions than the synthetic counterparts [START_REF] Kamoun | A review on polymeric hydrogel membranes for wound dressing applications: PVA-based hydrogel dressings[END_REF].

For more than two decades, poly(epsilon-caprolactone) (PCL) has drawn a lot of attention in a plethora of biomedical applications [START_REF] Woodruff | The return of a forgotten polymerpolycaprolactone in the 21st century[END_REF]. PCL displays several advantages including cost-effectiveness, good biocompatibility and mechanical stability and more controlled biodegradability than other polyesters such as poly(lactide) and poly(pactideco-glycolide) [START_REF] Choi | In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor (EGF)[END_REF][START_REF] Bayati | A dermal equivalent developed from adipose-derived stem cells and electrospun polycaprolactone matrix: an in vitro and in vivo study[END_REF]. Previous studies demonstrated the potential of PCL in tissue regeneration including wound repair [START_REF] Sundaramurthi | Electrospun nanofibers as scaffolds for skin tissue engineering[END_REF], Tra Thanh et al., 2018). However, PCL is highly hydrophobic and thus, it shows negligible ability to absorb wound exudates [START_REF] Hong | Fabrication of electrospun polycaprolactone biocomposites reinforced with chitosan for the proliferation of mesenchymal stem cells[END_REF]. In addition, electrospun PCL dressings lack functional groups that facilitate cell adhesion, which is an important feature when designing matrices for wound healing [START_REF] Zhang | Characterization of the surface biocompatibility of the electrospun PCL-collagen nanofibers using fibroblasts[END_REF].

Chitosan, a deacetylated derivative of chitin, presents a series of outstanding features including biodegradability, good biocompatibility, antimicrobial activity, non-toxicity and high versatility of chemical modification [START_REF] Dutta | Perspectives for chitosan based antimicrobial films in food applications[END_REF]. In addition, chitosan-based hydrogels are an appealing class of biomaterial to promote cell adhesion [START_REF] Croisier | Chitosan-based biomaterials for tissue engineering[END_REF] which may be related to its polycationic nature and ability to interact electrostatically with the negatively-charged cell membrane [START_REF] Nettles | Potential use of chitosan as a cell scaffold material for cartilage tissue engineering[END_REF]. Chitosan based hydrogels have been extensively investigated in wound healing applications [START_REF] Shamloo | Accelerated full-thickness wound healing via sustained bFGF delivery based on a PVA/chitosan/gelatin hydrogel incorporating PCL microspheres[END_REF][START_REF] Asfour | Chitosan based Pickering emulsion as a promising approach for topical application of rutin in a solubilized form intended for wound healing: In vitro and in vivo study[END_REF]. However, its use is challenging owing to a relatively high viscosity and limited aqueous solubility at pH >5.5 [START_REF] Ilyina | Enzymic preparation of acid-free-water-soluble chitosan[END_REF] and mechanical properties that can challenge dressing removal [START_REF] Nwe | The Mechanical and Biological Properties of Chitosan Scaffolds for Tissue Regeneration Templates Are Significantly Enhanced by Chitosan from Gongronella butleri[END_REF]. On the other hand, it represents an attractive component for the supplementation of biomaterials presenting limited cell adhesion.

Chitosan ascorbate (CA) is an organic salt formed by the reaction of chitosan with ascorbic acid in water and it shows a more pH-independent solubility profile, thus providing more flexibility in biomaterial processing and fabrication [START_REF] Sekar | Synthesis and characterization of chitosan ascorbate nanoparticles for therapeutic inhibition for cervical cancer and their in silico modeling[END_REF][START_REF] Rossi | Chitosan Ascorbate : A Chitosan Salt with Improved Penetration Enhancement Properties[END_REF], Elbarbary el al., 2015). For example, some studies showed that CA promotes periodontal tissue repair and regeneration owing to the reduced migration of inflammatory cells [START_REF] Xuan | An effectiveness validation: chitosan-ascorbate for periodontal tissue healing and regeneration in a rat periodontitis model[END_REF]. Hybrid natural/synthetic polymeric biomaterials can overcome the low mechanical properties and limited biological stability of natural biomaterials and the lack of cell adhesion moieties of PCL by capitalizing the advantageous features of each one of the components.

We hypothesized that the supplementation of electrospun PCL membranes with CA incorporated by physical infiltrationis a simple, reproducible and cost-effective strategy to improve cell adhesion and proliferation due to the overall reduction of the hydrophobicity. In addition, conferring moderate hydrophilicity to PCL membranes would augment their capacity to absorb exudates which highly desirable in wound regeneration. In this framework, this work describes the fabrication and characterization of a novel CA-supplemented PCL composite membranes for wound healing applications.

Experimental

Materials. PCL with molecular weight 80,000-120000 and tripolyphosphate (TPP) were purchased from Sigma Aldrich (St. Louis, MO, US). Chitosan (CS, degree of deacetylation of 94% and molecular weight of ~50,000 g/mol) was purchased from Glentham Life Sciences (Corsham, UK). Ascorbic acid (AA) was purchased from Fisher Scientific (Loughborough, UK). NaOH was purchased from Merck (NJ, US). Paraformaldehyde, (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) and 4',6-diamidino-2phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (USA). Phalloidin was purchased from Thermo Fisher Scientific (Paris, France). The Live/Dead assay kit was supplied by Sigma-Aldrich. Dulbecco's Modified Eagle's Medium (DMEM, Lonza, Basel, Switzerland) was used for for the culture of human mesenchymal stem cells (hMSCs).

Endothelial basic medium (Lonza) was used for human umbilical vein endothelial cell (HUVEC) culture. Media were supplemented with 10% foetal bovine serum (FBS, Lonza), 100 mg ml -1 fungizone (Fisher), 100 IU ml -1 penicillin (Sigma-Aldrich) and 200 mM Lglutamine (Sigma-Aldrich) on culture plates. All other reagents/solvents used in this study were from standard manufacturers and of analytical grade quality.

Synthesis and characterization of chitosan ascorbate.

For preparing CA, an exact amount of AA to obtain a 1:1 molar ratio with chitosan deacetylated amino groups was dissolved in distilled water under magnetic stirring for 30 min. Afterwards, chitosan was added to obtain a solution of CA under magnetic stirring for 48 h. The product was dialyzed in water, freezedried and characterized by Fourier Transform-Infrared Spectroscopy (FTIR), Proton-Nuclear Magnetic Resonance Spectroscopy ( 1 H-NMR), powder X-rays diffraction (PXRD) and differential scanning calorimetry (DSC).

FTIR samples were prepared at 25°C on KBr (Merck Chemicals GmbH, Darmstadt, Germany) disks and pressed to transparency. FTIR spectra were recorded in an Equinox 55 spectrometer (Bruker Optics Inc., Ettlingen, Germany) from 4000 to 400 cm-1 (32-64 scans with a resolution of 4 cm -1 ).

1 H-NMR spectra of the different products were recorded in a 400-MHz Bruker ® Avance III High Resolution spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) and Spin Works 4.0 software (University of Manitoba, Winnipeg, MB, Canada) using 5% w/v solutions in in D2O with the addition of 25 µL acetic acid-D4. Chemical shifts are reported in ppm using the signal of D2O (4.75 ppm) as internal standard.

PXRD patterns were obtained in an X-Ray Diffractometer MiniFlex, (Rigaku, Tokyo, Japan) under Bragg-Brentano geometry at a scanning rate of 1.25 o /min in the range of 0-600 (with intervals of 0.020) on a Perspex slide.

The thermal properties were analyzed in a DSC 2 STARe system equipped with a thermal analyzer with STARe Software V13 (Mettler-Toledo, Schwerzenbach, Switzerland) with an intracooler (Huber TC100) under a dry N2 atmosphere (flow of 20 mL min -1 ). For this, samples (5-15 mg) were sealed in 40 μL Al-crucible pans (Mettler-Toledo) and subjected to different heating-cooling cycles (10 °C min -1 ). For chitosan and CA, the treatment was as follows: (i) heating from 25 to 100°C (to erase the thermal history), (ii) isothermal heating at 100°C (30 min, to eliminate water traces), (iii) heating from 100 to 300°C, (iv) cooling from 300 to 25°C and (v) heating from 25 °C to 300 °C. For ascorbic acid, the treatment was as follows: (i) heating from 25 to 300°C and (ii) cooling from 300 to 25 °C. For chitosan and CA, the heating from 25 to 300°C in presented.

Preparation of chitosan ascorbate infiltrated PCL membranes. Electrospun PCL

membranes were fabricated according to our previous report [START_REF] Augustine | Electrospun poly(ε-caprolactone)-based skin substitutes: In vivo evaluation of wound healing and the mechanism of cell proliferation[END_REF]. Briefly, 15% w/v PCL (molecular weight of 80,000-120,000 g/mol) solutions were prepared in acetone. Then, 10 mL of this solution was loaded in a syringe with an 18-gauge needle and electrospun at a distance of 15 cm tip from the collector. An applied DC voltage of 18 kV and a flow rate of 2.5 mL•h-1 were maintained during the electrospinning process. After the completion of the electrospinning, deposited membranes were carefully removed and used for the fabrication of CA-loaded membranes.

To produce electrospun PCL membranes supplemented with CA, they were immersed in 1 M NaOH for 4 h to introduce surface hydrophilicity. Then, wet membranes were immersed in CA solution for 4 h to infiltrate the solution into the membrane pores and obtain hybrid PCL/CA composites. Four CA concentrations were used, 0.1% (PCL/CA-0.1), 0.5% (PCL/CA-0.5), 1% (PCL/CA-1) and 2% w/v (PCL/CA-2), to assess its effect on water swelling and cell adhesion. The CA matrix in the hybrid membranes was stabilized by ionotropic crosslinking with TPP (2.5% w/v in water) and formation of a hydrogel infiltrate. Fabricated composite membranes were dialyzed (regenerated cellulose dialysis membranes, nominal MWCO of 3500 g/mol) in ultrapure water for 2 days to remove unreacted reagents.

Characterization of the membranes. Membranes were characterized by High Resolution-Scanning Electron Microscopy (HR-SEM, Ultraplus, Zeiss, Oberkochen, Germany, acceleration voltage 5 kV, samples were coated with gold), X-Ray Diffractometer (XRD, MiniFlex, Rigaku, Tokyo, Japan, 2θ range 0-60 0 ) and FTIR (see above).

Water contact angles on the surface of the samples were measured by a Digital Contact Angle Measurement System equipped with a CCD camera (Phoenex-KGV-5000). PCL and PCL/CA membranes were placed on the stage of the machine. A small water droplet (Milli-Q-water) was carefully placed on the surface of the electrospun membrane by using the software-controlled system. A series of images of the solution droplet were automatically taken. Contact angle was measured from the captured images. All the experiments were carried out at 26°C and about 65% relative humidity. The experiment was repeated for three set of samples and the contact angle was expressed as mean ± SD (n = 3).

The swelling of the different membranes was measured by tracking the weight increase during incubation in phosphate buffer solution (PBS, pH = 7.4). For this, two-inch diameter disks of PCL and PCL/CA membranes were accurately weighed were placed in PBS (37°C) and at different time points, samples were taken out, carefully wiped with a lint free cloth to remove unabsorbed water and weighed. Then, each sample was freeze-dried and weighed again. Water uptake is expressed as an increase in weight percent according to Equation 1Swelling (%) = [(WW -DW)/DW] x 100

(1)

Where WW and DW are the wet and dry weight of the membrane.

Results are expressed as mean ± SD of three experiments.

Cell compatibility and cell adhesion.

The cell compatibility and adhesion were investigated with HUVECs and hMSCs. For this, fresh human umbilical cords were obtained after fullterm births (caesarean section or normal vaginal delivery) with informed consent using the guidelines approved by the University Hospital Centre of Nancy (France). hMSCs and HUVECs were isolated and amplified as previously reported [START_REF] El Omar | Immunomodulation of endothelial differentiated mesenchymal stromal cells: Impact on T and NK cells[END_REF]. hMSCs on passage 4 and HUVECs on passage 2 were seeded on PCL and PCL/CA membranes at 50,000 cells/cm 2 and cultured with appropriate medium for 24 h. To visualize cell attachment, samples were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% w/v Triton X-100 solution for 15 min and cell cytoskeletons were stained with phalloidin and nuclei were stained with DAPI. Then, the images were taken with a fluorescent microscope (Leica DMI 3000B, Munich, Germany). To assess cell compatibility of hMSCs and HUVECs on the developed membranes, Live/Dead and MTT assays were carried out according to the corresponding to protocols provided by the manufacturers (n = 3). From the fluorescent images of Live-Dead assay results, live and dead cells were counted and the percentage of viable cells was calculated from Equation 2.

Viable cells (%) = Live cells/Total cells x 100 (2)

Statistical analysis. The statistical analysis was performed using the un-paired Student's ttest and "One way ANOVA" with Dunnett's post-test performed using Graph-Pad Prism Version 6.04 (San Diego, CA, USA).

Results and Discussion

Synthesis of CA. CA was synthesized by the direct reaction of chitosan and ascorbic acid in water (Figure 1).

Figure 1. Scheme of the formation of CA.

After profuse dialysis to remove any residues and freeze-drying, the formation of the salt was confirmed by FTIR and 1 H-NMR. FTIR spectra of pure chitosan, ascorbic acid, and CA are shown in to its partial deacetylation [START_REF] Ma | Synthesis of cationic polyacrylamide by low-pressure UV initiation for turbidity water flocculation[END_REF](Ma et al., , Çetinus et al., 2007)). Ascorbic acid presents a strong characteristic peak at 1753 cm -1 of C=O stretching of the five-membered lactone ring system and 1672 cm -1 of C=C stretching (Yohannan Panicker et al., 2006). CA shows a shifting and weakening of the band at 3409 cm -1 due to the strong H bonding of C=O of ascorbic acid with amine groups in chitosan and from 2922 to 2846 cm -1 with a slight broadening (Figure 2A). The characteristic band of ascorbic acid at 1753 cm -1 shifted to 1720 cm -1 in the salt due to the weakening of C=O bond by an inductive effect. In addition, the band at 1672 cm -1 (C=C) shifted to 1590 cm -1 in CA (Figure 2A).

Moreover, a new band belonging to ammonium and iminium salt-like structure appeared at 2354 cm -¹ [START_REF] Ragamathunnisa | Spectroscopic study on Thiourea and Thiosemicarbazide in Nonaqueous media[END_REF].In 1 H-NMR, pure chitosan showed peaks at 3.0 (H-C-NH2) and 3.5-4.0 ppm (methylene on the backbone) [START_REF] Xiaolin | Synthesis and evaluation of chitosan-vitamin C complexes[END_REF] and AA at 4.8 (CH-ring), 4.02-4.07 (-CH) and 3.56-3.67 ppm (-CH2) (Figure 2B). The carboxylic group of AA reacted with the -NH2 of chitosan to form an ammonium carboxylate (Figure 2) that results in the shifting of the peak of AA from 4.8 to 4.5 ppm due to ionic interaction in the formation of a salt [START_REF] Xiaolin | Synthesis and evaluation of chitosan-vitamin C complexes[END_REF]. The 1 H-NMR spectrum of the salt presented the characteristic peak of primary amine at 3.00 ppm, indicating that despite the formation of CA, there were free amine groups for the crosslinking with TPP (Figure 2B), as described elsewhere [START_REF] Rossi | Chitosan ascorbate nanoparticles for the vaginal delivery of antibiotic drugs in atrophic vaginitis[END_REF]. This partial reaction between primary amine moieties and AA most probably stemmed from steric hindrance. In addition, we integrated the relative intensity of this peak at 3.0 ppm and compared it to that of pure CS. CA showed a decrease of approximately 23% in the relative intensity of this peak which indicates the reduction of number of free amine groups compared to CS. These results indicated the successful formation of the salt by the reaction between AA and amine group in CS. In addition, we characterized CA by PXRD and DSC.

Both analyses indicated the formation of a completely amorphous salt (Figure S1). flattened agglomerates of CA on the surface of fibers with an increase of the average diameter to 4.6 ± 2.2 µm (data not shown). DSC analysis of the composite membranes indicated the presence of CA which was evident from the thermal transitions observed from 150 to 280°C for all the PCL/CA specimens (Figure S2). The observed broad peaks might be due to the thermal degradation of CA [START_REF] Sarasam | Characterization of chitosan-polycaprolactone blends for tissue engineering applications[END_REF]. Moreover, we observed a 4-9% increase in the dry weight of the membranes after infiltration of CA which confirmed the successful infiltration of CA in the PCL/CA membranes. Surface hydrophilicity and swelling. The surface hydrophobicity can be estimated by measuring the contact angle. Results of water contact angle measurements of pure PCL and PCL/CA membranes obtained by the sessile drop technique are summarized in Table 1 and exemplified in Figure S3 for PCL/CA-2. Pure PCL showed a high contact angle of 104 ± 6 o that was consistent with the high hydrophobicity of the surface. NaOH treatment hydrophilized the surface and the contact angle decrease to 64 ± 5 o owing to a higher wettability, in good agreement with the literature [START_REF] Wang | Enhancing the hydrophilicity and cell attachment of 3D printed PCL/graphene scaffolds for bone tissue engineering[END_REF]. The mechanism is the partial hydrolysis of ester bonds of PCL on the fiber surface that increases the concentration of free hydroxyl and carboxyl groups [START_REF] De Luca | Chemical surface modification of poly-εcaprolactone improves Schwann cell proliferation for peripheral nerve repair[END_REF]. This hydrolysis is not detrimental of the mechanical properties. These groups played a key role to favor the adsorption of CA onto the membrane surface and its retention between inside the pores for the later stabilization by crosslinking. CA modification led to an additional decrease of the contact angle; e.g., PCL/CA-0.5 and PCL/CA-2 showed values of 53 ± 5 to 47 ± 8° owing to the presence of polar functional groups of chitosan on the surface [START_REF] Good | Contact angle, wetting, and adhesion: a critical review[END_REF]. These findings also confirmed the successful modification of the membrane. Similar results were reported for chitosan-coated PCL membranes [START_REF] Shalumon | Fabrication of chitosan/poly(caprolactone) nanofibrous scaffold for bone and skin tissue engineering[END_REF] and PCL/chitosan blend membranes [START_REF] Jin | Porous pcl/chitosan and nha/pcl/chitosan scaffolds for tissue engineering applications: fabrication and evaluation[END_REF]. The capacity to absorb fluids and exudates is a desired property of biomaterials envisioned as wound dressings, particularly of highly-exudating chronic wounds [START_REF] Mayet | A comprehensive review of advanced biopolymeric wound healing systems[END_REF]. In general, CA supplementation resulted in a substantial increase of the swelling capacity of the membranes (Figure 4). PCL membranes did not swell much with values that ranged between 10.8 ± 3.5% and 16.7 ± 2.8% along the entire assay (6 h). This limited weight gain stemmed from the unspecific retention of aqueous medium inside the small pores of the electrospun hydrophobic membrane. Pretreatment with NaOH increased the hydrophilicity of the membrane surface and thus, the swelling increased to 80.5 ± 14.8% to 127.6 ± 17.6%. Then, incorporation of CA and its crosslinking, increased the swelling up to approximately 800% owing to the formation of a swollen stable hydrogel [START_REF] Rossi | Chitosan ascorbate nanoparticles for the vaginal delivery of antibiotic drugs in atrophic vaginitis[END_REF]; as expected, the higher the CA concentration, the more substantial the swelling. These findings are noteworthy towards the application of these membranes in wound healing and are comparable to those reported for other PCL/chitosan systems [START_REF] Jin | Porous pcl/chitosan and nha/pcl/chitosan scaffolds for tissue engineering applications: fabrication and evaluation[END_REF]. The application of CA hydrogel infiltrated PCL membranes offers a highly absorbent wound dressing platform that can manage moderate to heavy exudate. Cell adhesion and cell viability studies. As we described above, we hypothesized that the modification of PCL membranes with CA would improve cell adhesion. To evaluate the cell adhesion properties of these membranes, we seeded hMSCs and HUVECs on the membranes infiltrated with different amounts of CA and compared the results with pure PCL counterparts. In general, cell adhesion and proliferation were higher in all the PCL/CA membranes than in bare PCL membranes with some changes based on the cell type. In the case of hMSCs, PCL/CA-0.5, PCL/CA-1 and PCL/CA-2 showed relatively similar adhesion, indicating that a relatively low amount of CA was sufficient (Figure 5A, upper row).

However, the difference in hMSC cell adhesion between bare PCL and PCL/CA membranes was marginal. Conversely, in the case of HUVECs, PCL/CA-2 membranes were the most efficient (Figure 5A, lower row). The observed differences in cell adhesion behaviour between hMSC and HUVEC on the membranes revealed the intrinsic difference in the inherent nature of the cells. HUVEC are recognized as inactive cells unless stimulated by an extracellular signal such as hypoxia, mechanical stimuli, or biochemical signals [START_REF] Gupta | Endothelial and stem cell interactions on dielectrophoretically aligned fibrous silk fibroinchitosan scaffolds[END_REF]. In this study, we showed that CA hydrogel coating might have also stimulated the proliferation of HUVEC, the effect being less pronunced for hMSC. Live-dead assay was also carried out to visualize the cells in the membranes (Figure 5B). The good cell In addition, the MTT cell metabolic assay confirmed that the membranes are cell compatible with a gradual increase in cell viability for higher CA concentrations (Figure 5E,F). These results confirmed the beneficial effect of CA supplementation on the cell compatibility and adhesion in vitro of both hMSCs and HUVECs, two cell types that play a fundamental role in wound healing. Future studies will asssess the performance of these membranes in murine models of partial wounds.

Conclusion

In this work, we report on the development and characterization of electrospun PCL membranes infiltrated with different concentrations of CA that result in the formation of a hydrogel filler. HR-SEM analysis confirmed the successful infiltration of CA within the electrospun PCL fibers. Water contact angle and swelling studies demonstrated the increase in hydrophilicity and exudate uptake capacity of the composite membranes. Finally, cell adhesion and compatibility using hMSCs and HUVECs showed better cell adhesion and cell viability on CA hydrogel-supplemented PCL membranes than on pristine counterparts.

Owing to the excellent exudate management and cell adhesion properties, the developed PCL/CA membranes are promising for wound healing applications.

Figure 2A .

 2A Figure 2A. Chitosan showed stretching vibration bands of -OH and -NH2 at 3465 cm -1 (Zhang et al., 2016) and -CH-at 2922and 2845 cm -1 (Halevas et al., 2017). Bands at 1645 (C=O stretching of amide I) and 1325 cm -1 (C-N stretching of amide III) indicated the presence of residual N-acetyl groups due

Figure 2 .

 2 Figure 2. (A) FTIR and (B) 1 H-NMR spectra of chitosan (CS), ascorbic acid (AA) and chitosan ascorbate (CA). Peaks at 3 ppm indicate the presence of free amine groups.

Figure 3 .

 3 Figure 3. High resolution-scanning electron microscopy micrographs of(A) pure PCL, (B) PCL/CA-0.5, (C) PCL/CA-1 and(D) PCL/CA-2 membranes.

Figure 4 .

 4 Figure 4. Swelling of the different hybrid PCL/CA membranes. Untreated and NaOH-treated (PCL-NaOH) membranes were used as controls.

  membranes and especially of PCL/CA-0.5, PCL/CA-1 and PCL/CA-2 was confirmed by the presence of only few dead cells stained in red by propidium iodide. The percentage of living cells was calculated and presented in Figure5C,D. All the membranes infiltrated with CA showed higher percentage of live cells than pure and NaOH-treated PCL.

Figure 5 .

 5 Figure 5. Interaction of MSCs and HUVECs with PCL membranes. (A) Fluorescent images of MSCs and HUVECs seeded on PCL, NaOH-treated PCL(PCL-NaOH) and PCL/CA membranes with CA content between 0.5% and 2% w/w, (B) live-dead assay of MSCs and HUVECs cultured on PCL, NaOH-treated PCL and PCL/CA membranes. Green cells are living cells, whereas red coloured cells are dead cells. (C) and (D) are the percentage of living HUVECs and MSCs on the membranes, respectively. (E) and (F) represent the cell viability of HUVECs and MSCs estimated by the MTT assay, respectively.

Table 1 .

 1 Water contact angle measurements on pure PCL and PCL/CA membranes.

	Sample	Contact angle (Degrees)
	PCL	104 ± 6
	NaOH-treated PCL 64 ± 5
	PCL/CA-0.5	53 ± 5
	PCL/CA-1	48 ± 4
	PCL/CA-2	47 ± 8