Mousseaux

Emilie Bollache
email: emilie.bollache@imed.jussieu.fr

PhD Nadjia Kachenoura

PhD, Ioannis BARGIOTAS a,b,c , MS Alain Giron

PhD Alain De Cesare

PhD Alban Redheuil

PhD Elie Mousseaux

How to estimate aortic characteristic impedance from magnetic resonance and applanation tonometry data?

Keywords: aortic characteristic impedance, velocity-encoded magnetic resonance, applanation tonometry, pulsatile load

How to estimate aortic characteristic impedance from magnetic resonance and applanation tonometry data?

Introduction

Structural and hemodynamic alterations of large conduit arteries, occurring with aging [START_REF] Laurent | Expert consensus document on arterial stiffness: methodological issues and clinical applications[END_REF][START_REF] Mitchell | Arterial and cardiovascular events: the Framingham Heart Study[END_REF][START_REF] Vlachopoulos | Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis[END_REF] as well as other aggravating factors and leading to arterial stiffness [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF], have been associated with increased cardiac and cerebral mortality [START_REF] Mitchell | Arterial and cardiovascular events: the Framingham Heart Study[END_REF][START_REF] Vlachopoulos | Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis[END_REF]. Proximal aortic characteristic impedance (ZcAo), defined as the pressure change generated by a given flow wave change in the absence of reflections [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF], is an index related to both local stiffness and geometry. It contributes to the pulsatile arterial load faced by left ventricle (LV) during ejection and has been shown to be an independent predictor of LV mass index [START_REF] Chirinos | Arterial pulsatile hemodynamic load induced by isometric exercise strongly predicts left ventricular mass in hypertension[END_REF]. Most studies, using invasive [START_REF] Binkley | Influence of positive inotropic therapy on pulsatile hydraulic load and ventricularvascular coupling in congestive heart failure[END_REF][START_REF] Gundel | Aortic input impedance in man: acute response to vasodilator drugs[END_REF][START_REF] Kelly | Noninvasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF][START_REF] Kromer | Aortic input impedence and neurohormonal activation in patients with mild to moderate chronic congestive heart failure[END_REF][START_REF] Laskey | Characteristics of vascular hydraulic load in patients with heart failure[END_REF][START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF][START_REF] Merillon | Aortic input impedance in heart failure: comparison with normal subjects and its changes during vasodilator therapy[END_REF][START_REF] Murgo | Aortic input impedance in normal man: relationship to pressure wave forms[END_REF][START_REF] Nichols | Input impedance of the systemic circulation in man[END_REF][START_REF] Nichols | Effects of age on ventricular-vascular coupling[END_REF][START_REF] Pepine | Aortic input impedance in heart failure[END_REF][START_REF] Ting | Arterial hemodynamics in human hypertension[END_REF] and non-invasive [START_REF] Kelly | Noninvasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF][START_REF] Cholley | Differential effects of chronic oral antihypertensive therapies on systemic arterial circulation and ventricular energetics in African-American patients[END_REF][START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF] techniques, estimated ZcAo in humans in the frequency domain as the aortic input impedance averaged over high frequencies to minimize the effects of reflected waves.

Other studies used simpler and faster time domain methods to calculate ZcAo, based on the early systolic part of pressure and flow waveforms, where reflections effects are supposedly negligible. Such studies used either invasive data from animals [START_REF] Dujardin | Characteristic impedance of the proximal aorta determined in the time and frequency domain: a comparison[END_REF][START_REF] Levy | Cardiac hypertrophy and characteristic impedance in spontaneously hypertensive rats[END_REF][START_REF] Li | Time domain resolution of forward and reflected waves in the aorta[END_REF] and humans [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF], or applanation tonometry of the carotid artery combined with Doppler echocardiography of the LV outflow tract (LVOT) [START_REF] Chirinos | Arterial pulsatile hemodynamic load induced by isometric exercise strongly predicts left ventricular mass in hypertension[END_REF][START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF][START_REF] Mitchell | Hemodynamics of increased pulse pressure in older women in the community-based Age, Gene/Environment Susceptibility-Reykjavik Study[END_REF][START_REF] Mitchell | Omapatrilat reduces pulse pressure and proximal aortic stiffness in patients with systolic hypertension: results of the conduit hemodynamics of omapatrilat international research study[END_REF][START_REF] Mitchell | Determinants of elevated pulse pressure in middle-aged and older subjects with uncomplicated systolic hypertension: the role of proximal aortic diameter and the aortic pressure-flow relationship[END_REF][START_REF] Mitchell | Arterial stiffness, pressure and flow pulsatility and brain structure and function: the Age, Gene/Environment Susceptibility--Reykjavik study[END_REF][START_REF] Sweitzer | Increases in central aortic impedance precede alterations in arterial stiffness measures in type 1 diabetes[END_REF], and showed good agreement between temporal estimates and the reference frequency domain values [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF][START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF][START_REF] Dujardin | Characteristic impedance of the proximal aorta determined in the time and frequency domain: a comparison[END_REF].

Cardiovascular magnetic resonance (CMR) is already used in clinical routine for reference LV systolic and global function evaluation and several studies also demonstrated its usefulness in the non-invasive estimation of aortic stiffness indices [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Dogui | Consistency of aortic distensibility and pulse wave velocity estimates with respect to the Bramwell-Hill theoretical model: a cardiovascular magnetic resonance study[END_REF][START_REF] Dogui | Measurement of aortic arch pulse wave velocity in cardiovascular MR: comparison of transit time estimators and description of a new approach[END_REF][START_REF] Grotenhuis | Reduced aortic elasticity and dilatation are associated with aortic regurgitation and left ventricular hypertrophy in nonstenotic bicuspid aortic valve patients[END_REF][START_REF] Redheuil | Reduced ascending aortic strain and distensibility: earliest manifestations of vascular aging in humans[END_REF]. Although phase-contrast (PC)-CMR sequences enable an accurate measurement of blood flow velocities with high temporal resolutions [START_REF] Firmin | The application of phase shifts in NMR for flow measurement[END_REF][START_REF] Gatehouse | Applications of phase-contrast flow and velocity imaging in cardiovascular MRI[END_REF], to our knowledge, only one study used CMR data to calculate ZcAo in the time domain [START_REF] Segers | Aortic reflection coefficients and their association with global indexes of wave reflection in healthy controls and patients with Marfan's syndrome[END_REF], but from aortic areas estimated from steady-state free-precession (SSFP) sequences, rather than aortic flows. Accordingly, our aims were 1) to non-invasively estimate ascending aorta characteristic impedance in the time domain in healthy normotensive and hypertensive subjects using a semi-automated analysis of aortic PC-CMR flow and carotid applanation tonometry pressure curves, based on 7 methods previously reported in the literature, 2) to test which methods are able to characterize hypertensive patients as compared against controls with a similar mean age, and 3) to investigate which methods are better associated with the reference frequency domain characteristic impedance, as well as with carotid pulse pressure.

Methods

Study population

We studied 77 healthy subjects free from overt cardiovascular disease (31 women; mean age: 43±16 years [19-79 years]) and 16 patients with hypertension (as defined by either systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or current treatment for hypertension; 5 women, mean age: 61±9 years [50-81 years]).

The study protocol was approved by the institutional review board, and all participants were informed and provided signed consent in accordance with the declaration of Helsinki.

Data acquisition

Cardiovascular magnetic resonance

Each subject underwent CMR imaging of the thoracic aorta using a 1.5 Tesla GE system (Signa, General Electric Medical Systems, Waukesha, Wisconsin, USA) with cardiac phased array coil (eight channels).

First, axial cine images were acquired, during breath-holding, using a fast retrospectively gated gradient echo SSFP sequence, in the mid-ascending aorta perpendicular to the aorta at the level of the center of the right pulmonary artery (Figure 1 View sharing was also used, resulting in an effective temporal resolution of 15 ms. The imaged structure was always at the centre of the acquired image to minimize PEwraparound effects.

Blood pressures

First, an oscillometric sensor cuff was used to assess blood pressure at the brachial artery (Vital Signs Monitor, Welch Allyn Inc, USA) in the magnet simultaneously to aortic CMR acquisitions. Three measurements were averaged.

Then, applanation tonometry of right carotid artery was performed using the Pulse Pen device (Diatecne, Milano, Italy) [START_REF] Salvi | Validation of a new non-invasive portable tonometer for determining arterial pressure wave and pulse wave velocity: the PulsePen device[END_REF] immediately after CMR acquisitions. At least two acquisitions of over 10 consecutive waveforms were performed with a temporal resolution of 2 ms. Pressures were calculated after rescaling tonometric measurements by brachial mean and diastolic pressures measured during CMR acquisitions [START_REF] Laurent | Expert consensus document on arterial stiffness: methodological issues and clinical applications[END_REF], to account for potential arterial condition changes between CMR and tonometric examinations. Carotid pressure waveforms were averaged for each subject over several cardiac cycles while visually excluding irregular cardiac cycles, which presented a mismatch between pressures at the beginning and the end of the cycle. On average, 7 [START_REF] Vlachopoulos | Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis[END_REF][START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF][START_REF] Chirinos | Arterial pulsatile hemodynamic load induced by isometric exercise strongly predicts left ventricular mass in hypertension[END_REF][START_REF] Binkley | Influence of positive inotropic therapy on pulsatile hydraulic load and ventricularvascular coupling in congestive heart failure[END_REF][START_REF] Gundel | Aortic input impedance in man: acute response to vasodilator drugs[END_REF][START_REF] Kelly | Noninvasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF][START_REF] Kromer | Aortic input impedence and neurohormonal activation in patients with mild to moderate chronic congestive heart failure[END_REF][START_REF] Laskey | Characteristics of vascular hydraulic load in patients with heart failure[END_REF][START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF] cardiac cycles were taken into account to calculate carotid pressure waveforms. Carotid pulse pressure (cPP) was calculated as the difference between carotid systolic and diastolic blood pressures.

Data analysis and characteristic impedance estimation

Contours of the ascending aorta (AA) were automatically detected for all phases of the cardiac cycle on both SSFP and PC modulus images (Figure 1.B) using the validated ArtFun software (U.678 Inserm / UPMC) [START_REF] Herment | Automated segmentation of the aorta from phase contrast MR images: validation against expert tracing in healthy volunteers and in patients with a dilated aorta[END_REF], which has been previously used in various studies on large populations [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Redheuil | Reduced ascending aortic strain and distensibility: earliest manifestations of vascular aging in humans[END_REF][START_REF] Corden | Body fat is associated with reduced aortic stiffness until middle age[END_REF]. Such segmentation resulted in AA lumen area variations from SSFP data and AA flow curves from PC data, after superimposition of modulus contours on velocity images (Figure 1.C).

Estimation of local aortic pulse wave velocity

Local AA pulse wave velocity was calculated from AA distensibility as PWVAO = 1 / √(ρ • AA distensibility), according to the Bramwell-Hill model [START_REF] Dogui | Consistency of aortic distensibility and pulse wave velocity estimates with respect to the Bramwell-Hill theoretical model: a cardiovascular magnetic resonance study[END_REF][START_REF] Westenberg | Bramwell-Hill modeling for local aortic pulse wave velocity estimation: a validation study with velocity-encoded cardiovascular magnetic resonance and invasive pressure assessment[END_REF], where ρ = 1059 kg.m -3 is blood density and AA distensibility is calculated as (As -Ad) / (Ad • cPP), As and Ad being respectively systolic and diastolic AA lumen areas.

Estimation of aortic characteristic impedance

A custom software, developed and integrated to an interface in Matlab (Mathworks, Natick, MA, USA), was used to superimpose tonometry carotid pressure and PC-CMR AA flow waveforms that were oversampled with a 1 ms temporal step. Then, to account for the distance between carotid and aortic measurement locations, early systolic up-slopes of the pressure and flow waveforms were interpolated using linear regressions, and feet of the two resulting lines were shifted to the beginning of the cardiac cycle [START_REF] Chirinos | Arterial pulsatile hemodynamic load induced by isometric exercise strongly predicts left ventricular mass in hypertension[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF][START_REF] Dujardin | Characteristic impedance of the proximal aorta determined in the time and frequency domain: a comparison[END_REF]. In addition, to take into account potential changes in heart rate between CMR and tonometric acquisitions that would induce different systolic and diastolic durations, the systolic and diastolic phases of the two waveforms were individually temporally interpolated to the mean duration value. Prior to this interpolation, systolic and diastolic phases were manually defined by the dicrotic notch for pressure curves and the first zero crossing for flow curves. Finally, automated peak and minima detection, area under curve and time derivatives calculation (Figure 2.A-C) were integrated to the software, to estimate characteristic impedance, using 7 previously described methods:

1) ZcAoQmax = (PQmax -cDBP) / Qmax [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF], where cDBP is carotid diastolic blood pressure, Qmax is peak flow and PQmax is pressure magnitude at peak flow (Figure 2.A);

2) ZcAoQ95%max = (PQ95%max -cDBP) / (Q95%max -Qmin) [START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF] where Q95%max and Qmin are flow at 95% of its peak value and at its minimal value, respectively, and PQ95%max is pressure magnitude at 95% of peak flow (Figure 2.A);

3) ZcAoPi = (Pi -cDBP) / Qmax [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF], where Pi is pressure at inflection point corresponding to the initial upstroke of the reflected pressure wave (Figure 2.A); [START_REF] Chemla | Towards new indices of arterial stiffness using systolic pulse contour analysis: a theoretical point of view[END_REF], where ES is systolic duration and SV is stroke volume (Figure 2.A); 5) ZcAoslopes = Pslope / Qslope [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF], where Pslope and Qslope are pressure and flow early systolic up-slopes, respectively (Figure 2.A); 6) ZcAoderiv = P'max / Q'max [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF] where P'max and Q'max are peaks of pressure and flow time derivatives, respectively (Figure 2.B). 7) Zcloop was derived from pressure-flow loop as the slope of the linear interpolation of its early systolic part (Figure 2.C) [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF].

4) ZcAoSV = ES • (Pi -cDBP) / 2•SV
Of note, original pressure and flow curves, before temporal shift and resizing, were used for methods 5 and 6.

Pressure and flow curves were also used to estimate in the frequency domain aortic input impedance magnitude, calculated for each harmonic as the ratio between fast Fourier transform moduli of pressure and flow (Figure 2.D). Reference aortic characteristic impedance (ZcAoF) was then calculated by averaging input impedance between the frequency of its first local minimal value and 15 Hz [START_REF] Mitchell | Measurement of aortic input impedance in rats[END_REF], while excluding noisy magnitudes that are out of the range defined by input impedance mean magnitude ± 2 times its standard deviation, over this frequency range.

Statistical analysis

Mean values and standard deviations (SD) were provided for continuous variables.

For comparison of baseline characteristics, central blood pressures, aortic diameter, pulse wave velocity and characteristic impedance between "young" (below 50 years) and "elderly" healthy subjects (above 50 years), as well as between the hypertensive patients and the elderly normotensive subjects with a similar mean age, a nonparametric Mann-Whitney test was used. For comparisons on the whole group of healthy subjects of time domain characteristic impedance against their reference frequency counterpart as well as carotid pulse pressure, linear regression was used.

For all comparisons, Pearson correlation coefficients were provided. Further significance of the difference between such dependent correlations was also evaluated using an Hotelling-Steiger test [START_REF] Sheskin | Handbook of Parametric and Nonparametric Statistical Procedures: Third Edition[END_REF]. Briefly, given three variables X1, X2 and X3 and rij the correlation coefficient obtained for comparison between Xi and Xj, the Hotelling-Steiger test is

) ( 3 1 2 ) 1 )( 1 ( ) ( r r r R n n r n r r t         
, where n is sample size and

Results

Baseline characteristics, central blood pressures as well as aortic diameter, pulse wave velocity and characteristic impedance estimated in the frequency and time domains are summarized in Table 1 for young and elderly healthy subjects as well as hypertensive patients. By design, there was no significant difference in age between elderly healthy subjects and patients with hypertension. Also, gender distribution was the same in the three groups. All subjects had a body mass index BMI <30 kg/m², except for two hypertensive patients who had BMI of 30.7 kg/m² (70 year-old male) and 31.1 kg/m² (72 year-old male). Central blood pressures were within normal range for healthy subjects, and systolic, diastolic as well as mean blood pressures were significantly higher for elderly subjects when compared to young subjects.

Furthermore, as expected, both ascending aortic diastolic diameter and pulse wave velocity were increased in old subjects. An increasing trend was observed in hypertensive patients as compared to the elderly group for aortic pulse wave velocity and blood pressures, but did not reach statistical significance except for pulse pressure. Finally, while aortic characteristic impedance was not different between young and elderly healthy subjects whichever temporal or frequency methods are used, it was significantly increased in hypertensive patients when compared to the elderly subjects, when using the reference frequency domain method as well as temporal methods based on 95% of peak flow and up-slopes and time derivatives peaks ratios. Such changes in characteristic impedance are illustrated on the aortic input impedance spectra averaged over the groups of young healthy subjects, elderly healthy subjects and elderly hypertensive subjects provided in Figure 3. When studying gender-related differences in aortic characteristic impedance, all frequency and time domain methods resulted in no change in young healthy subjects, while in the elderly healthy subjects higher values were observed in women compared to men.

Figure 4 illustrates such gender-differences in characteristic impedance when estimated using the reference frequency method as well as the temporal method based on 95% of flow peak, which is the most commonly reported time domain method.

Over the 77 healthy subjects, all temporal aortic characteristic impedance estimates

were significantly correlated to central pulse pressure, with a correlation coefficient slightly superior for the method based on up-slopes ratio (Table 2). However, this superiority was not significant. For comparisons of the 7 temporal ZcAo against ZcAoF reference values estimated in the frequency domain, Pearson correlation coefficients as well as Bland-Altman mean biases and limits of agreement are provided in Table 2.

All relationships were significant with again a slight overall superiority in terms of correlation coefficient when using the method based on up-slopes. While association between the up-slopes method and the frequency domain reference was superior than associations obtained for methods 1, 3, 4 and 7 (ZcAoQmax: p=0.0002; ZcAoPi: p=0.0001; ZcAoSV: p<0.0001; ZcAoloop: p=0.002, respectively), they were equivalent to those of methods based on 95% of peak flow and time derivatives peaks ratio. Finally, Bland-Altman analysis indicated an overall underestimation of time domain methods when compared to the frequency domain reference and this underestimation was lower in terms of mean bias for the time derivatives peaks ratio method.

Discussion

Characteristic impedance of the proximal aorta is defined as the ratio between pressure wave change and the corresponding flow change, in the absence of reflections [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF]. Thus, it contributes to the pulsatile component of LV afterload, and was shown to be an independent predictor of LV mass index [START_REF] Chirinos | Arterial pulsatile hemodynamic load induced by isometric exercise strongly predicts left ventricular mass in hypertension[END_REF]. The majority of previous studies estimated human ZcAo in the frequency domain [START_REF] Binkley | Influence of positive inotropic therapy on pulsatile hydraulic load and ventricularvascular coupling in congestive heart failure[END_REF][START_REF] Gundel | Aortic input impedance in man: acute response to vasodilator drugs[END_REF][START_REF] Kelly | Noninvasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF][START_REF] Kromer | Aortic input impedence and neurohormonal activation in patients with mild to moderate chronic congestive heart failure[END_REF][START_REF] Laskey | Characteristics of vascular hydraulic load in patients with heart failure[END_REF][START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF][START_REF] Merillon | Aortic input impedance in heart failure: comparison with normal subjects and its changes during vasodilator therapy[END_REF][START_REF] Murgo | Aortic input impedance in normal man: relationship to pressure wave forms[END_REF][START_REF] Nichols | Input impedance of the systemic circulation in man[END_REF][START_REF] Nichols | Effects of age on ventricular-vascular coupling[END_REF][START_REF] Pepine | Aortic input impedance in heart failure[END_REF][START_REF] Ting | Arterial hemodynamics in human hypertension[END_REF][START_REF] Cholley | Differential effects of chronic oral antihypertensive therapies on systemic arterial circulation and ventricular energetics in African-American patients[END_REF][START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF], which is considered to be the reference method. Other studies estimated human ZcAo in the time domain using early systolic parts of pressure and flow waveforms, before reflections arrival, from either invasive pressure and flow data [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF] or applanation tonometry carotid pressure combined with LVOT flow measured using Doppler echocardiography [START_REF] Chirinos | Arterial pulsatile hemodynamic load induced by isometric exercise strongly predicts left ventricular mass in hypertension[END_REF][START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF][START_REF] Mitchell | Hemodynamics of increased pulse pressure in older women in the community-based Age, Gene/Environment Susceptibility-Reykjavik Study[END_REF][START_REF] Mitchell | Omapatrilat reduces pulse pressure and proximal aortic stiffness in patients with systolic hypertension: results of the conduit hemodynamics of omapatrilat international research study[END_REF][START_REF] Mitchell | Determinants of elevated pulse pressure in middle-aged and older subjects with uncomplicated systolic hypertension: the role of proximal aortic diameter and the aortic pressure-flow relationship[END_REF][START_REF] Mitchell | Arterial stiffness, pressure and flow pulsatility and brain structure and function: the Age, Gene/Environment Susceptibility--Reykjavik study[END_REF][START_REF] Sweitzer | Increases in central aortic impedance precede alterations in arterial stiffness measures in type 1 diabetes[END_REF]. However, in the majority of Doppler echocardiography-based studies, flow was calculated as LVOT maximal velocities multiplied by LVOT [START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF][START_REF] Mitchell | Omapatrilat reduces pulse pressure and proximal aortic stiffness in patients with systolic hypertension: results of the conduit hemodynamics of omapatrilat international research study[END_REF][START_REF] Mitchell | Determinants of elevated pulse pressure in middle-aged and older subjects with uncomplicated systolic hypertension: the role of proximal aortic diameter and the aortic pressure-flow relationship[END_REF][START_REF] Sweitzer | Increases in central aortic impedance precede alterations in arterial stiffness measures in type 1 diabetes[END_REF] or aortic [START_REF] Mitchell | Hemodynamics of increased pulse pressure in older women in the community-based Age, Gene/Environment Susceptibility-Reykjavik Study[END_REF][START_REF] Mitchell | Arterial stiffness, pressure and flow pulsatility and brain structure and function: the Age, Gene/Environment Susceptibility--Reykjavik study[END_REF] area, measured on a single phase of the cardiac cycle, and used as a surrogate for aortic flow. Besides, LVOT area was estimated from diameter while neglecting its non-circular shape [START_REF] Garcia | Comparison between cardiovascular magnetic resonance and transthoracic Doppler echocardiography for the estimation of effective orifice area in aortic stenosis[END_REF]. In addition, Doppler studies using LVOT flow instead of aortic flow to assess aortic impedance assume that such flows are comparable during early systole, despite the differences in geometry and elastic properties between the two locations. Indeed, such similarity in LVOT and aortic flows has never been verified and the few CMR studies which showed both LVOT and aortic velocity profiles highlighted obvious differences in waveforms in both early and late systole [START_REF] Garcia | Comparison between cardiovascular magnetic resonance and transthoracic Doppler echocardiography for the estimation of effective orifice area in aortic stenosis[END_REF]. CMR, with its velocity-encoded sequences, is already considered to be the reference for LV volumes including stroke volume, myocardial mass and thus global systolic function evaluation. In addition, it has been recently used to estimate aortic stiffness indices [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Dogui | Consistency of aortic distensibility and pulse wave velocity estimates with respect to the Bramwell-Hill theoretical model: a cardiovascular magnetic resonance study[END_REF][START_REF] Dogui | Measurement of aortic arch pulse wave velocity in cardiovascular MR: comparison of transit time estimators and description of a new approach[END_REF][START_REF] Grotenhuis | Reduced aortic elasticity and dilatation are associated with aortic regurgitation and left ventricular hypertrophy in nonstenotic bicuspid aortic valve patients[END_REF][START_REF] Redheuil | Reduced ascending aortic strain and distensibility: earliest manifestations of vascular aging in humans[END_REF], providing direct measurements of local aortic elasticity, as well as time-varying aortic blood flow.

Therefore, we hypothesized that carotid pressure tonometry data combined with such aortic PC-CMR flow data would enable an accurate estimation of ZcAo, providing supplementary data on LV load especially regarding the contribution to its pulsatile component.

To our knowledge, this is the first study to report the temporal estimation of ZcAo using aortic PC-CMR and carotid tonometry data. Another original feature of our study is that seven previously presented methods of characteristic impedance estimation in the time domain [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF][START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF][START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF][START_REF] Chemla | Towards new indices of arterial stiffness using systolic pulse contour analysis: a theoretical point of view[END_REF] that could be easily integrated to a clinical workflow were tested on a group of 93 subjects including healthy volunteers and asymptomatic patients with hypertension. Such methods were based either on peak flow [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF], 95% of peak flow [START_REF] Mitchell | Pulsatile hemodynamics in congestive heart failure[END_REF], pressure at the inflection point [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF], stroke volume [START_REF] Chemla | Towards new indices of arterial stiffness using systolic pulse contour analysis: a theoretical point of view[END_REF], pressure and flow time derivatives peaks [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF], systolic up-slopes [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF] or early systolic slope of the pressure-flow loop [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF]. For all methods, the resulting values were within the physiological range for both the healthy and hypertensive populations. Indeed, when comparing to studies including healthy subjects with a similar age range, Ting et al. [START_REF] Ting | Arterial hemodynamics in human hypertension[END_REF] reported a mean value of 112 dyne.s/cm 5 We observed no differences in aortic characteristic impedance between young and elderly healthy subjects, regardless of the method. This finding is not inconsistent with literature since discrepancies have been previously reported regarding agerelated variation of such index. Indeed, while its increase with age has been demonstrated in 45 healthy subjects [START_REF] Nichols | Effects of age on ventricular-vascular coupling[END_REF], a population-based study including 2026 healthy subjects [START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF] found steady impedance values with age in women and a decrease in men. These differences could be explained by the methods used for impedance calculation: characteristic impedance based on aortic velocity could be excessively increased, since systolic peak velocity decreases with aging as the aorta dilates, resulting in unchanged aortic flow with age. Also, when considering the theoretical water hammer model relating aortic characteristic impedance to aortic pulse wave velocity (PWV) and area (ZcAo = ρ • PWV / area, where ρ is blood density) [START_REF] Merillon | Relationship between physical properties of the arterial system and left ventricular performance in the course of aging and arterial hypertension[END_REF], we can hypothesize that ZcAo might not increase in elderly subjects due to the parallel age-related increase of both aortic PWV and area. Thus, the age-related dilation of the proximal aorta might express a remodeling process to normalize pulsatile load when aorta stiffens [START_REF] Mitchell | Hemodynamic correlates of blood pressure across the adult age spectrum: noninvasive evaluation in the Framingham Heart Study[END_REF]. Finally, the previously described differences in aortic characteristic impedance between men and women, which are more pronounced in elderly subjects [START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF], were found in our population, demonstrating the reliability of our measurements.

To compare and further assess the reliability of the 7 time domain methods, we tested the ability of characteristic impedance to separate between patients with hypertension and controls. First, values obtained in our hypertensive subjects were close albeit slightly lower than characteristic impedance previously averaged on untreated hypertensive patients (166 dyne.s/cm 5 ) [START_REF] Ting | Arterial hemodynamics in human hypertension[END_REF] using catheterization, despite higher blood pressures in this latter study (mean aortic systolic/diastolic pressures: 168/99 mmHg).

In addition, similar to previous literature [START_REF] Ting | Arterial hemodynamics in human hypertension[END_REF], we found that characteristic impedance was significantly increased in patients with hypertension when compared to the elderly healthy volunteers with a similar age, for the reference frequency method and temporal methods based on 95% of peak flow as well as up-slopes and time derivatives peaks ratios.

Moreover, all time domain methods were significantly correlated to central pulse pressure indicating the pulsatile feature of characteristic impedance and to the reference frequency domain characteristic impedance. Despite these good correlations, time domain methods underestimated characteristic impedance when compared to the frequency domain method. While these underestimations were in line with results of a previous study [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF], they differ from those of Segers et al. [START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF]. Such slight discrepancies between studies could be explained by differences in methods used for impedance estimation. Indeed several frequency ranges for characteristic impedance estimation were proposed in the literature [START_REF] Binkley | Influence of positive inotropic therapy on pulsatile hydraulic load and ventricularvascular coupling in congestive heart failure[END_REF][START_REF] Kelly | Noninvasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF][START_REF] Kromer | Aortic input impedence and neurohormonal activation in patients with mild to moderate chronic congestive heart failure[END_REF][START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF][START_REF] Merillon | Aortic input impedance in heart failure: comparison with normal subjects and its changes during vasodilator therapy[END_REF][START_REF] Cholley | Differential effects of chronic oral antihypertensive therapies on systemic arterial circulation and ventricular energetics in African-American patients[END_REF][START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF].

Furthermore, differences between time and frequency domain impedance estimates might be due to the fact that time domain methods rely only on early systolic upslopes, while the frequency domain method takes into account the entire cardiac cycle. Furthermore, associations between frequency domain impedance and time domain methods based on 95% of peak flow, derivative peaks and systolic up-slopes were slightly inferior to those previously reported (r=0.96 for the derivative peaks method and r=0.94 for the up-slopes method) in the invasive study presented by Lucas et al. [START_REF] Lucas | Comparison of time domain algorithms for estimating aortic characteristic impedance in humans[END_REF]. Such slight difference might be explained by the fact that authors in this latter study studied characteristic impedance in 134 patients from 1 month to 64 years (mean age: 13.5 years) with a congenital septal defect before and/or after closure of the septal defect (262 datasets), resulting in a wide range of characteristic impedance values (approximately between 20 and 1600 dyne.s/cm 5 ). Correlations obtained for the three aforementioned time domain impedance methods were equivalent to the association reported by Segers et al. on 2026 healthy subjects (mean age: 46 years) (r = 0.82) [START_REF] Segers | Noninvasive (input) impedance, pulse wave velocity, and wave reflection in healthy middle-aged men and women[END_REF]. The equivalence of the associations of the presented time domain methods with the reference method when comparing against studies on large populations and/or on a large range of characteristic impedance suggests the accuracy of our PC-CMR based impedance measurements that were performed on a relatively small population (frequency domain characteristic impedance ranged between 40 and 310 dyne.s/cm 5 ).

In addition to their slight superiority in terms of discrimination between hypertensive patients and controls with a similar mean age, and of correlations against carotid pulse pressure or frequency domain reference values, methods based on up-slopes and time derivatives peaks have the advantages of being performed on raw pressure and flow curves, before temporal shift and resizing of the curves, minimizing potential errors created by the fact that central blood pressure and flow were not measured at the same site.

Indeed, a first limitation of our study is that pressure and flow were measured at different arterial sites and not simultaneously, which is required for an accurate estimation of characteristic impedance. However, such simultaneous measurements in the aorta can be performed only using invasive techniques, which cannot be acquired in healthy volunteers. To minimize such errors, pressure was measured using applanation tonometry at the carotid artery which is considered as a good surrogate for central blood pressure [START_REF] Van Bortel | Non-invasive assessment of local arterial pulse pressure: comparison of applanation tonometry and echo-tracking[END_REF] and has been widely used to derive relevant parameters that are strongly associated with cardiovascular events [START_REF] Vlachopoulos | Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis[END_REF]. Moreover, brachial blood pressure measurements acquired simultaneously to PC-CMR aortic imaging were used to calibrate the carotid pressure waveform, based on the assumption that diastolic and mean pressures remain constant between central and peripheral arteries [START_REF] Kelly | Noninvasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF][START_REF] Borow | Noninvasive estimation of central aortic pressure using the oscillometric method for analyzing systemic artery pulsatile blood flow: comparative study of indirect systolic, diastolic, and mean brachial artery pressure with simultaneous direct ascending aortic pressure measurements[END_REF]. Another limitation inherent to PC-CMR acquisitions is errors in velocity and flow measurements related to phase-offset. Such errors were minimized using a 50% rectangular field of view centered on the aorta but can be alternatively corrected using techniques presented in previous studies [START_REF] Chernobelsky | Baseline correction of phase contrast images improves quantification of blood flow in the great vessels[END_REF]. In addition, our study focused on comparison of various techniques all based on the same data, reducing the effect of such phase-offset errors.

Conclusion

Aortic characteristic impedance estimated in the time domain using PC-CMR and applanation tonometry data along with the straightforward and fast computation derivatives peaks, up-slopes and 95% of peak flow methods that can be easily integrated to a clinical workflow, was able to distinguish patients with hypertension from controls with a similar age and was in good agreement with carotid pulse pressure and with the frequency domain characteristic impedance reference. Such parameter of the pulsatile load facing the LV during its ejection might be complementary to the emerging CMR aortic stiffness and geometry indices [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF] to characterize LV-aortic coupling, especially in disease such as arterial hypertension.

  .A), using the following typical scan parameters: repetition time (TR) = 3.4 ms, echo time (TE) = 1.4 ms, flip angle = 50°, views per segment = 6, slice thickness = 6 mm, pixel spacing = 0.76x0.76 mm², matrix = 224x192 and temporal resolution = 10 ms after applying a view sharing technique. Then, the same slice location was used to acquire PC images, using a 2D throughplane velocity-encoded sequence with retrospective gating during breath-holding (Figure 1.B and C). Averaged acquisition parameters were: TR = 7.4 ms, TE = 3.0 ms, flip angle = 20°, number of excitation = 1, views per segment = 2, slice thickness = 8 mm, pixel spacing = 1.64x1.64 mm², acquisition matrix = 256x128, encoding velocity = 200 cm/s. To minimize background offsets and so that acquisition duration remained compatible with breath-holding, a 50% rectangular field of view was used.

Figure 1 -

 1 Figure 1 -A. Illustration on a sagittal view of the positioning (red line) of CMR axial

Figure 2 -

 2 Figure 2 -Semi-automated measurement of time domain (A-C) and frequency

Figure 3 -

 3 Figure 3 -Input impedance spectra averaged over the groups of young healthy

Figure 4 -

 4 Figure 4 -Aortic characteristic impedance values obtained in our healthy subjects

Table 2 -

 2 Comparisons of time domain aortic characteristic impedance against pulsepressure and the frequency reference values.

		Carotid pulse pressure	Frequency domain Zc Ao
		r	r	Bland-Altman biases (dyne.s/cm 5 )
	1) Zc AoQmax	0.50*	0.69*	-9 [-84;65]
	2) Zc AoQ95%max	0.49*	0.82*	-18 [-81;44]
	3) Zc AoPi	0.50*	0.65*	-3 [-81;75]
	4) Zc AoSV	0.44 †	0.65*	-29 [-106;49]
	5) Zc Aoslopes	0.52*	0.86*	-25 [-80;30]
	6) Zc Aoderiv	0.49*	0.82*	-2 [-62;57]
	7) Zc Aoloop	0.33 †	0.73*	-14 [-86;59]
	Zc Ao : aortic characteristic impedance; Time domain Zc AoQmax : based on peak flow;
	Zc AoQ95%max : based on 95% of peak flow; Zc AoPi : based on pressure at the inflection point;
	Zc AoSV : based on stroke volume; Zc Aoslopes : based on systolic up-slopes; Zc Aoderiv : based on
	time-derivatives peaks; Zc Aoloop : based on early systolic slope of the pressure-flow loop.
	Pearson correlation coefficients and Bland-Altman biases (defined as the averaged temporal
	value -frequency value) as well as limits of agreement (defined as [mean bias-1.96xSD;
	mean bias+1.96xSD]) are provided.
	* indicates p<0.0001 and † indicates p<0.005.

Acknowledgment

Ioannis Bargiotas was co-funded through Greek State Scholarship Foundation (IKY) academic year 2011-2012, by the funds of European Social Fund (ESF) and NSRF, 2007-2013.

Tables

 Table 1 -Subjects baseline characteristics, central blood pressures, aortic geometry and stiffness indices as well as aortic characteristic impedance estimated in frequency and time domains averaged for healthy young (below 50 years) and elderly (above 50 years) subjects as well as hypertensive patients.


Prof Laurent Beaugerie

Fabrice Carrat

Philippe Seksik

Jean-Frédéric Colombel

Laurent Peyrin- Biroulet

Laurent Beaugerie
email: laurent.beaugerie@aphp.fr

Frédéric Colombel

Janssen

The effects of aminosalicylates or thiopurines on the risk of colorectal cancer in inflammatory bowel disease

Keywords: aminosalicylates, thiopurines, inflammatory bowel disease, colorectal cancer, chemoprevention, CESAME CRC, colorectal cancer; aminosalicylates, thiopurines, inflammatory bowel disease, colorectal cancer, chemoprevention, CESAME

Background: Whether aminosalicylates or thiopurines reduce the risk of colorectal cancer (CRC) in inflammatory bowel (IBD) disease is controversial. Aim: To assess simultaneously the chemopreventive effect of aminosalicylates or thiopurines in a case-control study nested in the CESAME observational cohort that enrolled consecutive patients with IBD between May 2004 and June 2005. Patients were followed up to December 2007. Methods: Study population comprised 144 case patients who developed CRC from the diagnosis of IBD (65 and 79 cases diagnosed respectively before and from 2004, starting year of the prospective observational period of CESAME) and 286 controls matched for gender, age, IBD subtype and year of diagnosis, and cumulative extent of colitis. Exposure to aminosalicylates or thiopurines was defined by an exposure to the treatment during the year of the diagnosis of cancer. The propensity of receiving 5-ASA and thiopurines was quantified by a composite score taking into account patient and IBD characteristics. The role of aminosalicylates or thiopurines was assessed by multivariate analysis. Propensity scores and the history of primary sclerosing cholangitis were entered into the multivariate model for adjustment.

Results: By multivariate analysis adjusted for propensity, a significant protective effect of exposure to drugs during the year of cancer was found for aminosalicylates (OR=0.587, 95%CI: 0.367-0.937, P = 0.0257), but not for thiopurines (OR=0.762, 95%CI: 0.432-1.343, P = 0.3468).

Conclusion:

In a case-control study nested in the CESAME cohort, a significant decrease in the risk of CRC in IBD was associated with exposure to aminosalicylates, not to thiopurines.

CESAME, Cancers Et Surrisque Associé aux Maladies inflammatoires intestinales En

France; CI, confidence interval; IBD, inflammatory bowel diseases; SD, standard deviation; UC, ulcerative colitis;

Introduction

Colorectal cancer (CRC) is the third most common cancer in the world. 1 Patients with inflammatory bowel disease (IBD) may develop sporadic CRC and CRC complicating inflammation of colonic mucosa. 2 The resulting epidemiologic figure is that patients with IBD have an overall two-fold increase in the risk of CRC compared with age and gender-matched individuals from the general population. 3 This relative risk is much more elevated (from 6 to 9) in patients suffering from IBD associated with primary sclerosing cholangitis, 4 and in patients with longstanding extensive colitis, defined by IBD duration superior to ten years and an estimated cumulative extent of colonic inflammation of 50% or greater. 5 Various markers of chronic histologically active colonic disease are also independently associated with an increased risk of CRC. 6 Detecting and removing early neoplastic lesions during colonoscopy surveillance and the use of chemopreventive agents are the two theoretical approaches for reducing the risk of CRC in patients with IBD. Given the evidence for cases of inflammationdriven CRC in IBD, all anti-inflammatory drugs used on a long-term basis in IBD, including aminosalicylates, immune-modulators and biologics are candidates for chemoprevention. In addition, it has been suggested that aminosalicylates could have various specific inhibiting effects on CRC carcinogenesis at a molecular level. 7 Aminosalicylates used in IBD, that include mesalazine, sulfasalazine, olsalazine and balsalazide, were first reported as potentially chemopreventive in individuals studies and in a first reference meta-analysis restricted to ulcerative colitis. 8 This conclusion was confirmed later in two updated meta-analyses, one pooling patients with ulcerative colitis and Crohn's disease, 9 and the other restricted to patients with ulcerative colitis, 10 but not in a meta-analysis restricted to non-referral populations of patients with IBD. 11 Data on the effect of exposure to thiopurines on the risk of CRC in IBD are more limited and conflicting. The first studies, published from 1990 to 2010 came mainly from referral centers and failed to demonstrate a chemopreventive effect, [12][13][14][15][16][17] whereas the converse was true for the majority of more recent studies, including studies from medico-administrative databases, 18 observational cohorts 19 and referral centers. 6 Two meta-analyses have been published up to now, one with negative results, 20 and the other suggesting a chemopreventive effect of thiopurines. 21 We decided to assess simultaneously the chemopreventive effect of aminosalicylates (primary objective) or thiopurines (secondary objective) in the CESAME (Cancers Et Surrisque Associé aux Maladies inflammatoires intestinales En France) observational cohort. CESAME is a prospective national French observational cohort that included 19 486 patients who were enrolled between May 2004 and June 2005, and followed up to December 2007. History of prior CRC was part of the baseline data recorded at entry, and all cases of incident CRC were subsequently recorded. We had no details regarding exposure to aminosalicylates in the general CESAME project, so we performed a nested case-control study, with retrospective study of case and control source medical files, using a specific report form.

Methods

CESAME cohort design and population

Details on the design and demographics of the cohort are available elsewhere. 22 In brief, from May 2004 to May 2005, 680 French gastroenterologists (38.4% with a fulltime hospital practice, 24.3% with a mixed public/private practice, and 37.3% with a full-time private practice) enrolled, on an unpaid basis, 19 486 consecutive IBD patients from their individual practices. There were no exclusion criteria.

Data were collected on an electronic case report form. The patients' demographic characteristics, IBD type, date of diagnosis, cumulative disease location (small bowel, colon (more or less than 50% of the total mucosal area affected, estimated from macroscopic or microscopic findings), or anus), previous history of cancer, and exposure to immune-suppressive therapy were recorded at the time of inclusion in the cohort. The gastroenterologists were asked to report all cases of high-grade dysplasia, cancer or death among their patients during the follow-up period, and to provide information on each surviving patient, obtained during a final visit between 1 January and 31 December 2007. They were also asked to record all changes in immune-suppressive treatment status at interim visits. The follow-up period ended on 31 December 2007. Follow-up was complete (i.e. included the final visit) in 16 459 cases (84.5%), partial (interim visits but no final visit) for 588 cases (3.0%), and nonexistent for 2439 cases (12.5%). The median follow-up of the overall population was

Selection of cases and controls

We used a matched case-control study to assess the link between exposure to salicylates and thiopurines and the risk of colorectal cancer in patients with IBD, with propensity scores to adjust for measured confounding factors related to exposure to treatment. Patients of the case-control study were recruited between May 2004 and December 2007. Cases were patients who had a history of colorectal cancer at entry in the cohort or who subsequently developed a colorectal cancer during the follow up.

Colorectal cancer was defined as malignant colorectal lesions not limited to the epithelium (i.e. excluding high-grade dysplasia). The date of colorectal cancer was considered as the date on which histological proof of colorectal cancer was obtained.

We excluded eight patients who developed colorectal cancer before the diagnosis of IBD. We aimed to select 2 controls for every case from the same source population who had no prior or current diagnosis of colorectal cancer on the date of the colorectal cancer in the case. Controls were individually matched to cases by age (up to 10 years older or younger), gender, IBD (Crohn's disease on one side, ulcerative colitis or IBD unclassified on the other), duration of disease (up to 5 years longer or shorter) and presence of an extensive colitis (estimated cumulative proportion of mucosal area macroscopically or microscopically affected by IBD >50%). The selection of controls in the cohort was realized by an independent statistician using the greedy matching algorithm which minimized a global distance criteria between cases and potential controls. 23 Matching criteria were checked by a trained monitor on the basis of source medical files. Eligible controls who underwent a proctocolectomy during the exposure study period (n=13) were secondarily excluded from the study, and replaced by the same number of controls according to the same selection rules.

Definition of exposures

In the specific case-control report form filled-in according to data of the source medical files, we recorded treatment intake on at least one occasion during a period from IBD onset to the date when colorectal cancer was ascertained in case and their matched controls, the years of first and last intake (from which we derived exposures to treatment the year when cancer was diagnosed), and the proportion of time under treatment during the five years preceding colorectal cancer diagnosis. Demographic characteristics, smoking status, and history of primary sclerosing cholangitis were noted. Number of IBD flares, surgical procedures and medical visits over the last 5 years preceding cancer occurrence in cases and their matched controls were recorded on a semi-quantitative scale and then dichotomized according to predefined threshold in the analyses. Medical data of the specific case-control report form were systematically reviewed by two senior gastroenterologists (LB & PS).

Statistical analysis.

We estimated that 120 cases and 240 controls would be necessary to confirm, with more than 80% power, a decrease of 50% of the risk of colorectal cancer with salicylates, assuming that the exposure to salicylates in the control group was between 40% and 80%. Assuming exposure to thiopurine therapy of 20% in the control group, we estimated that 200 cases and 400 controls would be necessary to confirm, with more than 80% power, a decrease of 50% of the risk of colorectal cancer with thiopurines.

We used univariate conditional logistic regression model and the Wald Chi-Square test to compare patients with colorectal cancer with their matched controls. In order to address confounding by indication, we adjusted our analyses of treatment exposures on the propensity scores, i.e. the probability of being treated with salicylates or thiopurines according to the observed covariates. To account for temporal change in the indication of treatments, we built two separate logistic models for the propensity score according to the date of cancer, before 2004, or in2004 or after. The propensity score for receiving a treatment was the predicted probability obtained from the logistic equation given the covariates values. Covariates tested in the propensity models were age, sex, IBD type, duration of IBD, extensive colitis, active smoking, ≥1 surgical procedure over the 5 years preceding the cancer date.

Among several methods for taking into account propensity, adjustment, matching, stratification (most often by quintiles) or weighting (i.e. inverse probability treatment weighting, IPTW) can be used. In our particular case, we chose to use propensity predicted probabilities as adjustment covariates rather that matching, stratification or weighting, because our analysis was already matched (with strata defined by a case and matched controls).

We first explored the univariate association between exposure to treatments or other covariates with colorectal cancer using conditional logistic regression with adjustment on propensity scores. A multivariate conditional logistic regression model was then built by entering exposure to treatments, propensity scores and all covariates with a P-value <0.20 in univariate analysis. All analyses were performed with SAS v9.4 software (SAS institute Inc, Cary, NC).

Results

Study population

The study recruited 144 cases and 286 controls, since two cases were matched with only one control. Baseline characteristics of cases and their matched controls are presented in Table 1. Mean age of patients was 52.5 (standard deviation (SD) = 15.7) years, 178 (41%) were female, 161 (37%) had Crohn's disease, 337 (78%) had an extensive colitis and IBD duration was on average 15.2 (SD = 10.7) years. Sixtyfive cases occurred before 2004 (range 1961 -2003) and 79 cases occurred in 2004 or after (range 2004-2007). A primary sclerosing cholangitis was found more frequently in cases (12%) than in matched controls (2% -P < 0.001). Over the 5 years preceding the date of cancer, there was no difference between cases and controls regarding the proportion of patients with more than one flare per year or the proportion of patients with at least one medical visit per year, however, the proportion of patients with history of at least one surgical procedure was lower in cases (17%) than in controls (25%, P = 0.0327)

Exposure to IBD drugs and propensity scores

Based on source medical files, exposure to salicylates or thiopurines the year of cancer was missing in 4 (3%) cases (3 < 2004, 1 >= 2004) and 6 (2%) controls (5 < 2004, and 1 >= 2004), so comparisons were made between 140 cases and 280 controls (Table 2). Exposure to salicylates treatment during the year of cancer did not differ between cases and their respective controls (51% vs. 58%, P = 0.1369). The mean duration of exposure to salicylates was 8.8 ± 9 (mean + standard deviation) years in cases and 10.7 ± 8 years in controls (P = 0.07). Among patients exposed to salicylates the year of cancer, 135 controls (83%) and 56 cases (80%) had been exposed for more than two years before the year of cancer. No difference was found for exposure to thiopurines during the year of cancer (22% vs 23%, P = 0.7978).

However, exposure to thiopurines at any moment preceding the cancer was lower in cases (31% vs. 41%, P = 0.0356). No difference was found between cases and controls for anti-TNF therapy or other immune-suppressive therapy, including corticosteroids, during the year of cancer. Eleven cases and three controls -all with primary sclerosing cholangitis -were treated with ursodeoxycholic acid. Among case and control patients exposed or non-exposed to aminosalicylates in the year of cancer, 97% and 95% respectively underwent a colonoscopy within the 5 years preceding the diagnosis of cancer in cases (and the corresponding index date in controls). These percentages were 83% and 75% for the 2-year period preceding the diagnosis of cancer, and 14% and 20% for the 5 to 2-year period before the diagnosis of cancer. Exploration of factors associated with exposure to salicylates during the year of cancer (Table S1) showed that, before 2004, the probability of being treated with salicylates was lower in men (OR= 0.494, 95%CI 0.263-0.927, P = 0.0282). In 2004 or after, treatment with salicylates was positively associated with age (OR = 1.034 per one year increase, 95%CI 1.015-1.054, P < 0.001), and negatively associated with Crohn's diseases (OR = 0.412, 95%CI 0.222-0.765, P = 0.0049) and with history of colonic surgery (OR = 0.373, 95% CI 0.183-0.763, P = 0.0068). Association of salicylates treatment with primary sclerosing cholangitis did not reach statistical significance either before 2004 (OR = 2.364, 95%CI 0.556-10.053, P = 0.2442) or in 2004 or after (OR = 4.813, 95%CI 0.923-25.11, P = 0.0623). When this analysis was repeated for thiopurine therapy (Table S2), we found that, before 2004, the probability of being treated with thiopurine was negatively associated with history of colonic surgery (OR = 0.231, 95%CI 0.063-0.838, P = 0.0258) and positively associated with Crohn's disease (OR = 8.343, 95%CI 3.236-21.50, P < 0.001). In 2004 or after, propensity score for thiopurine therapy was negatively associated with age (OR = 0.963 per one year increase, 95%CI 0.942-0.984, P < 0.001), and with history of colonic surgery (OR = 0.350, 95%CI 0.152-0.804, P = 0.0134) and positively associated with Crohn's disease (OR = 5.129, 95%CI 2.487-10.58, P < 0.001).

The derived propensity scores for salicylate treatment were higher in cases than their respective matched controls (58.3% vs. 54.3%, P < 0.001) but no significant difference was found as regards propensity score for thiopurines (24.0% vs. 22.4%, P = 0.0721).

Effect of salicylates and thiopurines

By univariate analysis adjusted on the propensity score for receiving salicylates treatment, a protective effect of exposure to salicylates during the year of cancer was found (OR = 0.567, 95%CI 0.359-0.896, P = 0.0150). Detailed analyses stratified by year of cancer (<2004, >=2004), by IBD subtype and according to presence of longstanding extensive colitis (Figure 1) showed that the most protective effect was found in patients with longstanding extensive colitis before 2004 (OR=0.345, 95%CI 0.148-0.801, P = 0.0133) and in patients with ulcerative colitis and longstanding extensive colitis (OR=0.302, 95%CI 0.135-0.675, P = 0.0036). There was no significant preventive effect in the subset of patients with Crohn's disease (OR=0.736, 95%CI 0.348-1.556, P = 0.4223).

By univariate analysis adjusted on the propensity score for receiving thiopurines, no association was observed between thiopurines treatment and colorectal cancer (OR = 0.828, 95%CI 0.488-1.403, P = 0.4826). This was also the case for all stratified analyses (Figure 2). Multivariate analysis adjusted on propensity scores for receiving salicylates or thiopurines confirmed the independent association of salicylates treatment with cancer (OR = 0.587, 95%CI: 0.367-0.937, P = 0.0257), and the increased risk associated with primary sclerosing cholangitis (OR = 8.321, 95%CI: 1.172-59.10, P = 0.0342); no other covariate met the statistical level of significance (Table 3).

Discussion

Using a propensity-adjusted case-control study nested in the CESAME cohort, covering the 1961-2007 period, we confirmed the overall chemopreventive effect of exposure to salicylates in the risk of CRC in IBD, but we failed to confirm this effect for thiopurines.

In 1994 was published the first individual case-control study suggesting a chemopreventive effect of sulfasalazine on the risk of colorectal cancer. 24 In 2004 was presented during the Digestive Disease Week the results of the first metaanalysis of case-control and cohort studies in ulcerative colitis, suggesting a halving of the risk of CRC in patients with a regular exposure to salicylates, at an average daily dose of 1.2 g mesalamine equivalents or more. 8 In this context, it is plausible that gastroenterologists were progressively more prone from the late 90s to prescribe salicylates in patients with ulcerative or Crohn's colitis at high risk of CRC, partly or purely for the purpose of chemoprevention, and particularly after communication of results of the first positive meta-analysis. This propensity bias could lead to an overrepresentation of exposure to salicylates in patients at higher intrinsic risk of CRC, that could in turn mask the real chemopreventive effect of aminosalicylates in casecontrol and cohort studies.

Therefore, in our case-control study, we decided to adjust the main analyses on the propensity for receiving aminosalicylates. In addition, in order to account for temporal change in the behaviour of prescribers, we used two separate logistic models for building the propensity score before 2004 and from 2004. We observed that, before adjustment on propensity, the crude percentages of patients were not different between cases and controls, whatever the expression of the exposures to salicylates (ever use, percentage of time of use within the 5-year period before cancer or any use in the year of diagnosis of cancer). There was a change in the main result regarding aminosalicylates after adjustment on propensity and other prognosis factors, with a final significant 41% reduction in the risk of CRC in patients with IBD exposed to aminosalicylates in the multivariate model. We believe that this observation is of methodological interest and should be taken into account in further case-control or cohort studies exploring the impact of drugs that are known or suspected candidates for decreasing a risk.

The impact of exposure to aminosalicylates on the risk of CRC was clear in patients with ulcerative colitis, particularly in those with longstanding extensive colitis. This result is in line with most individual studies and meta-analyses that addressed the question of the chemopreventive effect of aminosalicylates and included patients with ulcerative colitis. One individual population-based study, covering a relatively recent historical period (1995-2008) had a negative result, but in this study there was not stratification for the extent of colitis, and there was no use of methods to control indication bias no adjustment for propensity. 25 A meta-analysis coming from the same group distinguished referral and non-referral populations, and found no chemopreventive signal in the non-referral populations. 11 However, in this metaanalysis, CESAME data were classified as coming from a referral population. Twothird of the CESAME investigators were gastroenterologists with partial or pure private practice outside the hospital, and the observed incidences of CRC in CESAME were identical to those reported in meta-analyses from population-based studies. 2 So we believe that the CESAME design is much closer to that of populationbased studies than that of referral-centre studies.

It can be speculated that patients who have a good adherence to treatment with aminosalicylates also have a better adherence to endoscopic surveillance, this latter phenomenon representing the real cause of decrease in colorectal cancer incidence.

However, in our study, almost all patients exposed or non-exposed to aminosalicylates underwent at least one colonoscopy within the 5 years preceding the index date, which excludes this bias.

Growing evidence suggests that epidemiology and carcinogenesis of CRC in ulcerative colitis and Crohn's colitis are the same. 2 However, we failed in our study to reproduce in patients with Crohn's disease the favourable effect of exposure to salicylates demonstrated in patients with ulcerative colitis. We must be prudent before drawing a negative conclusion in Crohn's disease because of methodological limitations. Patients with Crohn's disease who are at high risk of CRC and good candidates for chemoprevention are those with longstanding extensive colitis. These patients represent a small subset of patients with Crohn's disease, and their separate analysis leads to underpowered and uncertain multiple sub-stratifications. In addition, few patients with Crohn's disease are now chronically exposed to salicylates, and aminosalicylates are often prescribed for the sole purpose of chemoprevention in patients at highest risk of colorectal cancer, leading to a high propensity bias. Finally, we have limited global view in this field because, to date, there is no meta-analysis on the chemopreventive effect of aminosalicylates restricted to Crohn's disease or Crohn's colitis.

We also failed to confirm a chemopreventive effect of thiopurines in this case-control study that covered the 1961-2007 period, while we reported in a previous study a reduction of the risk in patients with longstanding extensive colitis exposed to thiopurines in the 2004-2007 period. 5 First, our case-control study was adequately powered for salicylates, but not for thiopurines. Second, we can speculate that, if thiopurines act via their anti-inflammatory effect, the efficacy signal would become apparent if patients exposed to thiopurines have a milder 'mean' colonic inflammation than the patients who are not exposed. The use of thiopurines has been restricted up to the early 2000s to a subset of patients with the most severe inflammation. In the study by Matula et al, patients exposed to 6-mercaptopurine were more often exposed to corticosteroids than those non exposed, suggesting that chronic inflammation was not lower, but greater, in patients treated with thiopurines than in those not treated with thiopurines. 12 Following the diffusion of thiopurines, it is plausible that this figure has been inverted and opens an historical window more favourable to demonstrate the efficacy of thiopurines. 5,18,19 Unfortunately, we have no tool to validate this hypothesis in the absence of markers of intestinal inflammation in exposed vs. non-exposed patients in most studies.

We must point out some limitations of our study. The assessment of exposure to drugs was mainly retrospective. The cases of CRC that were diagnosed before the prospective observational period of the CESAME cohort did not provoke death by the enrolment in the cohort, leading to an over-representation of relatively benign cases of CRC within this period. Finally, in our multivariate model, we were not able to adjust for some lifestyle factors that are established risk factors for CRC (such as alcohol consumption or lack of physical exercise), and for chronic histological activity of colonic inflammation.

In conclusion, our study is confirmative of the favourable impact of chronic exposure of salicylates on the risk of CRC in IBD, with a maximal magnitude in patients with longstanding extensive ulcerative colitis.

Other information Funding 22 done

Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the present article is based *Give information separately cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies.

Note

  35 months (inter-quartile range [IQR], 29-40].The protocol was approved by the institutional review boards of the French National Society of Gastroenterology, Association François Aupetit (the French IBD patient association), and Groupe d'Etude Thérapeutique des Affections Inflammatoires du Tube Digestif (GETAID). The study was authorized by the French Data Protection Agency (CNIL, registration number #04-1239, dated 6 July 2004). The patients' specific written informed consent was not required for this observational study.

Figure 1 .Figure 2 .

 12 Figure 1. Multivariate-adjusted exploratory analyses of the association between

  ** Propensity score to receive thiopurines (during the YoC)

STROBE

  Statement-checklist of items that should be included in reports of observational studies Indicate the study's design with a commonly used term in the title or the abstract (b) Provide in the abstract an informative and balanced summary of what was done and what was found of study design early in the paper Setting 5 done Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection Cohort study-Give the eligibility criteria, and the sources and methods of (b) Cohort study-For matched studies, give matching criteria and number of exposed and unexposed Case-control study-For matched studies, give matching criteria and the number of controls per case Variables 7 Done Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if applicable Data sources/ measurement 8* done For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability of assessment methods if there is more than one group Bias 9 Done Describe any efforts to address potential sources of bias Study size 10 done Explain how the study size was arrived at Quantitative variables 11 done Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why Statistical methods 12 Done (a) Describe all statistical methods, including those used to control for confounding (b) Describe any methods used to examine subgroups and interactions (c) Explain how missing data were addressed (d) Cohort study-If applicable, explain how loss to follow-up was addressed (e) Describe any sensitivity analyses Continued on next page Report numbers of individuals at each stage of study-eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (b) Give reasons for non-participation at each stage (c) Consider use of a Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential confounders (b) Indicate number of participants with missing data for each variable of interest (c) Cohort study-Summarise follow-up time (eg, average and total amount) Outcome data 15* done Cohort study-Report numbers of outcome events or summary measures over time Main results 16 done (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and why they were included (b) Report category boundaries when continuous variables were categorized (c) If relevant, consider translating estimates of relative risk into absolute risk for a the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias Interpretation 20 done Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar studies, and other relevant evidence Generalisability 21 N/A Discuss the generalisability (external validity) of the study results

Table 1 . Patient baseline characteristics

 1

	Cases	Controls	P -value
	(n=144)	(n=286)

Table 2 .

 2 Exposure to IBD drugs in cases and controls

Table 3 .

 3 Multivariate analysis of factors associated with colorectal cancer, adjusted on propensity scores for receiving salicylates or thiopurines.

		23 (8)	0.7552
		14 (5)	0.5911
		1.6 +/-7.8	0.6426
		16 (6)	0.7578
		7 (3)	0.8232
		1.2 +/-8.6	0.9754
		8 (3)	0.6709
		1 (0)	0.9917
	3 +/-3.4	0.2 +/-1.8	0.6793
	46 (33)	115 (41)	0.0642
	35 (25)	77 (28)	0.5372
	18.4 +/-34.6	20.0 +/-34.8	0.5755
	92 (66)	208 (74)	0.0331
	31 (22)	55 (20)	0.6549
	13.8 +/-26.7	14.0 +/-24.5	0.9752
	13 (9)	4 (1)	0.0011
	11 (8)	3 (1)	0.0025
	5.0 +/-19.6	0.9 +/-8.9	0.0239
	3 (2)	20 (7)	0.0409
	1 (1)	11 (4)	0.0783
	0.4 +/-3.2	1.9 +/-11.3	0.1897

:

  An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is available at www.strobe-statement.org.

Table S1 .

 S1 Propensity for being treated with aminosalicylates the year of cancer according to

	covariates and calendar periods
		<2004		>=2004
	Covariates	OR	p-value	OR	p-value
	Age (per yr increase)	1.02 (0.99-1.04)	0.19	1.03 (1.02-1.05)	<0.001
	Male gender (vs female)	0.49 (0.26-0.93)	0.03	1.02 (0.55-1.88)	0.96
	Crohn's disease (vs	0.81 (0.42-1.56)	0.52	0.41 (0.22-0.77)	0.004
	ulcerative colitis or IBD
	unclassified)
	Duration of IBD (per year	1.00 (0.96-1.03)	0.78	1.03 (0.99-1.07)	0.10
	increase)
	Extensive colitis	1.29 (0.63-2.67)	0.49	0.62 (0.30-1.31)	0.22
	Primary sclerosing	2.36 (0.56-10.1)	0.24	4.81 (0.92-25.1)	0.06
	cholangitis (Yes vs No)
	Active smoking (Yes vs No) 0.97 (0.29-3.27)	0.96	0.91 (0.37-2.25)	0.83
	>= 1 surgical procedure	0.64 (0.30-1.39)	0.26	0.37 (0.18-0.76)	0.007
	over the 5 years

Table S2 .

 S2 Propensity for being treated with thiopurines the year of cancer according to covariates and calendar periods

		<2004		>=2004
	Covariates	OR	p-value	OR	p-value
	Age (per yr increase)	1.00 (0.97-1.03)	0.93	0.96 (0.94-0.98)	<0.001
	Male gender (vs female)	0.44 (0.18-1.08)	0.07	1.08 (0.55-2.14)	0.82
	Crohn's disease (vs	8.34 (3.24-21.5)	<0.001	5.13 (2.49-10.6)	<0.001
	ulcerative colitis or IBD
	unclassified)
	Duration of IBD (per year	1.04 (0.99-1.09)	0.13	1.01 (0.97-1.05)	0.56
	increase)
	Extensive colitis	2.07 (0.68-6.31)	0.20	1.58 (0.71-3.51)	0.27
	Primary sclerosing	0.23 (0.02-2.44)	0.22	0.55 (0.12-2.45)	0.44
	cholangitis (Yes vs No)
	Active smoking (Yes vs No) 0.96 (0.18-5.22)	0.96	0.81 (0.32-2.05)	0.66
	>= 1 surgical procedure	0.23 (0.06-0.84)	0.03	0.35 (0.15-0.80)	0.01
	over the 5 years

ACKNOWLEDGEMENTS

Collaborators of the CESAME Study group are listed in Supporting information, Appendix S1 FUNDING: The CESAME project was supported by grants from Programme Hospitalier de Recherche Clinique National (AOM05157), Association François Aupetit, Délégation Inter-régionale de la Recherche clinique Ile de France-AP-HP, Ligue contre le Cancer, and Fonds de Recherche de la Société Nationale Française de Gastro-entérologie. The nested case-control study on the effect of aminosalicylates or thiopurines on the risk of colorectal cancer was supported by an unconditional grant from Ferring Pharmaceuticals.

grants from Biocodex, sponsored travel from Merck-MSD and Takeda. Jean-

Supporting Information

Table S1. Propensity for being treated with aminosalicylates the year of cancer according to covariates and calendar periods Table S2. Propensity for being treated with thiopurines the year of cancer according to covariates and calendar periods Appendix S1. List of collaborators of the CESAME Study Group AUTHORSHIP Guarantor of the article: Fabrice Carrat. Author contributions: LB and FC were jointly responsible for the study concept, design and implementation. LB and FS were responsible for the reviewing of medical data extracted from source patient files. FC Appendix S1

Collaborators of the CESAME Study Group

The following investigators participated in the Study, listed from highest to lowest number of patients enrolled in the cohort: Jean-Frédéric Colombel; Jacques Cosnes; Jean-Pierre Gendre; Marc Lémann; Xavier


Mehwish Younas

Fabrice Carrat

Corinne Desaint

Odile Launay

Professor Pierre Corbeau
email: pierre.corbeau@igh.cnrs.fr

Sanae Finge

Immune activation, smoking, and vaccine response

Keywords: Hepatitis B virus, HIV infection, tobacco, inflammation

Understanding the factors that fuels inter-individual variability in vaccine response is important to improve vaccine efficiency. Here we tested the hypothesis that chronic immune activation might impair vaccine response. To this aim, we studied whether immune activation observed in HIV-1-infected patients [START_REF] Psomas | One of the immune activation profiles observed in HIV-1-infected adults with suppressed viremia is linked to metabolic syndrome : the ACTIVIH study[END_REF] might reduce hepatitis B virus (HBV) vaccine efficiency.

We chose to quantify in peripheral blood soluble CD40L to evaluate platelet, endothelial and T cell activation, IgG, IgA, and IgM as a sign of B cell activation, soluble CD14 and CD163 to measure macrophage/monocyte activity, interleukin-6, soluble TNF receptor I (sTNFRI), and interferon gamma-induced protein 10 to estimate inflammation. We analyzed these markers before vaccination in 124 HIV-1-infected adults who received 3 intramuscular injections of GenHevac B Pasteur 20 µg in the course of ANRS HB03 trial [START_REF] Launay | Safety and immunogenicity of 4 intramuscular double doses and 4 intradermal low doses vs standard hepatitis B vaccine regimen in adults with HIV-1: a randomized controlled trial[END_REF]. Eighty-four (68%) patients were responders i.e. with anti-hepatitis B surface antigen (HBs) titers higher or equal to 10 mIU/mL one month after the third vaccine injection.

Among the tested markers, sTNFRI was the only one higher in non-responders than in responders (3.27±1.58 ng/mL and 2.50±1.13 ng/mL, p=0.016, Figure 1). The anti-HBs titers at week 28 tended to be inversely correlated with baseline sTNFRI levels (r=-0.168, p=0.061).

A multivariate analysis adjusted on all the factors previously shown to be predictive (female sex, lower age, no active smoking, higher CD4 count and undetectable HIV-1 RNA at baseline [START_REF] Launay | Safety and immunogenicity of 4 intramuscular double doses and 4 intradermal low doses vs standard hepatitis B vaccine regimen in adults with HIV-1: a randomized controlled trial[END_REF]) and on nadir CD4 count confirmed the independent link between sTNFRI and vaccine response (odds-ratio (OR) 0.65 [95% CI 0.46-0.93], p = 0.019). Smoking did not remain associated with vaccine response when sTNFRI was introduced in the model (OR for smoking 0.46 [0.20-1.04]; p=0.06; OR for sTNFR1 0.67 [0.49-0.91], p=0.01), suggesting that the link between smoking and impaired response is mediated by immune activation. sTNFRI levels were higher in smokers than in non-smokers (3.10±1.36 ng/mL and 2.57 ±1.29 ng/mL, p=0.028, Figure 1). Moreover, the association between sTNFRI levels and smoking remained significant after adjustment on age, sex, CD4 count, nadir CD4 T count and undetectable HIV RNA (p=0.037 in a multiple linear regression model). We show here that sTNFRI, a marker of inflammation, is predictive for vaccine response. Our data are in line with the observation that response to influenza vaccine is correlated with plasma TNFa levels [START_REF] Parmigiani | Impaired antibody response to influenza vaccine in HIV-infected and uninfected aging women is associated with immune activation and inflammation[END_REF]. This might open the way in the future for tailored vaccination adapted to each patient's immune activation level. Of note, in clinical settings of long-lasting inflammation as chronic infections, autoimmune diseases, and aging, poor vaccine immunogenicity is often observed. Another finding of our work is the link between smoking and inflammation. This concurs with the link reported between T cell activation and tobacco smoking [START_REF] Valiathan | Tobacco smoking increases immune activation and impairs T-cell function in HIV infected patients on antiretrovirals: a cross-sectional pilot study[END_REF]. It raises the interesting hypothesis that tobacco might be pathogenic via immune activation.

Figure legend .

 legend Figure legend. Mean levels of sTNFRI in responders and non-responders to HBV