Samuel Groeschel

Lucie Hertz-Pannier

Matthieu Delion

Sébastien Loustau

Béatrice Husson

Manoelle Kossorotoff

Cyrille Renaud

Sylvie Nguyen

The Tich

Stéphane Chabrier

Mickaël Dinomais

| Lucie Hertz-Pannier

| Matthieu Delion

| S Ebastien Loustau

| B Eatrice Husson

Sylvie Nguyen The Tich

| St

Ephane Chabrier

Mickael Dinomais
email: mickael.dinomais@les-capucins-angers.fr

Mickael Dinomais

Association of transcallosal motor fibres with function of both hands after unilateral neonatal arterial ischemic stroke

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

AIM The objective of this study was to investigate the involvement of the motor fibres of the corpus callosum after unilateral neonatal arterial ischemic stroke (NAIS) of the middle cerebral artery territory and the relationship to both ipsilesional and contralesional hand function.

METHOD Using high-resolution structural magnetic resonance imaging (MRI), functional MRI, and magnetic resonance diffusion-tractography, we compared the midsagittal area of the motor part of the corpus callosum (defined by the fibres connecting the precentral gyri) between 33 7-year-old children after unilateral NAIS and 31 typically developing 7-year-old children. Hand motor performance was assessed by the box and blocks test.

RESULTS Children after NAIS showed on average significantly smaller motor corpus callosum area compared to typically developing children (p<0.001, without differences of the nonmotor corpus callosum area). In addition, there was a significant positive association between the motor part of the corpus callosum and both contralesional (Pr(>|t|)=0.034) and ipsilesional hand motor performance (Pr(>|t|)=0.006) after controlling for lesion volume and sex. In a post-hoc analysis the additional contribution of corticospinal tract damage was evaluated.

INTERPRETATION Compared to typically developing children, children after NAIS exhibited a smaller motor part of their corpus callosum associated with reduced contralesional but also ipsilesional manual dexterity. These results indicate that the affection of transcallosal motor fibres in unilateral NAIS might be of functional relevance and an important part of the involved structural network that should be elucidated in further studies.

in children after unilateral neonatal arterial ischemic stroke (NAIS).

Neuroimaging data showed that unilateral early brain injury can lead to different patterns of motor system reorganization depending on the type and time of lesion occurrence. [START_REF] Holmstrom | Hand function in relation to brain lesions and corticomotor-projection pattern in children with unilateral cerebral palsy[END_REF][START_REF] Staudt | Reorganization after pre-and perinatal brain lesions[END_REF] Patients with prenatal or perinatally acquired unilateral lesions to either the motor cortex or the corticospinal tract may develop ipsilateral corticospinal pathways, which allow the unaffected hemisphere to control the paretic hand (ipsilateral reorganization of motor The corpus callosum is the largest white matter pathway in the brain connecting bi-directionally homotopic cortical areas between the two hemispheres. The motor part of the corpus callosum can be defined as the part connecting the primary motor areas (M1) in the precentral gyri [START_REF] Hofer | Topography of the human corpus callosum revisitedcomprehensive fiber tractography using diffusion tensor magnetic resonance imaging[END_REF] and is important for unimanual and bimanual motor performance through inhibitory and excitatory effects. [START_REF] Wahl | The human motor corpus callosum[END_REF][START_REF] Gooijers | Interactions between brain structure and behavior: the corpus callosum and bimanual coordination[END_REF] Although the association between structural integrity of the corpus callosum and motor performance has been demonstrated in adults with stroke, [START_REF] Li | The microstructural status of the corpus callosum is associated with the degree of motor function and neurological deficit in stroke patients[END_REF][START_REF] Wang | Degeneration of corpus callosum and recovery of motor function after stroke: a multimodal magnetic resonance imaging study[END_REF] this relationship remains unexplored control). The presence of mirror movements reflects this interhemispheric reorganization. [START_REF] Staudt | Two types of ipsilateral reorganization in congenital hemiparesis: a TMS and fMRI study[END_REF] This type of interhemispheric reorganization is often observed following unilateral periventricular brain lesions, 8 but can also be observed less often following perinatal ischemic stroke. [START_REF] Van Der Aa | Neonatal neuroimaging predicts recruitment of contralesional corticospinal tracts following perinatal brain injury[END_REF] Models for understanding developmental neuroplasticity of the motor system after early unilateral lesions suggest interactions between the ipsilesional and contralesional motor cortex [START_REF] Juenger | Microstructure of transcallosal motor fibers reflects type of cortical (re-)organization in congenital hemiparesis[END_REF] as the key to clinical function, but the structural connection between the motor cortices (i.e. the corpus callosum) remains an understudied element of such models. The same models have also recently been translated in positive neuromodulation clinical trials, suggesting high translational relevance. [START_REF] Zewdie | contralesional corticomotor neurophysiology in hemiparetic children with perinatal stroke: developmental plasticity and clinical function[END_REF][START_REF] Kirton | Transcranial direct current stimulation for children with perinatal stroke and hemiparesis[END_REF] NAIS represents 'an ideal human model' for understanding developmental motor plasticity. [START_REF] Kirton | Life after perinatal stroke[END_REF] Having more mechanistic studies on how the two motor cortices structurally interact could add more knowledge on how to design 'advance personalized' neuromodulation therapies for children having suffered from NAIS. [START_REF] Kirton | Transcranial direct current stimulation for children with perinatal stroke and hemiparesis[END_REF] An association between the size of the corpus callosum and the motor performance of both hands after early unilateral brain lesions would support the importance of the corpus callosum for bilateral motor performance. In this work, we examined the relationship between structural changes of the corpus callosum, especially of its motor part, and the motor performance of both hands after unilateral NAIS.

METHODS Participants

As part of the AVCnn study, [START_REF] Husson | Motor outcomes after neonatal arterial ischemic stroke related to early MRI data in a prospective study[END_REF][START_REF] Chabrier | Obstetrical and neonatal characteristics vary with birthweight in a cohort of 100 term newborns with symptomatic arterial ischemic stroke[END_REF] 33 7-year-old children (13 females, 20 males; mean age 7y 3mo [SD 2.46mo], range 6y 11mo-7y 11mo) with a history of unilateral NAIS of the middle cerebral artery (MCA) territory participated in a follow-up investigation with 3T magnetic resonance imaging (MRI) and motor evaluation. The participants in this study were the same as in our previous study, [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF][START_REF] Dinomais | Does contralesional hand function after neonatal stroke only depend on lesion characteristics?[END_REF] except for five participants where neither diffusionweighted imaging nor box and blocks test (BBT, see below) data were available. The recruitment criteria of the participants were given in mode details in the previous cohort description. [START_REF] Husson | Motor outcomes after neonatal arterial ischemic stroke related to early MRI data in a prospective study[END_REF][START_REF] Chabrier | Obstetrical and neonatal characteristics vary with birthweight in a cohort of 100 term newborns with symptomatic arterial ischemic stroke[END_REF][START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF][START_REF] Dinomais | Does contralesional hand function after neonatal stroke only depend on lesion characteristics?[END_REF][START_REF] Chabrier | Multimodal outcome at 7 years of age after neonatal arterial ischemic stroke[END_REF] Further sample characteristics are presented in Table I.

Motor performance of the ipsilesional and contralesional hands was assessed with the BBT, a reliable and validated tool for measuring gross manual dexterity in children. [START_REF] Mathiowetz | Box and block test of manual dexterity: norms for 6-19 years olds[END_REF] For each child after NAIS, the maximum number of cubes was defined that were transferred by the ipsilesional and the contralesional hand from one compartment to the other in one minute.

The presence of mirror movements was also assessed 8 using video-recorded examination. Children after NAIS exhibiting contralateral involuntary finger movements that were reproducible, phasic, and exceeding the amplitude of 10mm at the fingertips during voluntary unimanual movements (repetitive fist clenching, repetitive index finger-to-thumb opposition, and sequential finger-to-thumb opposition) were considered having mirror movements. This definition of mirror movements 8 corresponds to grades 3 to 4 (i.e. 3=strong and unsustained repetitive movement, 4=movement equal to that performed by the intended hand) in the criteria proposed by Woods and Teuber. [START_REF] Woods | Mirror movements after childhood hemiparesis[END_REF] In addition, MRI data of a control group of 31 typically developing children were acquired, who had no neurological history and normal neurological examination (17 males, 14 females; mean age 7y 8mo [SD 6.88 mo], range 6y 9mo-8y 9mo).

Written informed consent from typically developing children/patients/parents was obtained and the study protocol was approved by the local Ethics Committee (eudract-number-2010-A00976-33).

MRI acquisition and processing

All images were acquired on the same 3.0 Tesla scanner (Siemens Trio, 12-channel head coil, at Neuropsin-CEA-Saclay, France).

As described before, [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF][START_REF] Dinomais | Does contralesional hand function after neonatal stroke only depend on lesion characteristics?[END_REF] a high-resolution 3D T1weighted image was obtained covering the whole brain (176 sagittal slices, repetition time 2300ms, echo time 4.18ms, field of view 256mm, flip angle=9°, voxel size 19191mm [START_REF] Gooijers | Interactions between brain structure and behavior: the corpus callosum and bimanual coordination[END_REF] ) as well as a diffusion-weighted dual spin-echo planar imaging sequence with 30 diffusion-weighted directions and a diffusion-weighting of b=1000s/mm 2 (repetition time 9500ms, echo time 86ms, 40 slices, voxel size 1.87591.87593mm 3 ). Diffusion-weighted images were processed using the MRtrix software package [START_REF] Tournier | MRtrix: diffusion tractography in crossing fiber regions[END_REF] (www.mr trix.org), reconstructing the transcallosal motor pathways using probabilistic tractography based on constrained spherical deconvolution. [START_REF] Tournier | Robust determination of the fibre orientation distribution in diffusion MRI: non-negativity constrained super-resolved spherical deconvolution[END_REF] Functional localization of primary motor cortices (M1s) Functional MRI (fMRI) was used for functional identification of both M1s (for tractography of transcallosal fibres). An echo planar imaging sequence was used to acquire functional series (repetition time 2500ms, echo time 30ms, 40 axial sequential slices of 3.0mm slice thickness, in plane matrix=64964, yielding a voxel size of 39393mm 3 ), with 60 functional volumes per session.

The fMRI paradigm was implemented in block design (first practised by the child in a mock scanner) with two conditions (20s each, repeated 5 times), checked and video recorded during the acquisition: (1) repetitive hand opening and closing at a frequency of 1Hz (=motor task condition, guided by 1Hz metronome sounds over headphones);

(2) rest condition. Two identical functional sessions were What this paper adds

• Unilateral neonatal arterial ischemic stroke (NAIS) in middle cerebral artery territory affects transcallosal motor fibres.

• Affection of transcallosal motor fibres after NAIS is associated with both contralesional and ipsilesional gross manual dexterity as assessed by box and block test.

• Unilateral lesions in the developing brain may influence dexterity of both hands through involvement of corpus callosum.

containing the motor part of the corpus callosum (=mCC), indicated in Figure 1. Both the mCC and the whole corpus callosum (=wCC) areas were measured manually in the midsagittal plane (defined as the medial plane, including the third ventricle, after analysis on the three planes) by consensus of two expert raters blinded to clinical information (Fig. 1), [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF] using ITK-SNAP software (www.itksnap.org). [START_REF] Yushkevich | User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability[END_REF] In addition, the total intracranial (midsagittal) area was measured to correct for the variability of brain size across participants. Hence, final mCC and wCC were normalized by brain size (ratio). The rest of the corpus callosum (=rCC) was calculated as follows: rCC=wCC-mCC.

Measuring the midsagittal areas of the transcallosal fibres using high-resolution 3D T1-weighted images (1mm slice thickness) rather than diffusion tensor imaging parameters (3mm slice thickness) has the great advantage of reducing partial volume effects from cerebrospinal fluid contamination of the ventricles, especially in case of corpus callosum atrophy in children after NAIS [START_REF] Jones | Twenty-five pitfalls in the analysis of diffusion MRI data[END_REF][START_REF] Vos | Partial volume effect as a hidden covariate in DTI analyses[END_REF] and therefore making the results more meaningful. It would be almost impossible to disentangle those partial volume effects from real changes in diffusivity when interpreting changes in FA, diffusion tensor (DTI) eigenvalues, or mean diffusivity in a thin structure like the corpus callosum with performed, one for each hand. fMRI during active motor tasks may indicate shift in M1 activation toward the unaffected hemisphere when moving the paretic hand. [START_REF] Mackey | Upper limb function and cortical organization in youth with unilateral cerebral palsy[END_REF] The acquisition was successful in 29 out of 33 children after NAIS and in 30 out of 31 typically developing children. Single-subject fMRI analysis [START_REF] Fadiga | Functional magnetic resonance imaging: measuring versus estimating[END_REF] was done using standard functionality in the SPM8 software (www.fil.ion.ucl.ac.uk/ spm) running in Matlab R2011a (MathWorks, Natick, USA). A statistical parametric map was calculated for each participant and for each hand using the framework of the general linear model (significance p<0.001 uncorrected).

Each participant's 3D-T1-weighted image and fMRI activation maps were co-registered to the individual fractional anistropy (FA) image using FSL FLIRT tool (http:// fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSL) [START_REF] Jenkinson | Improved optimization for the robust and accurate linear registration and motion correction of brain images[END_REF]

Corpus callosum delineation and measurements

To identify the motor fibres connecting primary motor areas, the precentral gyri were defined anatomically as described before, [START_REF] Groeschel | Identification and interpretation of microstructural abnormalities in motor pathways in adolescents born preterm[END_REF] using overlaid anatomical and FA images with additional confirmation by fMRI activation of the hand motor paradigm as described above. [START_REF] Groeschel | Identification and interpretation of microstructural abnormalities in motor pathways in adolescents born preterm[END_REF] The transcallosal motor fibres were projected on to the co-registered 3D-T1-weighted images to identify the voxels additional atrophy, given the relatively low-resolution, diffusion-weighted data. [START_REF] Groeschel | Identification and interpretation of microstructural abnormalities in motor pathways in adolescents born preterm[END_REF][START_REF] Vos | Partial volume effect as a hidden covariate in DTI analyses[END_REF] Statistics Area measurements (mCC, rCC, and wCC) were compared between children after NAIS and typically developing children using a parametric unpaired t-test.

A linear model (analysis of covariance [ANCOVA]) was conducted to determine the relationship between the BBT scores (separately for ipsilesional and contralesional hand) and cross-sectional areas of the corpus callosum (mCC and rCC) controlling for lesion volume (obtained from our previous work) [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF] and sex, see Table I. The principal assumptions of a linear model are the independence, normality, and the homoscedasticity of the error, which were tested over the residuals (and found to hold true). Lesion volume and sex were put as nuisance covariables in our statistical model due their possible influence on motor outcomes following NAIS. [START_REF] Golomb | Cerebral palsy after perinatal arterial ischemic stroke[END_REF] Exact levels of significance are reported. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using R (www.r-project.org).

Post-hoc analysis of correlation with corticospinal tract

To assess the additional involvement of the corticospinal tract in hand motor function after NAIS, we correlated DTI values (for both ipsilesional and contralesional side) at the level of the posterior limb of the internal capsule with the BBT scores using Pearson correlation coefficient. The DTI values were calculated as part of our previous work, [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF] where the corticospinal tract was delineated using probabilistic tractography and the measurement region in the posterior limb of the internal capsule was extracted using equally distributed levels along the tract.

RESULTS

Midsagittal area of corpus callosum (Fig. 2)

The area of wCC was smaller in children after NAIS (mean 0.03678 [SD 0.005365], 95% confidence interval [CI] 0.03488-0.03869) than in typically developing children (mean 0.04048 [SD 0.004016], 95% CI 0.03901-0.04196, p=0.029). When focusing on the motor part of the corpus callosum, mCC was on average clearly smaller in children after NAIS (mean 0.003934 [SD 0.0008429], 95% CI 0.003636-0.004233) than in typically developing children (mean=0.005867, SD 0.0008010, 95% CI 0.005574-0.006161, p<0.001), and the difference between the two groups was more pronounced (p<0.001) than for wCC (p=0.029).

By contrast, there was no statistical difference between the two groups concerning the rCC (mean 0.004 [SD 0.001], 95% CI 0.004 for NAIS; mean 0.03462 [SD 0.003667], 95% CI 0.03327-0.03596 for typically developing children, p=0.11). of neurophysiological signalling and associated with the motor performance of both sides of the human body. [START_REF] Fling | Fundamental differences in callosal structure, neurophysiologic function, and bimanual control in young and older adults[END_REF] The current study further supports the importance of these interhemispheric interactions for motor function through transcallosal motor fibres after early brain lesions, in line with earlier studies in adults with stroke. [START_REF] Li | The microstructural status of the corpus callosum is associated with the degree of motor function and neurological deficit in stroke patients[END_REF][START_REF] Wang | Degeneration of corpus callosum and recovery of motor function after stroke: a multimodal magnetic resonance imaging study[END_REF] Early injury to the developing white matter leads to structural changes of the corpus callosum in children with periventricular leukomalacia, a major form of preterm brain injury, [START_REF] Hoon | Diffusion tensor imaging of periventricular leukomalacia shows affected sensory cortex white matter pathways[END_REF][START_REF] Koerte | Anisotropy of transcallosal motor fibres indicates functional impairment in children with periventricular leukomalacia[END_REF] which were related to gross motor function. [START_REF] Davatzikos | Correlation of corpus callosal morphometry with cognitive and motor function in periventricular leukomalacia[END_REF] Furthermore, the reduction in size of the corpus callosum was associated with poor motor performance in children with neonatal encephalopathy. [START_REF] Van Kooij | Corpus callosum size in relation to motor performance in 9to 10-year-old children with neonatal encephalopathy[END_REF] Our study shows that early unilateral focal lesions, also affecting grey matter, have an impact on white matter structures remotely from the primary infarct later during childhood, with long lasting functional consequences.

The size reduction of the motor part of the corpus callosum in children after NAIS might relate to secondary axonal degeneration following the primary ischemic neuronal cell body damage and resulting in a loss of transcallosal motor fibres (diminution of axon density) and/or subsequently reduced myelin thickness. Interestingly, corpus callosum area measurements have been associated with axon density. [START_REF] Evangelou | Regional axonal loss in the corpus callosum correlates with cerebral white matter lesion volume and distribution in multiple sclerosis[END_REF] In the acute phase after NAIS, pre-Wallerian degeneration of axons remote from the infarct site has been demonstrated in the corticospinal tract, [START_REF] Husson | Motor outcomes after neonatal arterial ischemic stroke related to early MRI data in a prospective study[END_REF][START_REF] Dinomais | Does contralesional hand function after neonatal stroke only depend on lesion characteristics?[END_REF] visible as a reduced ADC on diffusion imaging, often followed by peduncular atrophy, both being highly predictive of the further development of unilateral cerebral palsy. Similar to the corticospinal tract, the reduced size of the mCC might result from Wallerian degeneration.

The association between focal grey matter injuries and the topographic distribution of Wallerian degeneration in the corpus callosum has been described previously. [START_REF] De Lacoste | Topography of the human corpus callosum[END_REF] In this (historical) post-mortem study, the authors showed degeneration of the motor part of the corpus callosum in case of cortical lesions located in the MCA territory. Our finding of the selective involvement of the motor part of the corpus callosum in the case of NAIS in the MCA territory confirms the early findings, [START_REF] De Lacoste | Topography of the human corpus callosum[END_REF] in line with the topography of the corpus callosum with axons traversing the corpus callosum to their (cortical) origin. [START_REF] Lebel | Agerelated regional variations of the corpus callosum identified by diffusion tensor tractography[END_REF] Our finding that the ipsilesional corticospinal tract damage correlated with the involvement of transcallosal motor fibres underlines that unilateral NAIS in MCA territory damages a broader structural network including fibres of both the corticospinal tract and the mCC, probably due to the location of the infarct. The damage of the ipsilesional structural corticospinal tract might be related more directly to the contralesional hand motor dysfunction in these children; [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF] whereas the involvement of the transcallosal motor fibres (mCC) could be responsible for the ipsilesional hand motor dysfunction, with only marginal contribution of the contralesional corticospinal tract. For children with reorganization of motor control to the contralesional hemisphere (ipsilateral motor reorganization), the contribution of the

Hand motor performance and corpus callosum

The mean BBT scores for the contralesional hand were 28.39 cubes per minute (SD 9.40; 95% CI of mean: 25.06-31.73) and for the ipsilesional hand 32.18 cubes per minute (SD 6.32; 95% CI 29.94-34.42). Only two participants (2/33, 6% of our sample, participants #24 and #34) exhibited mirror movements.

In children after NAIS, the ANCOVA analysis (F(4, 28) =3.062, p=0.033) showed a positive association between the BBT score of the contralesional hand and mCC area (t=2.233, Pr(>|t|)=0.034) by contrast to rCC area (t=0.304, Pr(>|t|)=0.76) after controlling for lesion volume (t=À0.784, Pr(>|t|)=0.44) and sex (t=0.657, Pr(>|t|)=0.52). The Durbin-Watson test (DW=2.2354, p=0.80) showed absence of autocorrelation of the residuals.

The ANCOVA analysis (F(4,28)=2.903, p=0.040) also found a positive association between the BBT score of the ipsilesional hand and mCC area (t=2.954, Pr(>|t|)=0.0063) but not with rCC area (t=0.217, Pr(>|t|)=0.83) after controlling for lesion volume (t=1.491, Pr(>|t|)=0.15) and sex (t=0.467, Pr(>|t|)=0.64). The Durbin-Watson test (DW=2.4198, p=0.92) again dismissed correlation of the residuals.

Hand motor performance and corticospinal tract

There was a significant positive correlation of the ipsilesional corticospinal tract with the contralesional hand function (contralesional BBT scores with FA r=0.41 [p=0.017], negative correlations with apparent diffusion coefficient (ADC) r=À0.46 [p=0.007], with radial r=À0.45 [p=0.009], and axial diffusivity r=À0.46 [p=0.007]), but no significant correlation (p>0.1) with ipsilesional BBT scores. In addition, the contralesional corticospinal tract did not show a significant correlation of FA and eigenvalues with the ipsilesional BBT scores, only the ADC values were negatively correlated (r=À0.46, p=0.008). Also, there was no significant correlation of contralesional corticopsinal tract values with contralesional BBT scores (p>0.1).

To investigate whether the corticospinal tract damage of the ipsilesional corticospinal tract correlates with the mCC damage as expected due to their same anatomical origin (MCA stroke territory), we correlated the mCC area with corticospinal tract results in the posterior limb of the internal capsule. We found significant positive correlation (Pearson's r=0.40 [p=0.001]) for FA and negative correlations (Pearson's r=À0.42 for ADC [p=0.001], r=À0.38 for radial diffusivity [p<0.01], and r=À0.48 for axial diffusivity [p<0.001]).

DISCUSSION

The present study underlines the importance of the motor part of the corpus callosum for bilateral hand motor function after unilateral NAIS. During movement, activity of one M1 is modulated by the activity of the opposite M1 through inhibitory and/or excitatory effects transmitted by motor transcallosal fibres. [START_REF] Wahl | The human motor corpus callosum[END_REF] The structural integrity of these fibres is hereby crucial for the interhemispheric exchange contralesional corticospinal tract in the ipsilesional hand motor function could be more important. Further studies are needed to test this in this sub-population.

While reporting an association between transcallosal motor fibres and hand motor function, it is of importance to keep in mind that with our study design we could not prove causality, nor did we investigate the extended structural network of fibre pathways known to be involved in hand motor dysfunction after unilateral NAIS. Such network analyses would require a whole-brain structural analysis and additional neurophysiological measures in order to understand potential causal effects. [START_REF] Zewdie | contralesional corticomotor neurophysiology in hemiparetic children with perinatal stroke: developmental plasticity and clinical function[END_REF] Using fMRI, however, we were able to add more information about the functional motor network in our cohort, demonstrating that M1 functional localization remained in the affected hemisphere in all patients but one (participant #24) who had unilateral cerebral palsy (and mirror movements) and where fMRI peak activation following paretic hand movement was located in the precentral gyrus of the unaffected hemisphere. Note that participant #34 also exhibited mirror movements, but that fMRI peak activation following paretic hand movement was in the precentral gyrus of the affected hemisphere. No other patients showed clinical or neuroimaging evidence of reorganization of motor control to the unaffected hemisphere (ipsilateral reorganization), with the known limitation that active motor fMRI tasks also partly activate the primary somatosensory area by afferent somatosensory information. However, removing the two participants from our analysis did not change the significance of results in our study. Regarding the statistical method used here, it must be noted that due to the historical follow-up cohort (AVCnn study), no a priori power calculation has been done. Still, our very homogenous cohort of participants with this rare condition reduces (e.g. age-related) between-subject variability and suggests the observed effect to be valid and relevant, also given the relatively small sample size. [START_REF] Friston | Ten ironic rules for non-statistical reviewers[END_REF] Our results have further important clinical and methodological implications. When assessing a child after unilateral NAIS in clinical practice or in a research setting, both the contralesional and the ipsilesional hands should be evaluated. And, even more importantly, in future therapeutic trials for children with unilateral hemiparesis following NAIS (e.g. using transcranial direct current stimulation) [START_REF] Kirton | Transcranial direct current stimulation for children with perinatal stroke and hemiparesis[END_REF] the target could not only be the contralesional primary motor cortex, but as well its supposedly nondamaged counterpart of the ipsilesional hemisphere. The same remark holds true for the type of noninvasive neuromodulation used (i.e. inhibitory or excitatory stimulation).

CONCLUSION

After NAIS, contralesional hand motor performance is related not only to structural abnormalities of the ipsilesional grey matter and connected white matter structures [START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF][START_REF] Dinomais | Does contralesional hand function after neonatal stroke only depend on lesion characteristics?[END_REF] but also to changes in transcallosal motor fibres. Interestingly, the changes in this part of the corpus callosum also influence motor performance of the ipsilesional hand. Therefore, early unilateral brain lesions influence the motor performance of both hands during childhood. This study thus underlines that unilateral NAIS is a focal early brain injury with global structural effects on the developing brain by influencing brain structures remote from the infarct.

Figure 1 :Figure 2 :

 12 Figure1: Constrained-spherical, deconvolution-based probabilistic tractography was used to identify the transcallosal motor fibres (in red) connecting the primary motor cortex (middle), which was delineated anatomically as described previously.[START_REF] Groeschel | Identification and interpretation of microstructural abnormalities in motor pathways in adolescents born preterm[END_REF] In addition, functional magnetic resonance imaging activation of hand movements was used as additional guidance for identification of the precentral gyri. The midsagittal area of the corpus callosum (green) and its motor part (yellow, as defined by the transcallosal fibres) were traced manually. [Colour figure can be viewed at wileyonlinelibrary.com].

Table I :

 I Sample characteristics and individual results for hand motor function, measurements of corpus callosum, and lesion volumes

			Motor		Lesion	Contralesional	Ipsilesional	Ratio	Ratio	Ratio	Lesion
	No.	Sex	examination	Handedness	side	BBT	BBT	mCC	rCC	wCC	volume
	2	M	L hemi	R	R	25	36	0.00292	0.03309	0.03601	140.644
	3	M	N	L	L	22	28	0.00486	0.02474	0.02960	21.824
	4	M	N	R	R	25	31	0.00445	0.03309	0.03754	34.348
	5	M	N	L	L	44	42	0.00602	0.04905	0.05507	35.483
	6	F	N	R	L	30	30	0.00345	0.03150	0.03495	3.377
	7	F	N	R	R	23	23	0.00390	0.04039	0.04429	10.956
	8	M	L hemi	R	R	19	24	0.00335	0.02169	0.02504	42.55
	9	F	N	R	L	28	25	0.00324	0.02841	0.03165	38.398
	10	F	L hemi	R	R	26	34	0.00301	0.03008	0.03309	83.812
	11	M	N	R	L	39	29	0.00343	0.03630	0.03973	0.293
	12	F	L hemi	R	R	22	31	0.00373	0.03121	0.03494	127.433
	13	M	N	L	L	26	25	0.00352	0.02914	0.03265	1.924
	14	F	N	R	R	28	31	0.00353	0.02989	0.03342	10.128
	15	M	N	R	L	28	31	0.00401	0.03150	0.03551	48.26
	16	F	N	L	L	43	41	0.00468	0.03026	0.03494	0.686
	17	M	N	R	R	33	33	0.00416	0.03097	0.03513	2.935
	18	F	N	L	L	30	40	0.00412	0.03341	0.03753	84.98
	19	M	N	R	L	38	34	0.00330	0.03136	0.03467	2.982
	20	M	N	R	R	39	33	0.00425	0.03234	0.03659	14.321
	21	F	N	L	L	29	34	0.00312	0.02791	0.03103	23.923
	23	M	N	R	R	28	34	0.00472	0.03214	0.03686	15.072
	24	M	R hemi	L	L	10	26	0.00220	0.02781	0.03002	181.779
	25	M	N	L	L	37	42	0.00501	0.03716	0.04216	31.116
	26	M	R hemi	L	L	31	34	0.00348	0.03344	0.03692	123.848
	27	F	N	R	R	31	39	0.00463	0.03843	0.04306	1.027
	28	M	R hemi	L	L	40	42	0.00440	0.03005	0.03445	69.073
	29	F	R hemi	L	L	14	17	0.00438	0.03775	0.04213	8.597
	30	M	N	L	R	44	44	0.00466	0.03539	0.04005	0.652
	31	F	N	R	L	31	35	0.00552	0.03452	0.04004	19.284
	32	F	R hemi	L	L	17	27	0.00328	0.03254	0.03582	76.038
	34	M	R hemi	L	L	4	28	0.00315	0.03409	0.03724	8.905
	35	M	N	R	R	31	31	0.00464	0.03449	0.03912	1.698
	36	M	L hemi	R	R	22	28	0.00272	0.03987	0.04259	76.786

Participant numbers correspond to Table I in Dinomais et al.

[START_REF] Dinomais | Long term motor function after neonatal stroke: lesion localization above all[END_REF]

F, female; M, male; R, right; L, left; N, normal; hemi, hemiplegia; contraBBT, box and blocks test score for the contralesional hand; ipsiBBT, box and blocks test score for the ipsilesional hand; mCC, motor part of the corpus callosum; rCC, rest of the corpus callosum; wCC, whole corpus callosum.

ACKNOWLEDGEMENTS

The research was supported by the University Hospital of Angers (eudract-number-2010-A00976-33), the University Hospital of Saint-Etienne (eudract-number-2010-A00329-30), and the Fondation de l'Avenir (ET0-571). Sponsors of the study had no role in the study design data collection, data analysis, data interpretation, writing of the report, or decision to submit for publication. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. Trial registration numbers: PHRC r egional no. 0308052 and PHRC interr egional no. 1008026; EudraCT number 2010-A00329-30; ClinicalTrials.gov Identifier: NCT02511249. The authors have stated that they had no interests that might be perceived as posing a conflict or bias.

SUPPORTING INFORMATION

The following additional material may be found online:

Appendix S1: AVCnn Group members.


Martine Morzel

Francis Canon

Sylvain Guyot

Interactions between Salivary Proteins and Dietary Polyphenols: Potential Consequences on Gastrointestinal Digestive Events

Keywords: digestion, saliva, polyphenols, tannins, proline-rich proteins, mucins

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

■ INTRODUCTION

The health benefits of dietary polyphenols are tremendously well-documented, for example regarding cardiovascular health, 1 metabolic diseases, 2 or cognitive decline. [START_REF] Baba | Effect of Daily Intake of Green Tea Catechins on Cognitive Function in Middle-Aged and Older Subjects: A Randomized, Placebo-Controlled Study[END_REF] When it comes to digestion, the impact of polyphenols is described mainly in relation to inhibition of enzymes [START_REF] Mcdougall | The Inhibitory Effects of Berry Polyphenols on Digestive Enzymes[END_REF][START_REF] Sun | Inhibition of α-Amylase by Polyphenolic Compounds: Substrate Digestion, Binding Interactions and Nutritional Intervention[END_REF] or modulation of gut microbiota. [START_REF] Duarte | Polyphenols and Their Anti-Obesity Role Mediated by the Gut Microbiota: A Comprehensive Review[END_REF] Another aspect of polyphenols that is extensively studied is their contribution to the sensory properties of food: bitterness and astringency. For the latter, a key mechanism at the origin of this sensation resides in the ability of polyphenols to aggregate proteins including those forming the mucosal pellicle. Such binding may initiate changes in the friction forces at the surface of the oral mucosa and/or intracellular signaling leading to the release of a neurotransmitter. [START_REF] Canon | Perspectives on Astringency Sensation: An Alternative Hypothesis on the Molecular Origin of Astringency[END_REF] This mechanism is modulated by the presence in saliva of proteins presenting a high affinity for tannins. In fact, when polyphenols enter the digestive tract, it is expected that part of them will be in the form of salivary proteins-polyphenols complexes.

In parallel, the impact of oral physiology (mastication and salivation) on digestion is gaining interest. For example, in vitro digestion was applied to boli produced by in vivo mastication of protein-fortified sponge cakes [START_REF] Assad-Bustillos | Role of the Bolus Degree of Structure on the Protein Digestibility during in Vitro Digestion of a Pea Proteinfortified Sponge Cake Chewed by Elderly[END_REF] or to boli produced by in vitro simulated normal or deficient mastication of meat products [START_REF] Peyron | Deciphering the Protein Digestion of Meat Products for the Elderly by in Vitro Food Oral Processing and Gastric Dynamic Digestion, Peptidome Analysis and Modeling[END_REF] or pasta. [START_REF] Blanquet-Diot | Importance of Oral Phase in in Vitro Starch Digestibility Related to Wholegrain versus Refined Pastas and Mastication Impairment[END_REF] The impact of saliva on digestion has also been described, focusing on its enzymatic content, [START_REF] Pedersen | Salivary Functions in Mastication, Taste and Textural Perception, Swallowing and Initial Digestion[END_REF] antioxidant properties, [START_REF] Gorelik | Saliva Plays a Dual Role in Oxidation Process in Stomach Medium[END_REF] or the effect it may have on emulsions. [START_REF] Wang | Effect of Simulated Saliva Components on the in Vitro Digestion of Peanut Oil Body Emulsion[END_REF] The present article aims at bringing these different fields closer together. This narrative review does not intend to summarize the plethoric literature on the effect of polyphenols on digestion but is focused on the polyphenols-saliva interactions and how these may modulate the bioaccessibility or bioavailability of polyphenols themselves or of other nutrients. For that purpose, the review provides an updated description of the different classes of polyphenols and how they are modified by either food processing or digestion. A second part will deal with salivary proteins, especially their fate during digestion, their interactions with polyphenols, and how the resulting complexes may be affected by digestion. This will provide the necessary background to then consider the impact of such interactions (binding, complexation, and/or precipitation) on digestive processes. Some hypotheses have been tested experimentally and published, and they are reported in this article. We also report articles suggesting an impact of polyphenols-salivary proteins interactions, even when this was not tested specifically. Finally, we will also formulate novel hypotheses based on the current knowledge.

■ DIFFERENT CLASSES OF DIETARY POLYPHENOLS Polyphenols are very widespread secondary metabolites mainly found in higher plants and in some brown algae. They constitute a vast family of compounds with very diverse molecular structures. However, the term "polyphenol" is often used inappropriately in the literature, in particular by mistakenly considering as polyphenols compounds having in their structure only a single phenolic nucleus. Thus, a comprehensive definition for polyphenols was recommen-ded: [START_REF] Quideau | Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis[END_REF] The term "polyphenol" should be used to define plant secondary metabolites derived exclusively from the shikimatederived phenylpropanoid and/or the polyketide pathway(s), featuring more than one phenolic ring and being devoid of any nitrogen-based functional group in their most basic structural expression. Thus, very widespread simple phenolics such as hydroxybenzoic or hydroxycinnamic acids must not be considered as "true polyphenols".

With regard to this definition and based on biosynthetic pathways, several categories of polyphenols are distinguished. A special mention is made for flavonoids, of which there are more than 8000 different polyphenolic structures which are subdivided into a large series of subclasses (anthocyanins, flavanols, flavonols, flavones, etc.) according to the oxidation state and chemical functionalization scheme of the heterocycle. [START_REF] Andersen | Chemistry, Biochemistry and Applications[END_REF] Stilbenes (for instance, resveratrol) and lignans are two other less widespread polyphenol classes for which numerous papers related to their biological properties have been published. [START_REF] Landete | Plant and Mammalian Lignans: A Review of Source, Intake, Metabolism, Intestinal Bacteria and Health[END_REF][START_REF] Meng | Health Benefits and Molecular Mechanisms of Resveratrol: A Narrative Review[END_REF] The other method of polyphenol categorization refers to a specific physicochemical feature of certain polyphenolstheir tanning propertythat refers to the ability of some polyphenols to associate to proteins. Obviously, this property is of particular importance in the context of the present review focusing on polyphenol interactions with salivary proteins. Historically, the term "vegetable tannin" refers to a plant extract used for the conversion of animal skin into leather. Still today, the generally accepted definition for a "vegetable tannin" is the one first proposed by Theodore White, a chemist in the leather industry, [START_REF] White | The Chemistry of Vegetable Tannins -a Symposium[END_REF] and later completed by Swain and Bate-Smith. [START_REF] Swain | Flavonoids Compounds. Comparative Biochemistry[END_REF] Vegetable tannins are water-soluble phenolic compounds having a molecular weight between 500 and 3000 Da and having the ability to precipitate alkaloids, gelatin, and other proteins. Nevertheless, the upper value of molecular weight (3000 Da) must be now reconsidered since several recent studies have demonstrated that much higher molecular weight tannins are water-soluble and in general have even stronger capacity to precipitate proteins. [START_REF] Girard | Interaction Mechanisms of Condensed Tannins (Proanthocyanidins) with Wheat Gluten Proteins[END_REF][START_REF] Leppa | Distribution of Protein Precipitation Capacity within Variable Proanthocyanidin Fingerprints[END_REF] Tannins are classified into three categories as depicted in Figure 1. First, condensed tannins (proanthocyanidins) are flavanol dimers, oligomers, and polymers. They are divided into subclasses (procyanidins, prodelphinidins, and profisetinidins) depending on the nature of their flavanol constitutive units ((epi)catechin, (epi)gallocatechin, and fisetinidol, respectively). In some cases, a part of the flavanol units are esterified by gallic acid, thus forming galloylated proanthocyanidins. They are found in many edible plant materials, including fruits (apples, grapes, plums, red fruits, ...), some legumes (beans and lentils), nuts (almonds, hazelnuts, pecans, ...), grains (barley, sorgho, ...), and processed food (fruit juices, cocoa, wine, and ciders). [START_REF] Smeriglio | Proanthocyanidins and Hydrolysable Tannins: Occurrence, Dietary Intake and Pharmacological Effects[END_REF] Second, hydrolyzable tannins (gallo-and ellagitannins) are mainly gallic or ellagic acid polyesters of a polyol moiety, which is generally glucose. Simply considering ellagitannins, near to 1000 different hydrolyzable tannin structures have been identified. [START_REF] Quideau | Chemistry and Biology of EllagitanninsAn Underestimated Class of Bioactive Plant Polyphenols[END_REF] They are less widespread in comparison to condensed tannins; however, they are found in diverse sources, such as strawberries, raspberries, pomegranates, mangos, pecans, walnuts, and wines, in particular those aged in oak barrels. [START_REF] Quideau | Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis[END_REF][START_REF] Smeriglio | Proanthocyanidins and Hydrolysable Tannins: Occurrence, Dietary Intake and Pharmacological Effects[END_REF] Third, phlorotannins [START_REF] Meng | Phlorotannins: A Review of Extraction Methods, Structural Characteristics, Bioactivities, Bioavailability, and Future Trends[END_REF] are oligomers and polymers composed mainly of phloroglucinol units linked together by biaryl or/and diaryl ether bonds and are essentially found in brown algae.

Food Processing: Oxidation and Structural Changes of Polyphenols. During food processing, a part of native polyphenols is involved in biochemical and chemical reactions, undergoing significant changes in their molecular structure and, thus, generating new molecules that can be described as "secondary polyphenols". [START_REF] Tanaka | Chemistry of Secondary Polyphenols Produced during Processing of Tea and Selected Foods[END_REF] Those newly formed products Journal of Agricultural and Food Chemistry contribute to the organoleptic and nutritional properties of foods, as do native polyphenols. For instance, they contribute to the color and the flavor of various plant-based beverages such as tea, coffee, juices, ciders, and wines. [START_REF] Tanaka | Chemistry of Secondary Polyphenols Produced during Processing of Tea and Selected Foods[END_REF][START_REF] Cheynier | Polyphenols in Foods Are More Complex than Often Thought[END_REF] Among reactions involving polyphenols, oxidation is the one occurring the most frequently during plant raw material transformation. [START_REF] Liu | Reactivity of Flavanols: Their Fate in Physical Food Processing and Recent Advances in Their Analysis by Depolymerization[END_REF] From a chemical point of view, oxidation of polyphenols can be summarized by the ability of the phenolic nucleus to lose an electron or a hydrogen atom leading to the formation of reactive species. As a consequence, oxidation of polyphenols is also clearly the main reaction route which leads to the formation of many "secondary polyphenols" which can be described as "oxidation products".

The oxidation of catechol or pyrogallol groups present in the structures of phenolic compounds (tannins, flavonoids, hydroxycinnamic and hydroxybenzoic acids, etc.) leads to the formation of very reactive intermediate species such as quinones and semiquinones. Note that depending on the situation, these species can be either chemically generated in low concentrations but over long periods, as for example in the case of wine aging, or rapidly produced in large quantities under enzymatic catalysis by polyphenoloxidases and peroxidases during a precise step of the transformation process. For black tea, for example, this occurs during the bruising and rolling operations, [START_REF] Subramanian | Role of Polyphenol Oxidase and Peroxidase in the Generation of Black Tea Theaflavins[END_REF] while for apple juice or cider, this is observed during the crushing and pressing of the fruits. [START_REF] Nicolas | Enzymatic Browning Reactions in Apple and Apple Products[END_REF] The kinetic aspects of these oxidation steps strongly depend on environmental conditions such as the availability of oxygen, the pH, and the presence of transition metals capable of catalyzing oxidation. Thus, in a weakly acidic environment (which is the case with most plant foods), the autoxidation of phenolic compounds is all the more favored as the pH is close to neutrality.

Once formed, quinones and semiquinones then evolve rapidly according to various reaction pathways: (i) electron transfer reactions generating new quinone or radical species, (ii) intramolecular additions and rearrangements, and (iii) intermolecular additions with other phenolic or nonphenolic compounds. Finally, depending on the plant matrix considered (the nature, concentrations, and proportions of the different categories of polyphenols and other constituents present) and environmental conditions imposed by the transformation processes (pH, availability of oxygen, etc.), this multiplicity of reaction pathways logically leads to the generation of numerous oxidation products exhibiting structures whose molecular architectures are sometimes very different from those of their native phenolic precursors. Although our objective is not here to make an exhaustive review of these different structures of oxidation products, we can, however, cite a few relevant examples. Thus, the theaflavins from black teas with their specific benzotropolone nucleus are generated by oxidative coupling between epicatechin and epigallocatechins. [START_REF] Takemoto | Synthesis of Theaflavins and Their Functions[END_REF] Interestingly, B-type procyanidins can be converted into A-type either enzymatically [START_REF] Poupard | Characterization of Procyanidin B2 Oxidation Products in an Apple Juice Model Solution and Confirmation of Their Presence in Apple Juice by High-Performance Liquid Chromatography Coupled to Electrospray Ion Trap Mass Spectrometry: LC-ESI-MS/MS of Procyanidin B2 Oxidation Products[END_REF] or chemically [START_REF] Kondo | Conversion of Procyanidin B-Type (Catechin Dimer) to A-Type: Evidence for Abstraction of C-2 Hydrogen in Catechin during Radical Oxidation[END_REF] as a result of an intramolecular oxidative coupling reaction. Intra-and intermolecular autoxidative coupling of condensed tannins in wine was characterized in model solutions showing that intermolecular coupling was highly favored when tannins are more concentrated. [START_REF] Vernhet | Condensed Tannin Changes Induced by Autoxidation: Effect of the Initial Degree of Polymerization and Concentration[END_REF] Still in the field of wine chemistry, the coupling of anthocyanins with catechins and condensed tannins (proanthocyanidin oligomers) often proceeds through oxidative mechanisms leading to the formation of pyranoan-thocyanin derivatives that contribute to the color change during red wine aging. [START_REF] Rentzsch | Pyranoanthocyanins -an Overview on Structures, Occurrence, and Pathways of Formation[END_REF] In the case of apple condensed tannins, it has been shown that the structural modifications generated by oxidation (essentially additional intramolecular linkages) significantly modify some of their physicochemical properties, such as their solubility, their rigidity, and their solvation properties in water-ethanol mixtures. [START_REF] Zanchi | Conformation, and Solvation of Native and Oxidized Tannin Macromolecules in Water-Ethanol Solution[END_REF] Although little knowledge is currently available on this issue, it is expected that these structural modifications also affect the ability of tannins to interact with proteins, including salivary proteins. Recently, in relation to what could be the perception of astringency in juices made from some cider apple varieties, a series of dehydrodimers with original structures resulting from oxidative coupling of caffeoylquinic acid (i.e., chlorogenic acid) were studied for their ability to complex saliva proteins in model solutions. [START_REF] Castillo-Fraire | Interactions between Polyphenol Oxidation Products and Salivary Proteins: Specific Affinity of CQA Dehydrodimers with Cystatins and P-B Peptide[END_REF] Contrarily to what is generally expected in the case of tannin-salivary protein interactions, those dehydrodimers presented a low interaction with proline-rich proteins (PRPs) but revealed a specific interaction with statherins, P-B peptide, and cystatins. Obviously, this specific example does not allow establishment of general rules regarding the impact of oxidation of polyphenols on their ability to interact with salivary proteins, and further studies regarding catechins and condensed and hydrolyzable tannins are needed to better understand the structure-activity relationships that characterize those interactions.

Modification during Digestion. The stability and metabolization of polyphenols in the digestive tract depend on the class to which they belong and also vary mainly according to the gastrointestinal compartment considered. Polyphenols are relatively stable in an in vitro simulated gastric environment (acidic pH in the presence of pepsin) while they are much more degraded under the neutral pH conditions of the intestine, in the presence of pancreatin and bile salts, which are favorable to autoxidation reactions. [START_REF] Bermudez-Soto | Stability of Polyphenols in Chokeberry (Aronia Melanocarpa) Subjected to in Vitro Gastric and Pancreatic Digestion[END_REF][START_REF] Neilson | Catechin Degradation with Concurrent Formation of Journal of Agricultural and Food Chemistry pubs[END_REF][START_REF] Thomas-Valdes | Effect of Simulated Gastrointestinal Digestion on Polyphenols and Bioactivity of the Native Chilean Red Strawberry (Fragaria Chiloensis Ssp. Chiloensis f. Patagonica)[END_REF] For instance, catechins were significantly degraded under simulated digestion, and some of them, in particular those containing a pyrogallol group, were partly converted into dimers by autoxidative coupling. [START_REF] Neilson | Catechin Degradation with Concurrent Formation of Journal of Agricultural and Food Chemistry pubs[END_REF][START_REF] Martini | Bioactivity and Cell Metabolism of Dark Chocolate Phenolic Compounds after in Vitro Gastro-Intestinal Digestion[END_REF] Interestingly, those dimers were analogues of theasinensins, a class of polyphenols also found in mildly fermented tea. [START_REF] Neilson | Catechin Degradation with Concurrent Formation of Journal of Agricultural and Food Chemistry pubs[END_REF] Furthermore, regarding glycosylated flavonoids, the small intestine is favorable to some deglycosylation reactions. Indeed, human small intestine epithelial cells produce β-glucosidase enzymes able to hydrolyze quercetin glycosides into free quercetin, which is much more bioavailable. [START_REF] Nemeth | Deglycosylation by Small Intestinal Epithelial Cell Beta-Glucosidases Is a Critical Step in the Absorption and Metabolism of Dietary Flavonoid Glycosides in Humans[END_REF] However, considering the numerous studies published in this field, it seems that one of the most important biotransformations of dietary polyphenols occurs as a result of the biological action of the gut microbiota. [START_REF] Gonzalez-Sarrias | Non-Extractable Polyphenols Produce Gut Microbiota Metabolites That Persist in Circulation and Show Anti-Inflammatory and Free Radical-Scavenging Effects[END_REF][START_REF] Selma | Interaction between Phenolics and Gut Microbiota: Role in Human Health[END_REF] Thus, flavonols, 44 catechins, [START_REF] Clifford | Human Studies on the Absorption, Distribution, Metabolism, and Excretion of Tea Polyphenols[END_REF] and proanthocyanidins (i.e., condensed tannins) [START_REF] Bazzocco | Factors Affecting the Conversion of Apple Polyphenols to Phenolic Acids and Fruit Matrix to Short-Chain Fatty Acids by Human Faecal Microbiota in Vitro[END_REF][START_REF] Deprez | Polymeric Proanthocyanidins Are Catabolized by Human Colonic Microflora into Low-Molecular-Weight Phenolic Acids[END_REF] are largely converted into a series of small molecules corresponding to phenolic acids, with most of them being benzoic, phenylacetic, or phenyl propionic acids and their hydroxylated derivatives. In the case of catechins [START_REF] Takagaki | Catabolism of (+)-Catechin and (-)-Epicatechin by Rat Intestinal Microbiota[END_REF] and proanthocyanidins, [START_REF] Bourvellec | Procyanidin-Cell Wall Interactions within Apple Matrices Decrease the Metabolization of Procyanidins by the Human Gut Microbiota and the Anti-Inflammatory Effect of the Resulting Microbial Metabolome In Vitro[END_REF] a series of hydroxylated phenyl valeric acids are also produced. Interestingly, those metabolites are produced even from nonextractable polyphenols. [START_REF] Gonzalez-Sarrias | Non-Extractable Polyphenols Produce Gut Microbiota Metabolites That Persist in Circulation and Show Anti-Inflammatory and Free Radical-Scavenging Effects[END_REF] When they are highly polymerized, procyanidins are poorly metabolized. [START_REF] Bazzocco | Factors Affecting the Conversion of Apple Polyphenols to Phenolic Acids and Fruit Matrix to Short-Chain Fatty Acids by Human Faecal Microbiota in Vitro[END_REF] Regarding ellagitannins, they are partly metabolized into urolitin, [START_REF] Cerda | Identification of Urolithin A as a Metabolite Produced by Human Colon Microflora from Ellagic Acid and Related Compounds[END_REF] a specific tricyclic metabolite that exhibits phytoestrogenic activities. [START_REF] Larrosa | Ellagic Acid-Derived Metabolites Produced by Human Colonic Microflora, Exhibit Estrogenic and Antiestrogenic Activities[END_REF] ■ SALIVARY PROTEINS Whole saliva is defined as the biological fluid present in the mouth. The reader is directed to excellent reviews for further information on saliva functions and composition. [START_REF] Pedersen | Salivary Functions in Mastication, Taste and Textural Perception, Swallowing and Initial Digestion[END_REF][START_REF] Humphrey | A Review of Saliva: Normal Composition, Flow, and Function[END_REF][START_REF] Dawes | The Functions of Human Saliva: A Review Sponsored by the World Workshop on Oral Medicine VI[END_REF] One should note, however, that saliva flow and composition are highly variable between subjects and depend on many factors, such as age, [START_REF] Morzel | Human Infant Saliva Peptidome Is Modified with Age and Diet Transition[END_REF][START_REF] Vandenberghe-Descamps | Salivary Flow Decreases in Healthy Elderly People Independently of Dental Status and Drug Intake[END_REF] diet, [START_REF] Dawes | Effects of Diet on Salivary Secretion and Composition[END_REF][START_REF] Chambon | The Salivary Proteome Reflects Some Traits of Dietary Habits in Diabetic and Non-Diabetic Older Adults[END_REF] medication and/or pathologies, [START_REF] Proctor | Disease-Induced Changes in Salivary Gland Function and the Composition of Saliva[END_REF] or sensory stimulation during food intake. [START_REF] Neyraud | Effects of Different Tastants on Parotid Saliva Flow and Composition[END_REF][START_REF] Watanabe | The Effects of Different Foods and Concentrations of Citric Acid on the Flow Rate of Whole Saliva in Man[END_REF] In any case, apart from water, saliva is composed mainly of proteins. In fact, it contains thousands of different proteins and peptides. These are either secreted by the salivary glands or originate from the crevicular fluid (an exudate of plasma reaching the oral cavity through the gingival crevices), epithelial cells, oral microbiota, or food remnants. The estimation of the proportions of the different proteins suggests that the most abundant proteins are those secreted by salivary glands (mucins, α-amylase, cystatins, histatins, statherin, and PRPs) together with immunoglobulins and albumin. [START_REF] Messana | Facts and Artifacts in Proteomics of Body Fluids. What Proteomics of Saliva Is Telling Us?[END_REF][START_REF] Schenkels | Biochemical Composition of Human Saliva in Relation To Other Mucosal Fluids[END_REF] PRPs are of special interest for this review because of their high affinity for tannins. They represent up to 70% of proteins secreted by the parotid glands and 20-30% of proteins in whole saliva. [START_REF] Bennick | Salivary Proline-Rich Proteins[END_REF] Fragments of PRPs also represent a large part of the adult saliva peptidome [START_REF] Vitorino | Towards Defining the Whole Salivary Peptidome[END_REF] and the vast majority of oligopeptides identified in human infant saliva. [START_REF] Morzel | Human Infant Saliva Peptidome Is Modified with Age and Diet Transition[END_REF] The PRPs family is characterized by a high heterogeneity: the products of six genes are found in saliva as a complex mixture of entire, truncated, phosphorylated, glycosylated, and dimerized proteoforms. [START_REF] Manconi | The Intriguing Heterogeneity of Human Salivary Proline-Rich Proteins[END_REF] Fate of Salivary Proteins along the Digestive Tract. Except during the sleeping period, where salivary flow is virtually null, [START_REF] Dawes | Salivary Flow Patterns and the Health of Hard and Soft Oral Tissues[END_REF] saliva is constantly secreted and thus swallowed. It is estimated that an adult swallows around 0.6-1 L of saliva per day. [START_REF] Watanabe | The Effects of Different Foods and Concentrations of Citric Acid on the Flow Rate of Whole Saliva in Man[END_REF][START_REF] Richardson | Salivary Response to Food in Humans and Its Effect on Gastric Acid Secretion[END_REF] There are large variations in the reported protein concentration in whole saliva, which may depend on the assay used or the sampling conditions. Mean values are, for example, 0.6 mg•mL -168 in unstimulated saliva of individuals (n = 200) included in an observational cohort or 1.06 mg•mL -1 (n = 19), [START_REF] Henskens | Protein, Albumin and Cystatin Concentrations in Saliva of Healthy Subjects and of Patients with Gingivitis or Periodontitis[END_REF] 1.77 mg•mL -1 (n = 30), [START_REF] Chiba | Periodontal Condition, Changes in Salivary Biochemical Parameters, and Oral Health-related Quality of Life in Patients with Anorexia and Bulimia Nervosa[END_REF] or 2.2 mg• mL -1 (n = 13) [START_REF] Neyraud | Relationships between the Metabolome and the Fatty Acid Composition of Human Saliva; Effects of Stimulation[END_REF] in unstimulated saliva of healthy individuals. In any case, this implies that the total amount of protein originating from saliva would not exceed 2 g per day, well below the daily dietary protein intake.

Like any protein entering the digestive tract, it is expected that salivary proteins will be rapidly degraded by proteolytic enzymes. A study unexpectedly reported that secreted carbonic anhydrase 6 was present in mouse stools, with an apparent molecular weight on electrophoretic gels suggesting that it was intact or at least little degraded, [START_REF] Oleksiewicz | Identification of Stool Proteins in C57BL/6J Mice by Two-Dimensional Gel Electrophoresis and MALDI-TOF Mass Spectrometry[END_REF] but such a result would need confirmation. The particular case of proline-rich proteins (PRPs) also deserves some attention. Their unique composition in amino acids renders them particularly resistant to digestive hydrolysis. Indeed, the presence of proline residues after basic amino acids (Lys-Pro and Arg-Pro) precludes their cleavage by trypsin. For example, after a protein-free meal, oligopeptides in the mass range 500-5000 kDa were sought in mini-pig jejunal aspirates: 23 endogenous peptides were identified, among which 20 corresponded to PRP fragments of 8 to 22 amino acids (personal data).

Salivary Proteins Interacting Preferentially with Polyphenols. The presence of proteins which are effective precipitators of tannins has been reported in the saliva of some mammals. These proteins are called tannin-binding salivary proteins (TBSPs) and include mainly PRPs and histatins.

Their presence in saliva is thought to be related to diet, as they are abundant in species consuming high amounts of tannins, such as herbivores, lagomorphs, rodents, and omnivores, while they are absent in the saliva of mammals weakly exposed to tannin. [START_REF] Clauss | Tannin-Binding Salivary Proteins in Three Captive Rhinoceros Species[END_REF][74][START_REF] Shimada | Salivary Proteins as a Defense Against Dietary Tannins[END_REF] Hypotheses explaining the presence of TBSPs in mammalian saliva postulate that these proteins participate in the maintenance of oral homeostasis, counteract the effect of tannins, and/or trigger the sensation of astringency, which signals a high tannin concentration in food. In rats, PRP secretion is induced by a tannin-rich diet. [START_REF] Mehansho | Modulation of Proline-Rich Protein Biosynthesis in Rat Parotid Glands by Sorghums with High Tannin Levels[END_REF] This secretion precludes the antinutritional effect of tannins [START_REF] Mehansho | Modulation of Proline-Rich Protein Biosynthesis in Rat Parotid Glands by Sorghums with High Tannin Levels[END_REF] and improves the consumption of solutions containing tannins, [START_REF] Glendinning | Effect of Salivary Proline-Rich Proteins on Ingestive Responses to Tannic Acid in Mice[END_REF] suggesting that their presence reduces the intensity of unpleasant sensations (bitterness and/or astringency) induced by tannin. In agreement with these observations, PRPs protect the oral mucosal pellicle from aggregation by tannins. [START_REF] Ployon | Mechanisms of Astringency: Structural Alteration of the Oral Mucosal Pellicle by Dietary Tannins and Protective Effect of BPRPs[END_REF] Therefore, rather that inducing astringency, TBSPs can be considered as counterdefenses inherited from natural selection, which modulate astringency intensity by scavenging tannins.

PRPs have a higher capacity to protect proteins from aggregation by tannins than other salivary proteins. [START_REF] Mehansho | Modulation of Proline-Rich Protein Biosynthesis in Rat Parotid Glands by Sorghums with High Tannin Levels[END_REF] This capacity has been attributed to their intrinsically disordered conformation [START_REF] Canon | Stoichiometry, and Stability of Salivary Protein-Tannin Complexes by ESI-MS and ESI-MS/MS[END_REF] and to their tandem repeat sequences composed of proline clusters surrounded by flexible amino acids. [START_REF] Canon | Binding Site of Different Tannins on a Human Salivary Proline-Rich Protein Evidenced by Dissociative Photoionization Tandem Mass Spectrometry[END_REF] The rigid proline clusters provide stable binding sites to initiate the interaction with tannins, [START_REF] Canon | Aggregation of the Salivary Proline-Rich Protein IB5 in the Presence of the Tannin EgCG[END_REF][START_REF] Luck | Astringency and Proline-Rich Proteins[END_REF] while the flexibility of surrounding amino acids allows structural rearrangement, [START_REF] Canon | Binding Site of Different Tannins on a Human Salivary Proline-Rich Protein Evidenced by Dissociative Photoionization Tandem Mass Spectrometry[END_REF][START_REF] Canon | Folding of a Salivary Intrinsically Disordered Protein upon Binding to Tannins[END_REF] stabilizing the interaction through the establishment of additional hydrogen bonds between the peptide chain and tannins. [START_REF] Canon | Ability of a Salivary Intrinsically Unstructured Protein to Bind Different Tannin Targets Revealed by Mass Spectrometry[END_REF] This structural rearrangement may explain the higher affinity of full PRPs compared to proline-rich peptides. [START_REF] Charlton | Tannin Interactions with a Full-Length Human Salivary Proline-Rich Protein Display a Stronger Affinity than with Single Proline-Rich Repeats[END_REF] The several tandem repeat sequences also provide several binding sites, allowing several stoichiometries of interaction. [START_REF] Canon | Stoichiometry, and Stability of Salivary Protein-Tannin Complexes by ESI-MS and ESI-MS/MS[END_REF] Once soluble noncovalent complexes are formed, bound tannins, which are described as multidentate ligands, may bridge tannin•PRP complexes, leading to the formation of aggregates. [START_REF] Canon | Aggregation of the Salivary Proline-Rich Protein IB5 in the Presence of the Tannin EgCG[END_REF] A number of at least three tannins per protein is required to form aggregates. PRP-tannin aggregates grow with tannin concentration up to their precipitation. [START_REF] Canon | Aggregation of the Salivary Proline-Rich Protein IB5 in the Presence of the Tannin EgCG[END_REF] Beside PRPs, histatins have been reported to precipitate tannin more effectively than acidic PRPs (aPRPs) at pH 7.4, while acidic PRPs show a greater ability at pH 3.0. This difference may result from different mechanisms of interactions. Nevertheless, both histatins and aPRPs efficiently protected salivary amylase from tannin inhibition. [START_REF] Yan | Identification of Histatins as Tannin-Binding Proteins in Human Saliva[END_REF] Interaction of tannin with histatins has been reported to involved π-π stacking between the ring of aromatic amino acids and the phenolic ring of tannins. [START_REF] Shraberg | A Study of Procyanidin Binding to Histatin 5 Using Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and Molecular Simulations[END_REF] Other works have evidenced that aPRP, histatins, and statherins are more prone to be precipitated by tannins than basic PRP (bPRP) and glycosylated PRP (gPRP). [START_REF] Soares | Reactivity of Human Salivary Proteins Families Toward Food Polyphenols[END_REF] For bPRP, this effect could be due to Coulombic repulsion between the bPRP-tannin soluble complexes decreasing the formation of aggregates, as bPRPs are highly charged at the acidic pH used in this study. Regarding gPRP, the presence of glycosylation has been reported to increase the solubility of the gPRPtannin supramolecular edifices compared to nonglycosylated PRPs. [START_REF] Sarni-Manchado | Influence of the Glycosylation of Human Salivary Proline-Rich Proteins on Their Interactions with Condensed Tannins[END_REF] The presence of sugar chains may reduce the aggregation due to steric hindrance. Investigations on the affinity of TBSPs for tannins have revealed that bPRP and gPRP have a higher affinity for tannins than aPRP, [START_REF] Soares | Interaction of Different Classes of Salivary Proteins with Food Tannins[END_REF] illustrating that a difference should be made between the TBSPs' affinity for tannin and their propensity to be precipitated. The same study highlighted the influence of the D tannin structure on TBSP-tannin affinity, with changes of the ranking of TBSP affinity as a function of tannin structure. Another study also reported a higher affinity of bPRPs for flavan-3-ol than aPRPs. [START_REF] Ramos-Pineda | Synergistic Effect of Mixture of Two Proline-Rich-Protein Salivary Families (APRP and BPRP) on the Interaction with Wine Flavanols[END_REF] Mucins, which play an important role in the lubrification of the oral cavity, are other salivary proteins prone to interact with tannins. The interaction between the mucin MUC5B and tannins is followed by a structural reorganization of the mucin network [START_REF] Davies | Reorganisation of the Salivary Mucin Network by Dietary Components: Insights from Green Tea Polyphenols[END_REF] up to the formation of aggregates. MUC5B is the main salivary protein composing the mucosal pellicle, which is a thin layer of salivary proteins anchoring at the surface of the oral cells via interactions with the transmembrane mucin MUC1. [START_REF] Ployon | The Membrane-Associated MUC1 Improves Adhesion of Salivary MUC5B on Buccal Cells. Application to Development of an in Vitro Cellular Model of Oral Epithelium[END_REF] The aggregation of the mucosal pellicle by tannins leads to an increase of the friction forces at the surface of the oral mucosa. [START_REF] Ployon | Mechanisms of Astringency: Structural Alteration of the Oral Mucosal Pellicle by Dietary Tannins and Protective Effect of BPRPs[END_REF] This phenomenon is thought to be involved in the sensation of astringency and may induce the cleavage of the transmembrane MUC1, which is an external sensor of epithelial cells. [START_REF] Canon | Perspectives on Astringency Sensation: An Alternative Hypothesis on the Molecular Origin of Astringency[END_REF] Regarding the impact of the phenolic structure on tanninprotein interactions, the affinity of proanthocyanidins for proteins increases with the number of constitutive units and their level of galloylation. [START_REF] Silva | Interaction of Grape Seed Procyanidins with Various Proteins in Relation to Wine Fining[END_REF] The presence of a third hydroxyl group on the B-ring has also been reported to increase the affinity of tannins for proteins. [START_REF] Siebert | Effects of Protein-Polyphenol Interactions on Beverage Haze, Stabilization, and Analysis[END_REF] Indeed, the number of hydroxyl groups increases the stability of the tannin-protein interaction, [START_REF] Canon | Ability of a Salivary Intrinsically Unstructured Protein to Bind Different Tannin Targets Revealed by Mass Spectrometry[END_REF] via the establishment of hydrogen bonds, while their positions are also important. [START_REF] Bacon | Development of a Competition Assay for the Evaluation of the Binding of Human Parotid Salivary Proteins to Dietary Complex Phenols and Tannins Using a Peroxidase-Labeled Tannin[END_REF] The establishment of interactions also seems to depend of the polyphenol spatial configuration. [START_REF] Mcmanus | Polyphenol Interactions. 1. Introduction -Some Observations on the Reversible Complexation of Polyphenols with Proteins and Polysaccharides[END_REF] For example, molecules with flavan-3-ol monomers linked via C4-C6 linkages have a higher affinity for proteins than their isomers with C4-C8 linkages. [START_REF] Silva | Interaction of Grape Seed Procyanidins with Various Proteins in Relation to Wine Fining[END_REF][START_REF] Ezaki-Furuichi | Affinity of Procyanidins (Condensed Tannins) from the Bark of Rhaphiolepis Umbellata for Proteins[END_REF] Moreover, cis-2,3-flavan-3-ols have a slightly higher affinity for proteins than trans-2,3-flavan-3-ols. [START_REF] Bacon | Development of a Competition Assay for the Evaluation of the Binding of Human Parotid Salivary Proteins to Dietary Complex Phenols and Tannins Using a Peroxidase-Labeled Tannin[END_REF] Persistence of Salivary Proteins-Polyphenols Complexes along the Digestive Tract. In order to investigate preabsorption events in the gastrointestinal tract, polyphenols are sometimes subjected to in vitro digestion, which includes an oral phase. [START_REF] Laurent | Flavonoids from a Grape Seed Extract Interact with Digestive Secretions and Intestinal Cells as Assessed in an in Vitro Digestion/Caco-2 Cell Culture Model[END_REF] However, the most widely used and recognized protocols (e.g., ref 100) recommend the use of artificial saliva, which typically contains only amylase. Artificial saliva's protein composition is, therefore, far less complex than true saliva. The question of the resistance of complexes (specifically PRPs-tannins complexes) to digestion has, nevertheless, been addressed using in vitro approaches. Thus, insoluble complexes formed from basic PRPs and condensed tannins remained stable in conditions mimicking those in the stomach. 101 Similar observations were reported for histatintannin aggregates, with most aggregates remaining insoluble under in vitro conditions mimicking those of the stomach and of the small intestine without bile salts. 102 For both types of aggregates, the presence of bile salts in intestinal conditions partly solubilized the insoluble complexes. Whether tannins remained in the form of soluble complexes or were released was not investigated. More recently, it was shown that SPtannins complexes with a low degree of polymerization were disrupted (or at least dissolved) by gastric digestion while complexes made of salivary proteins and tannin tetramers and above were significantly less solubilized, which the authors interpreted as being more resistant to gastric digestion. 103 Finally, an in vivo study showed a 3-to 4-fold increase of fecal excretion of a hydrolyzable tannin (pentagalloyl glucose) in PRPs-secreting rats compared to rats which were not secreting PRPs. The authors, therefore, proposed that some PRPtannins complexes may be excreted in feces. 104 ■ IMPACT OF PP-SPS INTERACTIONS ON DIGESTIVE PROCESSES: EXPERIMENTAL EVIDENCE AND HYPOTHESES Digestive Enzymes' Activities. Sugars' Metabolizing Enzymes. Some studies report interactions of polyphenols with salivary amylase. Tea, but not tea depleted in tannins, was shown to reduce amylase activity in saliva, 105 and isolated human salivary amylase was inhibited by a gallotannin. 106 A recent study reported that some flavonoids (ECG and EGCG) bind very rapidly, within seconds, to human salivary amylase. 107 In the earlier articles, the relevance of amylase inhibition was described in relation to the development of caries, but the downstream consequences on digestion were not envisaged. This is probably the case because salivary amylase was traditionally not considered as a direct contributor to starch digestion except within the oral cavity. However, it was evidenced that the kinetics of the gastric pH decrease was compatible with preservation of salivary amylase activity for at least 20 min. 108 In in vitro dynamic conditions, 80% of starch was released (under solubilized or hydrolyzed forms) during the first 20 min of gastric digestion in the presence of salivary amylase, vs approximately 10% when saliva was replaced by water. Taken together, these articles suggest that inhibition of salivary amylase by tannins may have an impact on starch digestion in the gastrointestinal tract. Pancreatic amylase or intestinal α-glucosidases are also inhibited by polyphenols or phenolic extracts from various plant species, [109][110][111] which is rather considered as an opportunity to control glucose uptake and, therefore, manage type 2 diabetes in humans. 112 However, we found no report of the impact of tannin-binding salivary proteins (TBSPs) on the activities of such gastrointestinal enzymes. In contrast, it was shown that the brush border enzyme lactase was inhibited by EGCG but also that binding of EGCG to PRPs could alleviate this inhibition. 113 Lipases. Similarly to sugar metabolizing enzymes, the general trend described is a reduction of pancreatic lipase activity by polyphenols. 114,115 Gastric lipase has received less attention, but at least one study reports its inhibition by myricitrin-5-methyl ether, a star anise flavonoid. 116 Again, the literature does not describe the impact of the formation of salivary proteins-PP complexes on lipase activity.

Proteases. Due to the importance of protein metabolism on muscle mass, much research has focused on the impact of dietary polyphenols (particularly condensed tannins) on protein digestibility in animals. 117,118 The overall consensus is that diets rich in tannins have a negative impact on weight, but the underlying mechanisms (systemic or digestive) are not fully understood. Focusing on protein digestion, in vitro studies on gastric or gastrointestinal proteolysis sometimes differ in their conclusions, [119][120][121] even within the same study, depending on the protein substrate. 122 In fact, tannins may have different consequences on protein digestibility, as summarized by Butler. 117 On one hand, tannins may denature dietary protein substrates and, thus, facilitate the action of proteases. Contrarily, binding of tannins to protein substrates may hinder accessibility of proteases to the cleavage sites. Finally, and more generally for all hydrolytic enzymes, binding of tannins to the enzymes themselves could either modify the tridimensional structure of their catalytic sites (allosteric inhibition) or preclude access to the catalytic site by steric hindrance Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Review https://doi.org/10.1021/acs.jafc.2c01183 J. Agric. Food Chem. XXXX, XXX, XXX-XXX E (competitive inhibition) and, thus, directly inhibit their activity. For example, phenolic extracts from various plant species can drastically inhibit the activity of isolated digestive enzymes, for example, the proteolytic enzymes pepsin, trypsin, and chymotrypsin. 123,124 The activity of the brush border enzyme aminopeptidase N is also inhibited by some flavonoids of grape seeds. [START_REF] Laurent | Flavonoids from a Grape Seed Extract Interact with Digestive Secretions and Intestinal Cells as Assessed in an in Vitro Digestion/Caco-2 Cell Culture Model[END_REF] In this context, salivary proteins (particularly PRPs) have long been considered as major actors in the protection against the detrimental impact of tannins through binding of those molecules before they can interact with proteolytic digestive enzymes. This hypothesis is partly supported by animal studies: in rodents, a tannin-rich diet induces weight loss attributed to reduced food digestibility, which is continuous in hamsters but reversible after 3 days in rats and mice. In parallel, feeding on tannins induces in rats and mice a dramatic increase in salivary proline-rich proteins (PRPs) within 3 days, [START_REF] Mehansho | Modulation of Proline-Rich Protein Biosynthesis in Rat Parotid Glands by Sorghums with High Tannin Levels[END_REF] while this is not observed in hamsters. 125 Another study on rats demonstrated that diet supplementation in propanolol, which can suppress PRP production in saliva, aggravated the adverse effect of tannins on growth. 126 However, this study and others 104 suggested that the reduced apparent nitrogen digestibility induced by dietary tannins, and calculated using the nitrogen content in feces, may not be due to reduced digestibility of dietary protein but rather to increased nitrogen excretion related to higher secretion of PRPs. Shimada [START_REF] Shimada | Salivary Proteins as a Defense Against Dietary Tannins[END_REF] even proposed that tannins should not be regarded as inhibitors of digestion but rather as toxins with systemic impacts on growth and metabolism. In fact, it is plausible that both effects exist, depending on the type and structure of tannins.

Oxidation of Nutrients. It is admitted that the gastric environment is suitable for oxidation of dietary nutrients, particularly lipid peroxidation. It was demonstrated that, on average (pooling salivas of 7 volunteers), saliva could limit lipid peroxidation of turkey meat in simulated gastric fluid and that this effect was greatly enhanced in the presence of red wine polyphenols. [START_REF] Gorelik | Saliva Plays a Dual Role in Oxidation Process in Stomach Medium[END_REF] Unfortunately, the impact of polyphenols alone was not evaluated in this study, so it is unknown whether the effect is of an additive or synergetic type. Depending on their structure, polyphenols may chelate Fe 2+ ions, increasing their oxidation to Fe 3+ ions in the presence of oxygen. This mechanism decreases the quantity of Fe 2+ that can participate in the Fenton reaction and, thus, precludes the production of hydroxyl radicals. 127 This antioxidant activity of polyphenols may be limited by the presence of TBSP, which may compete with Fe 2+ for the binding to polyphenols. 128 However, and in contrast to this hypothesis, a study evaluated the oxidant scavenging activities (OSAs) of various polyphenols and polyphenol-containing beverages and noted a very large intensification of the OSA in the presence of human saliva. 129 The authors suggested that salivary proteins help solubilize lipophilic polyphenols and, thereby, increase their availability. Whether this translates into limitation of oxidation in the gastric compartment is not described.

Physical Modification of the Gastric and Intestinal Mucus. The structure of the mucus layer, particularly in the small intestine, can modulate the rate of nutrient absorption. For example, dietary fibers (alginate) can combine with the intestinal mucus and delay the transport of lipid digestion products. 130 Various polyphenols can interact with oral or gastrointestinal mucins. For example, the oral mucins MUC5B and MUC7 aggregate in the presence of EGCG. [START_REF] Davies | Reorganisation of the Salivary Mucin Network by Dietary Components: Insights from Green Tea Polyphenols[END_REF] Binding of EGCG to purified porcine gastric mucin was characterized and indicated a multilayer binding process. 131 Cross-linking of the GI purified mucins MUC5A and MUC2 by tea-derived galloylated polyphenols was also demonstrated. 132 Using a cell-based model of oral epithelium lined by a mucosal pellicle, [START_REF] Ployon | The Membrane-Associated MUC1 Improves Adhesion of Salivary MUC5B on Buccal Cells. Application to Development of an in Vitro Cellular Model of Oral Epithelium[END_REF] it was shown that the presence of bPRPs greatly moderated the impact of EGCG on the formation of MUC5B aggregates. [START_REF] Ployon | Mechanisms of Astringency: Structural Alteration of the Oral Mucosal Pellicle by Dietary Tannins and Protective Effect of BPRPs[END_REF] In contrast, such a formal demonstration of the impact of salivary proteins in the gastrointestinal tract has to our knowledge not been performed. Nonetheless, some observations are in line with this hypothesis. For example, in pigs fed a diet rich in shredded acorns, the cardiac gland region of the stomach became dark-brownish, and fluorescent microscopy revealed that tannins bonded to the gastric mucin, but no tissue damage was observed. This relative innocuity of acorn tannins was accompanied by an accumulation of endogenous proline in the stomach. Although the origin of this proline was not precisely determined, the authors highlighted the possibility that it may correspond to salivary PRPs. 133 Therefore, PP-SP interactions would not prevent binding to gastric mucins but would limit the disruption of the mucin layer. If similar protective phenomena occur toward intestinal mucins, this would most likely preserve the physical structure of the mucus layer and, in turn, the diffusion of nutrients and the rates of nutrient absorption.

Transport of Polyphenols across the Intestinal Epithelial Barrier. Some studies have focused on the transport of polyphenols across the intestinal epithelium, in most cases using cell culture methods. However, studies often use polyphenols in their original form and not as complexes with dietary or salivary proteins. An interesting study investigated whether a basic PRP (IB4) modulated such transport in Caco-2 cells. 134 It was found that the presence of IB4 reduced the transport of a hydrolyzable tannin (pentagalloyl glucose) in both the apical to basolateral and basolateral to apical directions and that this was concomitant with the formation of insoluble tannins-IB4 complexes. The study was further extended to another salivary protein (histatin 5) and to two polyphenols. 135 The presence of histatin 5 considerably reduced the transport of pentagalloyl glucose across cells but hardly affected the transport of quercetin. The authors, therefore, concluded that some salivary proteins may act as scavenger molecules as far as in the intestine, to limit the uptake of tannins. Another article documented that the cytotoxic effect of EGCG was higher on intestinal HT29 (non-mucin-secreting) than on HT29-MTX-E12 (mucinsecreting) intestinal cells and overall much higher than the cytotoxicity of EC, which has lower ability to bind proteins. The results suggested a binding of EGCG to the mucus which prevented absorption of the noxious polyphenol. The presence of β-caseins further reduced the EGCG cytotoxicity, and the authors pointed out that β-casein may be considered as a simple model to replicate the effect of salivary PRPs due to common characteristics such as richness in proline residues. 136 Therefore, by analogy and as in the previously quoted articles, PRP-EGCG interactions would represent a protective mechanism to limit tannin bioavailability.

Modifications of the Chyme Physical Properties. One of the functions of saliva is to produce a food bolus that is lubricated and cohesive and, thus, safe to swallow. Saliva can also induce in-mouth emulsification of oil or fat 137 or flocculation of emulsions. 138 A recent study documented the impact of proteins modeling salivary constituents (α-amylase, mucin, or a combination of both) on the particle size of peanut F olesome emulsions after in vitro oral and gastrointestinal digestion. Salivary constituents had no impact on the particle size distribution after the model oral phase but induced flocculation in gastric conditions, particularly in the presence of mucins. [START_REF] Wang | Effect of Simulated Saliva Components on the in Vitro Digestion of Peanut Oil Body Emulsion[END_REF] Given the high affinity of polyphenols for mucins as described above, it would be of interest to determine whether this effect is modified by the presence of polyphenols.

■ RESEARCH PROSPECTS

This rapid review of the literature highlighted that research on the consequences of polyphenols on digestive processes is active, and so is work on polyphenols-salivary proteins interactions. The impact of saliva on digestive processes is also an emerging field. However, only a limited number of studies have combined those topics. The few experimental works on the interactions salivary proteins-polyphenols (and even more specifically salivary proteins-tannins) all point at beneficial consequences on digestive events: such interactions would protect against the inhibition of digestive enzymes (amylase, lactase, and proteolytic enzymes), would limit the alteration of the gastric and intestinal mucosal integrity, and would reduce intestinal absorption of potentially toxic tannins (Figure 2).

Other potential consequences regarding oxidation of nutrients or the physical properties of the chyme in the gastric compartment (with, in turn, impact the digestibility or digestion kinetics) are only hypothetical and deserve further attention.

In any case, the studies focusing on the matter should carefully consider the ratio of the different interactors; that is, what is the proportion of polyphenols or tannins bound by salivary proteins, and how do these saliva proteins compete with proteins of the food matrix or with other host-derived proteins (in particular mucins of the gastrointestinal lining)? To our knowledge such data are not available in conditions mimicking digestion of a mixed meal containing polyphenols. One should also consider that salivary proteins-tannins complexes formed in the oral cavity will be modified downstream during digestion. Studies on absorptive events (in particular, those using cellular models) could, therefore, take advantage of the now well-recognized in vitro digestion models to prepare the materials that may be applied to cells. It is thanks to those improvements that the combined effects of polyphenol and salivation on digestion will be adequately characterized. Such results would be of interest when formulating nutritional recommendations on tannin-containing foods, particularly for specific populations affected by hyposalivation due to pathologies, medical treatments, or aging.

■ AUTHOR INFORMATION

Figure 1 .

 1 Figure 1. Three classes of vegetable tannins

  Martine Morzel -INRAE, Institut Agro, UMR STLO, F-35042 Rennes, France; orcid.org/0000-0002-3589-3641; Phone: +33 223 48 56 17; Email: martine.morzel@ inrae.fr; Fax: +33 223 48 53 50

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Francis Canon