Amel Halimi
email: halimi.amel.18@gmail.com

Achraf Boudiaf

Larbi Hemmouche

Aboubakr Medjahed

Djalel Eddine Tria

Abdelkhalek Henniche

Mohammed El

Amine Djeghlal

Thierry Baudin

Quasi-static and Dynamic Characterization of Ultrafine-Grained 2017A-T4 Aluminium Alloy Processed by Accumulative Roll Bonding

Keywords: Accumulative roll bonding (ARB), 2017A-T4 aluminium alloy, Quasi-static loading, Dynamic loading, Grain refinement

In this study, novel composite strips based on 2017A-T4 aluminium alloy (Al-Cu-Mg) produced by accumulative roll bonding (ARB) were developed. The microstructure and mechanical properties of the ultrafine-grained sheets under quasi-static and dynamic loadings were investigated. The initial microstructure characterization with Optical Microscope and Scanning Electron Microscope indicated that the ARBed sheets formed a compact material with homogeneous and identical thickness for the individual bonded layers. Besides, the presence of precipitates was identified in all the processed strips with diverse sizes, quantities and distribution. Moreover, from Electron Back Scatter Diffraction, the microstructure was noticeably refined with increasing the ARB cycles to reach 1.7 µm of the grain size at the fifth cycle. The microhardness measurement and the tensile test were carried out for both natural ageing and ARBed specimens. Accordingly, the tensile stress acts on the individual layers rather than the entire sample that conduct to a reduction in the overall properties for the ARBed strips. Furthermore, a stabilization in the mechanical properties for the three first ARB cycles was noted, whereas, the domination of the dynamic recrystallization was responsible for a significant drop after the fourth cycle which is considered as the transition state. The characteristics of the compression deformation were examined under dynamic and quasi-static loadings conditions by using the Split-Hopkinson Pressure Bar system and the universal testing machine, respectively. The strain hardening behaviour was investigated using the Hollomon analysis. It was found that the thermal softening played a crucial role when compared to the strain hardening for all the studied strips. Moreover, the strain rate under the dynamic loading has a minor effect on the stress flow of the ARBed sheets compared to the as-received material.

Introduction

The severe plastic deformation process (SPD) consists of applying a large plastic deformation to material through a top-down approach to produce ultrafine-grained structures (UFGs) from the initial coarse-grained material [START_REF] Lee | Developing superplastic properties in an aluminium alloy through severe plastic deformation[END_REF][START_REF] Valiev | Structure and properties of ultrafine-grained materials produced by severe plastic deformation[END_REF][START_REF] Azushima | Severe plastic deformation (SPD) processes for metals[END_REF]. That allows the obtaining of outstanding overall properties according to Hall-Petch relation [START_REF] Hansen | Hall-Petch relation and boundary strengthening[END_REF][START_REF] Abson | The Hall-Petch Relation and High-Temperature Subgrains[END_REF][START_REF] Furukawa | Microhardness measurements and the Hall-Petch relationship in an Al-Mg alloy with submicrometer grain size[END_REF], which relates to the hardening by refining the grain size.

There are many techniques of SPD [START_REF] Koizumi | Grain size effects in aluminium processed by severe plastic deformation[END_REF][START_REF] Guan | A Review on Grain Refinement of Aluminum Alloys: Progresses, Challenges and Prospects[END_REF][START_REF] Valiev | Compact nanostructured materials from severe plastic deformation[END_REF][START_REF] Valiev | Producing Compact Ultrafine-Grained Materials by Severe Plastic Deformation[END_REF], most of them have come from the adaptation of an existing industrial process such as Accumulative Roll Bonding (ARB) inspired from the rolling process [START_REF] Darmiani | Corrosion investigation of Al-SiC nano-composite fabricated by accumulative roll bonding (ARB) process[END_REF][START_REF] Sun | Cu/Zr nanoscaled multi-stacks fabricated by accumulative roll bonding[END_REF], Equal-Channel Angular Pressing (ECAP) derivated from the extrusion technique [START_REF] Maier | Annealing behaviour of ultrafine grained structure in low-carbon steel produced by equal channel angular pressing[END_REF][START_REF] Mostaed | Microstructure, mechanical behaviour and low temperature superplasticity of ECAP processed ZM21 Mg alloy[END_REF][START_REF] Besterci | Formation Of Ultrafine-Grained (UFG) Structure And Mechanical Properties By Severe Plastic Deformation (SPD)[END_REF], High-Pressure Torsion (HPT) from deformation by torsion and Constrained Groove Pressing (CGP) issue from the forging process [START_REF] Khodabakhshi | Hardness-strength relationships in fine and ultra-fine grained metals processed through constrained groove pressing[END_REF][START_REF] Zrnik | Ultrafine-grained structure development and deformation behaviour of aluminium processed by constrained groove pressing[END_REF][START_REF] Hosseini | Nanostructure and mechanical properties of 0-7 strained aluminium by CGP: XRD, TEM and tensile test[END_REF].

The ARB process is an effective way of producing ultrafine-grained (UFG) materials [START_REF] Kamikawa | Microstructure and Mechanical Properties of ARB Processed Aluminium with Different Purities[END_REF][START_REF] Saito | Novel Ultra-High Straining Process For Compact Materials-Development Of The Accumulative Roll-Bonding (ARB) Process[END_REF][START_REF] Faradonbeh | Friction Stir Welding of Al-B 4 C Composite Fabricated by Accumulative Roll Bonding: Evaluation of Microstructure and Mechanical Behaviour[END_REF][START_REF] Yousefieh | An investigation on the microstructure, texture and mechanical properties of an optimized friction stir welded ultrafine-grained Al-0.2 wt% Sc alloy deformed by accumulative roll bonding[END_REF][START_REF] Salimi | Evaluation of Mechanical Properties and Structure of 1100-Al Reinforced with Zro 2 Nano-Particles via Accumulatively Roll-Bonded[END_REF]. This process has been invented by Saito et al. [START_REF] Saito | Ultra-Fine Grained Compact Aluminum Produced By Accumulative Roll-Bonding (ARB) Process[END_REF][START_REF] Saito | Novel Ultra-High Straining Process For Compact Materials-Development Of The Accumulative Roll-Bonding (ARB) Process[END_REF] by adopting a repeated procedure of cutting, stacking and roll-bonding of plates for a desired number of cycles. Besides forming an ultrafine-grained microstructure from alloys and pure metals, this process can be adapted industrially for continuous production of substantial quantities of material contrary to the other SPD techniques [START_REF] Ramesh | Nanomaterials Mechanics and Mechanisms[END_REF][START_REF] Schmidt | Particle Based Alloying by Accumulative Roll Bonding in the System Al-Cu[END_REF].

Lee et al. [START_REF] Lee | Role of shear strain in ultragrain refinement by accumulative roll-bonding (ARB) process[END_REF] demonstrated that shear strain greatly affects the grain refinement by ARB. In their study, they founded a good correspondence between grain size and shear strain. Also, an increase of shear strain redundant with the number of ARB cycles was proved. However, the distribution of shear strain through the thickness became complicated by the number of cycles and location in thickness.

The microstructure of ARB processed material has a lamellar/pancake structure. This microstructure is due to the redundant shear strain during the ARB process by grain deformation, not by grain subdivision as in the other SPD techniques [START_REF] Tsuji | ARB (ACCUMULATIVE Roll-Bonding) and other new Techniques to Produce Compact Ultrafine Grained Materials[END_REF][START_REF] Huang | Microstructural evolution during accumulative roll-bonding of commercial purity aluminium[END_REF].

The elongated grains formed at large strains are surrounding by high angle grain boundaries (HAGB) and subdivides by low angle grain boundaries (LAGB). The spacing of lamellar boundaries decreased, and the misorientation angle across boundaries increased with increasing the number of ARB cycles. The microstructure of ARB processed material, especially the lamellar structure, affect the material plastic anisotropy. This microstructure influence was demonstrated by measuring the Lankford parameter, which increased with increasing the number of ARB cycles [START_REF] Skrotzki | Plastic anisotropy of ultrafine grained Al alloy AA6016 produced by accumulative roll bonding[END_REF].

Moreover, the UFG microstructure had an impact on the mechanical properties of ARB processed material [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Valiev | Fundamentals of Superior Properties in Compact NanoSPD Materials[END_REF]. Hardness, yield stress and tensile strength significantly increased proportionately to the number of ARB cycles, unlike the ductility which decreased from the first cycle. Lee [START_REF] Lee | Microstructures and mechanical properties of 6061 aluminium alloy processed by accumulative roll-bonding[END_REF] and Gashti [START_REF] Gashti | Effects of grain size and dislocation density on strain hardening behaviour of ultrafine grained AA1050 processed by accumulative roll bonding[END_REF] found that the rising of hardness had an inhomogeneous distribution through the thickness. This distribution exhibits a maximum value near the layers' surface due to the shear strain redundant produced from the friction between the rolls and strip surfaces under non-lubricated conditions and the wire brushing before rolling [START_REF] Tsuji | ARB (ACCUMULATIVE Roll-Bonding) and other new Techniques to Produce Compact Ultrafine Grained Materials[END_REF]. Besides, the strain hardening of ARB processed alloy was related to the grain size and dislocation density, where the strain hardening rate increased by the number of ARB cycles [START_REF] Gashti | Effects of grain size and dislocation density on strain hardening behaviour of ultrafine grained AA1050 processed by accumulative roll bonding[END_REF].

Several studies in UFG material were focused on mechanical properties enhancement in the quasi-static strain rate domain. However, various applications were related to dynamic loading conditions such as automobile and aeronautic industry. Therefore, the quasi-static mechanical characterization and dynamic properties of UFG materials must be considered and examined in more depth.

There are few studies that explored the dynamic behaviour of UFG aluminium alloys as AA7075 aluminium alloy produced by Expansion Equal Channel Angular Extrusion (Exp-ECAE) [START_REF] Shahbazi Karami | Investigating the Effects of Expansion Equal Channel Angular Extrusion (Exp-ECAE) on Dynamic Behavior of AA7075 Aluminum Alloy[END_REF], Al-26wt.%Si and Al-Zn-Mg alloy produced by Equal Channel Angular Extrusion (ECAP) [START_REF] Jiang | Dynamic Compression Properties of an Ultrafine-Grained Al-26 wt.% Si Alloy Fabricated by Equal-Channel Angular Pressing[END_REF][START_REF] Henrique | Influence of grain size on the flow properties of an Al-Mg-Sc alloy over seven orders of magnitude of strain rate[END_REF][START_REF] Afifi | Effect of ECAP processing on microstructure evolution and dynamic compressive behaviour at different temperatures in an Al-Zn-Mg alloy[END_REF], Al-Mg-Mn alloy produced by High-Pressure Torsion (HPT) [START_REF] Petrova | Grain Size Effects on Quasi-static and Dynamic Strength of Ultrafine-Grained Al-Mg-Mn Alloy Produced by High-Pressure Torsion[END_REF], AA2219 aluminium alloy produced by Multiaxial Forging at Cryogenic Temperature (MFCT) [START_REF] Azimi1 | Dynamic Failure Investigation in Ultrafine Grained AA[END_REF] and AA1075 aluminium alloy produced by Accumulative Roll Bonding (ARB) [START_REF] Ivanov | Quasi-quasi-static and shockwave loading of ultrafine-grained aluminium: effect of microstructural characteristics[END_REF]. In these works, the dynamic investigations were performed using the Split Hopkinson Pressure Bar (SHPB) and the shock wave loading technique at different strain rate varied between 500 and 10 5 s -1 . It was reported that the results of dynamic tests for UFG aluminium alloy proceeding by SPD reveal an increase of the dynamic yield stress and the flow stress with increasing the strain rate. Besides, the grain refinement improved the strain rate sensitivity of the alloy. The dynamic proprieties have not been fully explored yet for the ARB processed aluminium-copper based alloys [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Azimi1 | Dynamic Failure Investigation in Ultrafine Grained AA[END_REF].

In the present work, the microstructure evolution and its effects on both quasi-static and dynamic mechanical properties of ARB processed 2017A-T4 aluminium alloy at various numbers of cycles were studied. The mechanical properties of the as-received material in the natural ageing state are governed by the precipitates strengthening phenomenon. So, from this metallurgical state and proceeding by the ARB technic, the resulting UFG structure's influence and the proceeding conditions on the precipitates evolution so then on the mechanical behaviour were investigated.

Results and Discussions

The microstructural characterisation

The number of the individual layers (nc) after N cycles can be calculated as follows: Equation 1 Fig. 4 shows the optical micrographs of the processed specimens from the 1 st to the 5 th cycles. At each cycle, good bonding with no delamination between strips was under the present ARB conditions. In particular, for the fifth cycle (Fig. 4e), 31 interfaces should be observed (n c -1). However, only the last interface and some parts of unbonded interfaces are seen in the full thickness of the specimen.

It can be noticed that the individual layers were homogeneous, parallel and having an identical thickness (t). This thickness can be evaluated from equation 2. Equation 2Moreover, the interfacial bonding is visible in the samples' centre for each cycle, thus presenting poor bonding and the weakest link in the processed strips due to the application of a single plastic deformation. However, the other interfaces from the previous cycles are difficult to detect or almost indistinguishable when the discontinuities between the sheets were reduced. This results from the important accumulated plastic deformation with increasing the number of cycles. Herein, it can be concluded that the degree of the interfacial bonding quality has been improved to reach metallurgical adhesion between the processed interfaces [START_REF] Saito | Novel Ultra-High Straining Process For Compact Materials-Development Of The Accumulative Roll-Bonding (ARB) Process[END_REF][START_REF] Lee | Microstructures and mechanical properties of 6061 aluminium alloy processed by accumulative roll-bonding[END_REF]. Fig. 5 illustrates SEM micrographs of a typical cross-section of the ARBed specimens at different cycles. The presence of second-phase particles can be observed in all ARBed sample microstructure, with dissimilarity in size, density, and dispersion. Two types of precipitate: Al 2 Cu, Al 2 CuMg were identified by EDS analyses (Fig. 6); they are naturally present in the as-received 2017A aluminium alloy in the T4 state [START_REF] Sigli | Recent advances in the metallurgy of aluminum alloys. Part II: Age hardening Développements récents en métallurgie des alliages d'aluminium. Deuxième partie : durcissement par revenu[END_REF][START_REF] Hemmouche | Influence of heat treatments and anodization on fatigue life of 2017A alloy[END_REF][START_REF] Tiamiyu | Effects of temper condition on high strain-rate deformation of AA 2017 aluminum alloy in compression[END_REF], and the formation of this kind of precipitates is responsible for the strengthening properties known as precipitation hardening phenomenon [START_REF] Tiamiyu | Effects of temper condition on high strain-rate deformation of AA 2017 aluminum alloy in compression[END_REF]. Furthermore, these precipitate agglomeration zone was located in the last interface, particularly at the three last cycles (Fig. 5: c,d,e).

It is clearly observed that with increasing the number of ARB cycles these precipitates become finer, more distant and distributed inhomogeneously in the matrix. This can be explained by the introduction of a fragmentation mechanism during the deformation process, a dissolution phenomenon caused by heat treatment before rolling, in addition to the converting of the severe plastic deformation energy to thermal energy which leads to the formation of a state close to a solid solution or loss of coherency precipitates -matrix, and finally a continuous deformation process simultaneously with the matrix [START_REF] Deschamps | Precipitation kinetics in a severely plastically deformed 7075 aluminium alloy[END_REF]. Thus, the evolution of precipitates in aluminium alloy 2017A-T4 processed by ARB and the grain refinement mechanism are attributed to the combination of plastic deformation and thermal energy which controlled the motion of dislocations in the function of barriers (grain boundaries and precipitates). Fig. 7 shows the microstructural evolution of the processed specimens obtained from the EBSD analyses. It is clear that there is a grain refinement of ARBed material compared to the natural ageing (Fig. 7 a). The plastic deformation is not yet homogeneous in the first ARB cycle, leading to the non-uniform grain size distribution, as illustrated in fig.7b [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Gashti | Effects of grain size and dislocation density on strain hardening behaviour of ultrafine grained AA1050 processed by accumulative roll bonding[END_REF]. After that, the ultrafine-grained structure becomes more clear (Fig. 7: d, e andf) with equiaxed grains, particularly in the interface due to redundant shear strain and the agglomeration of precipitates (fig. 7b and7c) [START_REF] Lee | Microstructures and mechanical properties of 6061 aluminium alloy processed by accumulative roll-bonding[END_REF], and elongated grains in the rolling direction (RD) were formed in most areas of deformed samples and developed a lamellar microstructure. Also, the grain size (Table 3) and the lamellar boundaries spacing decrease with increasing the number of ARB cycles [START_REF] Huang | Microstructural evolution during accumulative roll-bonding of commercial purity aluminium[END_REF].

Mechanical characterisation: 2.2.1. Microhardness measurements

Fig. 8 presents the microhardness variations of the as-received alloy and the processed samples at different ARB cycles. The microhardness of the naturally aged alloy reached about 145 Hv. This high microhardness is attributed to the precipitates strengthening phenomena [START_REF] Sigli | Recent advances in the metallurgy of aluminum alloys. Part II: Age hardening Développements récents en métallurgie des alliages d'aluminium. Deuxième partie : durcissement par revenu[END_REF][START_REF] Hemmouche | Influence of heat treatments and anodization on fatigue life of 2017A alloy[END_REF][START_REF] Fares | Coupled effects of substrate microstructure and sulphuric acid anodizing on fatigue life of a 2017A aluminum alloy[END_REF][START_REF] Tiamiyu | Effects of temper condition on high strain-rate deformation of AA 2017 aluminum alloy in compression[END_REF]. After the first cycle, a remarkable decrease of around 42% was detected compared to the as-received sheets. Although the grain refinement was achieved, as reported in fig. 7, the processed strips still not hardened as in previous articles [START_REF] Hosseini | Nanostructure and mechanical properties of 0-7 strained aluminium by CGP: XRD, TEM and tensile test[END_REF][START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Petrova | Grain Size Effects on Quasi-static and Dynamic Strength of Ultrafine-Grained Al-Mg-Mn Alloy Produced by High-Pressure Torsion[END_REF][START_REF] Ivanov | Quasi-quasi-static and shockwave loading of ultrafine-grained aluminium: effect of microstructural characteristics[END_REF], this decrease can be assigned to the reduction of size, quantity and distribution of the precipitates caused by plastic deformation and thermal energy during the ARB process (Fig. 5) [START_REF] Deschamps | Precipitation kinetics in a severely plastically deformed 7075 aluminium alloy[END_REF], which reduce the obstacles against the movement of dislocations. Furthermore, during the first three ARB cycles, the microhardness values revealed practical stability attributed to the saturation in dislocation density caused by the high strains conditions. This hardening behaviour was previously reported in UFG materials manufactured by SPD [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Pasebani | Nano-grained 70/30 brass strip produced by accumulative roll-bonding (ARB) process[END_REF]. Besides, a slight drop in the microhardness was observed after the third cycle. This phenomenon is ascribed to the dynamic recrystallisation mechanism at high strains conditions, leading to reduced grain size and annihilation of dislocations [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF]. There is a competition between two main phenomena that control the hardening of ARBed sheets: the dislocation annihilation and dynamic recrystallisation mechanism associated with precipitates behaviour reported above.

Tensile test

The representative engineering stress-strain curves of the studied samples and the corresponding tensile data are presented in fig. 9 and summarised in table4, respectively. The Ultimate Tensile Strength (UTS) decreased from about 458 MPa for the naturally aged sample to reach about 370 MPa after the first cycle, which corresponds to a reduction of around 23%. In contrast, a slight increase in Yield Stress (YS) of around 6% was reported. Further, the processed samples exhibit a decrease in the YS and UTS values from the first cycle gradually to attain a significant drop (51% and 48%, respectively) after the fourth cycle. Besides, a slight increase in strength after five cycles was noticed.

On the other hand, the Total Elongation (TE) dropped from about 21% in the natural ageing state to about 5% after the second cycle. Nevertheless, after four ARB cycles, a rise in the TE was attained to about 16% and then fell again in the fifth cycle. Herein, the tensile results for all the studied samples were concordant with the obtained microhardness ones as presented above.

In this context, this variation in mechanical properties can be attributed to two main factors: the applied heat treatments and plastic deformation. Indeed, the heat treatment carried out before rolling leads to the microstructure recrystallisation, dislocations mobility activation, and precipitates evolution. Moreover, the plastic deformation gives rise to the generation of dislocations, thus inducing the work hardening. Furthermore, with the increase of the ARB cycle, the grain refinement evolution is responsible for reducing the dislocations mean free path and its number of activity. There is a vital competition between several mechanisms: work hardening, grain refinement and dynamic recrystallisation which control the dislocations state (generation or annihilation) or grain size [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF]. Consequently, as was reported, this competition affects the main strengthening precipitates when the dislocations are like the favourite nucleation sites for the major of these phases. Therefore, it alters the overall properties of the processed strips [START_REF] Deschamps | Precipitation kinetics in a severely plastically deformed 7075 aluminium alloy[END_REF][START_REF] Tiamiyu | Effects of temper condition on high strain-rate deformation of AA 2017 aluminum alloy in compression[END_REF].

Thus far, the grain refinement produced by SPD has been believed that it is the main reason for the strengthening of materials based on Hall-Petch relationship. Our results show that this understanding is not totally true. This conclusion was confirmed by Koizumi et al. [START_REF] Koizumi | Athermal strength of pure aluminum is significantly decreased by severe plastic deformation and it is markedly augmented by subsequent annealing[END_REF] where they reported that softening of industrial pure aluminum processed by ECAP was observed at the ultralow strain rate of ~ 10 -7 /s in uniaxial tensile tests despite the grain size refinement obtained. They conclude that the SPD processes decreases the athermal strength, and increases the viscosity of UFG materials.

In conclusion, the strength and ductility in the initial ARB stages are mainly influenced by establishing equilibrium between strain hardening, dislocations density and precipitates process. However, in the fourth cycle, which represents a transition state, the dynamic recrystallisation phenomenon was the dominant mechanism associated with grain refinement and precipitates state [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Deschamps | Precipitation kinetics in a severely plastically deformed 7075 aluminium alloy[END_REF].

-The fracture mechanism

The SEM micrographs of the tensile fracture surfaces for the as-received and processed specimens are presented in fig. 10. At low (L) magnification, the naturally aged sample (Fig. 10-a-L) exhibits a slight necking when the micro-crack initiation and propagation were stared from the outer surface until attaining the global fracture towards the sample centre. At high (H) magnification, the fractography (Fig. 10-a-H) exhibits a ductile fracture behaviour confirmed by a grey and fibrous appearance with the presence of deep dimpled structure developed from the nucleation, growth, and coalescence of microvoids following the classical void growth model [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF][START_REF] Pasebani | Nano-grained 70/30 brass strip produced by accumulative roll-bonding (ARB) process[END_REF].

After the first ARB cycle, the tensile fractography (Fig. 10-b-L) shows clearly the two individual layers with an apparent interface. From the second cycle, the last and the second last interfaces can be identified (Fig. 10-c-L, 10-d-L, 10-e-L). In contrast, the interfacial bonding between the previous cycle interfaces has enhanced that conduct it to become indistinguishable with an appropriate metallurgical adhesion. This leads to assuming that the last and the second last bonding interfaces present the weakest zone in the processed strips, where the initiation and propagation of the micro-cracks will take place [START_REF] Alvand | Nano/ultrafine grained AA2024 alloy processed by accumulative roll bonding: A study of microstructure, deformation texture and mechanical properties[END_REF]. Also, a slight necking was observed in some individual layers, which means that the plastic deformation is not homogeneous across the samples thickness. Herein, it can be stated that the severely deformed strips and the narrowing are not similar in the whole cross-section of the processed specimens where each layer supports a distinct strain hardening, which leads to forming the basis of some types of micro-defects, such as void agglomerations and micro-cracks that lead to theirs initiation and propagation from the outer surface, accordingly reduce the mechanical properties [START_REF] Pasebani | Nano-grained 70/30 brass strip produced by accumulative roll-bonding (ARB) process[END_REF]. As a consequence, the processed strips behave like a laminate composite, in which the applied stress affects the individual layers rather than the entire sample. This can illustrate the stress distribution and its variation with necking or cracking in some layers. Consequently, the ductility and the strength were lower than those of the compact material.

Whereas, at high magnification (Fig. 10-c-H, 10-d-H, 10-e-H), a variation in the dimples shape, size and orientation of the ARBed samples was perceived. Moreover, the micrographs exhibited that the average size and the number of dimples progressively decreased with increasing the number of ARB cycles when fine shear lips were detected due to the refining of grains, revealing that the failure type was a shear-ductile mixed mode [START_REF] Pasebani | Nano-grained 70/30 brass strip produced by accumulative roll-bonding (ARB) process[END_REF][START_REF] Medjahed | Evolution of Microstructure, Mechanical Properties, and Thermal Conductivity of an Al-Li-Cu-Mg-Zr Alloy Processed by Accumulative Roll Bonding (ARB)[END_REF].

The Compression test

The experimental compression true stress-strain curves for the studied specimens are shown in fig. 11. The obtained compression curves can be divided into two stages: an initial linear elastic stage followed by a compacting stage that exhibited a considerable strain hardening phenomenon conducting to a linear rising in stress up to 50% of compressive strain. The average values of the Compressive Yield Stress (CYS) and Ultimate Compressive Strength (UCS) are given in table 5.

According to these results, the natural ageing state has the maximum value of the UCS. In contrast, the ARBed samples showed stabilisation between 831 and 851 MPa for all cycles with a slight decrease to 773 MPa in the second cycle. Furthermore, for the three first cycles, the CYS value is constant and reaches an average of 214 MPa, but in the fourth cycle, an increase is observed up to 329 MPa which is the maximum value of CYS. However, the natural ageing state has a CYS value (278 MPa) lower than that of the fourth cycle and higher than the other cycles.

The quasi-static strength characteristics in the tensile and compression tests have a similar tendency. Both techniques confirmed the existence of a transient state in the fourth ARB cycle which exhibited the appropriate comprehensive mechanical properties resulting from the proper combination of the strain hardening, grain refinement, and high bonding quality. These mechanisms control the state of dislocations by controlling the number of barriers during the ARB process that influences the evolution of precipitates in the matrix and the refinement of grain size [START_REF] Tiamiyu | Effects of temper condition on high strain-rate deformation of AA 2017 aluminum alloy in compression[END_REF].

Mechanical behaviour under high strain rate

Fig. 12 presents the true stress-strain curves for the studied specimens tested under dynamic impact loading. An elastic deformation was noticed at the beginning of the curves, followed by a transition zone after the true yield stress and, finally, a plastic deformation at the end of the curves, which corresponds to the samples strain hardening phenomena.

The SHPB curves show that, on the one hand, the as-received material exhibited the highest flow stress. On the other hand, stabilisation and decreased flow stress were observed for the ARBed samples at different cycles compared to the naturally aged sample.

The curves in Fig. 13 revealed a strain hardening rate drop, which indicates the domination effect of the thermal softening over the strain hardening. The annihilation and rearrangement of dislocations can explain this drop due to the increase of temperature, resulting from the conversion from the plastic work into heat through the dynamic loading condition [START_REF] Tiamiyu | Effects of temper condition on high strain-rate deformation of AA 2017 aluminum alloy in compression[END_REF]. The thermal softening effect on the strain hardening behaviour under dynamic loading occurs in the true strain critical range between 0.01 and 0.05. From Fi.14, it can be stated that the naturally aged alloy presents the peak true stress value. Whereas, all curves have the same allure for the different ARBed specimens, presenting stability of the true stress between 300 and 350 MPa for the various true strain levels.

Fig. 15 exhibits the variation of the true stress for the studied samples as a function of the strain rate (4 x 10 -4 s -1 and 3200 s -1 ) for 10, 20 and 30% from the true strain.

In the ARBed material, the strain rate does not have a noteworthy influence on the mechanical behaviour compared to the naturally aged material, where there was a considerable decrease in the stress under the dynamic conditions. That can be attributed to the thermal softening that overcomes the strain hardening in the as-received material with coarse grains; in addition to the thermal energy converted from the plastic work at the dynamic loading, the dislocations mobility increases and therefore the stress flow decreases [START_REF] Shahbazi Karami | Investigating the Effects of Expansion Equal Channel Angular Extrusion (Exp-ECAE) on Dynamic Behavior of AA7075 Aluminum Alloy[END_REF][START_REF] Azimi1 | Dynamic Failure Investigation in Ultrafine Grained AA[END_REF]. However, the equilibrium between thermal softening and strain hardening was established in the ARBed samples due to their microstructure evolution (grain refinement and precipitates state) which leads to conclude that the strain rate does not have a significant influence on the stress flow. Furthermore, a slight increase of the flow stress at the dynamic condition in the fourth cycle was observed, which confirmed the transition state reported above at this ARB cycle due to interfaces bonding enhancement and the dynamic recrystallisation mechanism.

Based on the development of Hollomon law [START_REF] Maier | Annealing behaviour of ultrafine grained structure in low-carbon steel produced by equal channel angular pressing[END_REF][START_REF] Azimi1 | Dynamic Failure Investigation in Ultrafine Grained AA[END_REF], the strain hardening parameters for the studied strips showed in table 6 are extracted from the linear fit line of the logarithm of the true stress versus the logarithm of the true strain curves (Fig. 16) under dynamic loading. These results demonstrate the stability of the hardening for the ARBed strips in different cycles which is around 0.073-0.08 and 363-399 for n (strain-hardening exponent) and K (strength coefficient), respectively. However, the as-received material exhibits the highest hardening values with a 0.136 strain-hardening exponent and 675 strength coefficient.

Conclusion

In the present study, the UFGs 2017A aluminium alloy (Al-Cu-Mg) was successfully produced from the T4 temper condition (natural ageing) using the ARB process for up to five cycles. The deformation behaviour under both the quasi-static and dynamic loadings was studied. Based on the above investigations, the following conclusions can be drawn:

1-The optical micrographs show an excellent bonding between the individual layers for all the ARBed strips. The different layers were characterised as homogeneous, parallel and having identical thickness along with the interface with no delamination or cracks except in the last interface of the fifth cycle, where a slight loss of cohesion was observed.

2-

The SEM micrographs revealed the presence of precipitates (Al 2 Cu, Al 2 CuMg) with differences in shape and sizes. The configuration of these precipitates changed during the ARB process, which becomes finer and distant as a function of the number of cycles.

3-In the EBSD results a lamellar structure was observed in the three first ARB cycles due to severe plastic deformation. The spacing of lamellar boundaries decreased with increasing the number of ARB cycles. Beyond that, the lamellar structure changed to an ultrafine grain structure with an average grain size up to 1.7 µm at the fifth cycle.

4-The microhardness and strength (yield and ultimate tensile) measurements of all specimens exhibit that the natural ageing alloy had the highest values and a significant reduction in the ARB processed alloy. Saturation in microhardness was remarked in the three first ARB cycles, followed by a slight decrease thereafter. Moreover, the tensile test results revealed a decrease of strengthening until the fourth cycle and then it increased towards the same level as the first cycles.

5-

The fracture surface analyses of ARB processed material reveal that the failure mode was a shear ductile fracture. Moreover, it behaves like a laminate in which tensile stress acts on the individual layers rather than the compact material.

6-The mechanical characterisation by compression test was done at two strain rates: 3200 s - 1 (dynamic) and 4 x 10 -4 s -1 (quasi-static). The strain hardening behaviour of ARBed samples showed a lower strain hardening exponent and strain hardening rate compared to the natural ageing sample. The domination of thermal softening overstrain hardening was clearly observed at a high strain rate due to the conversion of kinetic energy to thermal energy. Besides, the influence of the strain rate on work hardening as a function of the number of ARB cycles was not important.

7-In addition to plastic deformation, thermal energy and dynamic recrystallisation phenomena, the evolution of precipitates during the ARB process into the 2017A-T4 aluminium alloy played an important role in the dislocations progression and thus on the decrease in the mechanical properties of the processed alloy, owing to the fact that the dislocations are like the favourite nucleation sites for the most of precipitates. Thus, there are other parameters besides grain size that influence the hardening of SPDed materials. The present work shows that the conditions of SPD process (heating conditions before roll bonding: T=350°C, t=15 min), composition and microstructure of started material (natural ageing state of 2017A aluminium alloy) play an important role on the mechanical behaviour of the materials grain refined.

Experimental Methods

The chemical composition of the as-received sheets of 2017A-T4 aluminium alloy (solution heat-treated and naturally aged) is listed in table 1:

In order to improve the interface bonding, the as-received strips for the ARB process with the dimensions of 300 mm × 150 mm × 4 mm were degreased in acetone and wire brushing before stacking; then, they were strongly fixed using steel wires. Subsequently, the sheets were heated at 300°C for 15 min to improve the workability, favourite high bonding quality and reduce the accumulative strain. Afterwards, the sheets were subjected to 50% reduction per cycle to a final thickness of 4 mm. The bonded strips were cut into two similar pieces; then, this procedure was repeated up to five cycles. The schematic illustration of the ARB process to produce strips up to 32 layers is shown in fig. 1. The ARB process was performed without lubricant on a laboratory rolling mill with a diameter of 500 mm at a roller speed of 600 rpm. The ARB process parameters are summarised in table 2.

The metallographic observation samples were prepared through conventional mechanical with Silicon Carbide Grinding papers and then electrolytic polishing using the A2 Struers electrolyte at 39 V for 20 s. The Olympus GX53 inverted optical microscope was employed to analyse the bonding quality between several prepared samples layers. To examine the evolution of the grain specifications at the different number of cycles, Electron Back Scattering Diffraction (EBSD) measurements were carried out using the TSL OIM software in FEG-SEM SUPRA 55 VP Scanning Electron Microscope (SEM) operating at 20 KV. An average of 5 Vickers micro-hardness measurements at different points were obtained by the HWDM-1 micro-hardness tester under the load of 300 g for 15 s. Tensile test specimens were cut along the rolling direction (RD) by wire electro-erosion technique according to ASTM Standard B 557M. All the tensile values were obtained from the average of three tests. The tensile test was performed at ambient temperature on an EZ20 testing machine at a crosshead speed of 0.1 mm/min. The dimensions of the tensile specimen are shown in fig. 2.

Cylindrical samples with 8 mm in diameter and 4 mm in length were prepared for the compression test at room temperature under quasi-static (strain rate of 4 x 10 -4 ) and dynamic (strain rate of 3200 s -1 ) loading conditions by using the universal testing machine and the Split-Hopkinson Pressure Bar system (SHPB) shown in fig. 3, respectively.

FiguresFig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 11

 12345678911 Figures

Fig. 13 Fig. 14 Fig. 15 Fig. 16

 13141516 Fig.[START_REF] Maier | Annealing behaviour of ultrafine grained structure in low-carbon steel produced by equal channel angular pressing[END_REF] The strain hardening rate curves

Table 1

 1 Chemical composition of 2017A aluminium alloy (wt.%)

	Tables

Table 2

 2 Roll-bonding parameters

	Heating	Roll diameter	Roll speed	Initial thickness (t 0 )	Reduction in each cycle Number of cycles
	300°C / 15 min	500 mm	600 rpm	4 mm	50 %	1 to 5

Table 3

 3 Grain size measurements of studied strips

	Specimen	Grain size (µm)
	Natural ageing	16.8 ± 1.9
	1 cycle	10.8 ± 0.8
	2 cycles	7.6 ± 0.4
	3 cycles	2.0 ± 0.3
	4 cycles	1.7 ± 0.2
	5 cycles	1.8 ± 0.2

Table 4

 4 Tensile properties data of studied samples

	Specimen	YS (MPa)	UTS (MPa)	TE (%)
	Natural ageing	282 ± 7.4	458 ± 12.3	20 ± 1.3
	1 cycle	301 ± 8.1	370 ± 11.4	09 ± 1.0
	2 cycles	277 ± 7.2	345 ± 10.9	05 ± 0.9
	4 cycles	199 ± 6.5	249 ± 9.6	16 ± 1.1
	5 cycles	275 ± 7.2	273 ± 10.1	07 ± 0.6

Table 5

 5 Results of the compression test

	Specimen	UCS (MPa)	CYS (MPa)
	Natural ageing	1078 ± 25	278 ± 2.1
	1 cycle	834 ± 21	215 ± 2.1
	2 cycles	773 ± 18	213 ± 2.0
	3 cycles	831 ± 19	213 ± 2.0
	4 cycles	851 ± 19	329 ± 2.5

Table 6

 6 Strain hardening parameters of studied samples

	Specimen	n	K
	Natural ageing	0.136	675
	1 cycle	0.080	399
	2 cycles	0.079	377
	3 cycles	0.083	394
	4 cycles	0.079	363
	5 cycles	0.073	381


A Coppa

Bruno Martin

J V Gauzentes

Donato Andueza

Prediction of cow diet composition and authentication of feeding specifications of Protected Designation of Origin cheese using mid-infrared spectroscopy on bulk milk

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.

(1) Université Clermont Auvergne, INRAE, VetAgro Sup, UMR 1213 Herbivores, F-63122 Saint-Genès-Champanelle, France (2) Pôle Fromager AOP Massif Central, 20 côte de Reyne, F-15000 Aurillac, France (3) Cantal Conseil Elevage, 26 rue du 139ème Régiment d'Infanterie -BP 239, F-15002 Aurillac (4) Agrolab's, 38 Rue de Salers F-15000 Aurillac, France (5) CNIEL, 42 rue de Châteaudun, F-75314 Paris, France RESUME L'aptitude de la spectroscopie dans le moyen infrarouge (MIR) appliquée sur des laits de mélange pour 1) prédire la composition de la ration des troupeaux laitiers et 2) authentifier des pratiques des cahiers des charges des fromages AOP Cantal et Laguiole, a été testée. Les spectres MIR de 7607 laits de mélange AOP et non AOP de la région ont été associés aux données d'enquêtes sur l'alimentation correspondantes (obtenues entre avril 2018 et mars 2019), afin de constituer la base des données. La composition de la ration était très variable, allant de rations 100% pâturage, à des rations à base d'ensilage de maïs (jusqu'à 74%). La base de données a été divisée en deux pour l'étalonnage (n = 6107) et la validation (n = 1500). Des modèles de régression « partial least squares » et des analyses discriminantes ont été utilisés pour prédire la composition de la ration en pourcentage de matière sèche (MS) et pour authentifier les critères d'alimentation inclus dans les cahiers des charges des deux AOP, respectivement. La proportion d'herbe pâturée dans la ration a été prédite avec un modèle caractérisé par un coefficient de détermination en validation externe (R 2 V) de 0,81 et une erreur standard de prédiction (SEP) de 11,7 %. Les proportions de pâturage + foin, d'ensilage de maïs, d'herbe conservée, de fourrages fermentés et d'herbe totale ont été prédites par des modèles ayant des R 2 V > 0,61 et SEP < 14,8%. Les modèles d'analyses discriminantes pour la présence du pâturage, la proportion de pâturage ≥ 50% et ≥ 57% ont montré des valeurs d'exactitude et de spécificité ≥ 90%. Des valeurs de sensibilité et de précision ≥ 80% ont été observées pour tous les modèles discriminants concernant la proportion du pâturage dans la ration. Les modèles discriminants pour la proportion de pâturage + foin ≥ 72%, herbe totale (fraiche et conservée) ≥ 50%, le pâturage + foin ≥ 25%, absence d'herbe fermentée, absence d'ensilage de maïs et ensilage de maïs ≤ 30% dans la ration ont montré des valeurs d'exactitude ≥ 80%, mais pour ces modèles soit la sensibilité, soit la précision ont été moins performantes. Les modèles construits pour tester le respect de l'ensemble des critères des cahiers des charges de chaque AOP ont montré des valeurs d'exactitude, spécificité, sensibilité et précision > 90%. Les modèles pour la prédiction du % des aliments dans la ration tout comme les modèles discriminants peuvent permettre de fournir, via la MIR, des indicateurs sur la composition de la ration des troupeaux laitiers utiles aux éleveurs, filières et aux consommateurs.

Prediction of cow diet composition and authentication of feeding specifications of

SUMMARY

The ability of mid-infrared spectroscopy (MIR) to predict indicators (i) of diet composition in dairy herds and (ii) for the authentication of the cow-feeding restrictions included in the specification of two Protected Designation of Origin (PDO) cheeses (Cantal and Laguiole) was tested on 7607 bulk milk spectra from 1355 farms located in the French Massif Central. For each milk sample, the corresponding cow diet composition was obtained through onfarm surveys between April 2018 and March 2019. Cow diet composition varied largely (from full grazing for extensive farming systems to corn silage-based diets). The database was divided into a calibration (n = 6107) and validation (n = 1500) sets. Partial least square regression and discriminant analysis were used to predict the proportion of different feedstuffs in the cows' diets and to authenticate the cow-feeding restrictions for the PDO cheese specifications, respectively. The pasture proportion in the cows' diets was predicted by MIR with an R 2 in external validation (R 2 V) = 0.81 and a standard error of prediction (SEP) of 11.7% dry matter. Pasture + hay, corn silage, conserved herbage, fermented forage and total herbage proportions were predicted with a R 2 V > 0.61 and a SEP < 14.8%. The discrimination models for pasture presence, pasture ≥ 50% and pasture ≥ 57% in the cows' diets achieved an accuracy and specificity ≥ 90%. A sensitivity and precision > 80% were observed for all models including pasture proportion in cow diet. An accuracy ≥ 80% was also observed for pasture + hay ≥ 72%, herbage ≥ 50%, pasture + hay ≥ 25%, absence of fermented herbage, absence of corn silage and corn silage ≤ 30% in the cows' diets, but for several models, either the sensitivity or precision was lower than the accuracy. Models built with respect to all the criteria of the feeding restrictions of PDO cheese specifications achieved an accuracy, specificity, sensitivity and precision > 90%. Both the regression and discriminant MIR models for bulk milk can provide useful indicators of cow diet composition and PDO cheese specifications to producers and consumers.

INTRODUCTION

Authentifier et certifier les pratiques d'élevage inscrites dans les cahiers des charges constitue un enjeu récurrent pour les filières fromagères, notamment pour les produits sous Appellation d'Origine Protégée (AOP). En pratique, les contrôles mis en place reposent sur des visites dans les élevages. Ces contrôles étant très couteux et chronophages, il serait intéressant de disposer d'outils analytiques permettant, si ce n'est de les remplacer, au moins de mieux les cibler. Parmi les techniques d'authentification des pratiques d'alimentation des troupeaux applicables sur les produits laitiers, la spectroscopie dans le moyen infrarouge (MIR) est prometteuse du fait de son coût limité et de sa rapidité de mise en oeuvre. Plusieurs auteurs ont validé l'intérêt de la MIR pour discriminer des laits ou des fromages issus de troupeaux de vaches laitières recevant des rations alimentaires très contrastées (Coppa et al., 2012 ;Andueza et al., 2013 ;Valenti et al., 2013), mais les techniques proposées n'ont jamais été testées à grande échelle pour discriminer des situations moins contrastées. L'intérêt de l'application de la MIR pour les filières fromagères AOP réside également dans la capacité de cette technique à discriminer des laits selon une grande diversité de critères alimentaires précisés dans les cahiers des charges, dans un objectif d'authentification et d'appui au contrôle. L'étude présentée avait pour objectif de tester l'aptitude de la MIR appliquée sur des laits de tank pour 1) prédire la composition de la ration alimentaire des troupeaux laitiers et 2) authentifier le respect des critères du cahier des charges relatifs à l'alimentation du troupeau dans deux fromages AOP : le Cantal et le Laguiole.

MATERIEL ET METHODES

ECHANTILLONNAGE DES LAITS ET ENQUETES EN FERME

Les spectres MIR ont été obtenus sur 7607 laits de mélange issus des départements du Cantal et de l'Aveyron (partie Nord). Les spectres étaient issus d'un seul appareil (FT-Plus équipement, Foss, Hillerød, Danemark), du laboratoire Agrolab's à Aurillac. Les échantillons utilisés étaient ceux analysés en routine pour le paiement du lait. Ils ont été associés aux données des constats d'alimentation correspondants, relevées entre avril 2018 et mars 2019 par les services de Conseil Elevage dans 1355 fermes. Les données utilisées étaient uniformément réparties au cours des mois de l'années, afin de correctement détecter les changements saisonniers dans l'alimentation du troupeau au sein de chaque ferme.

REGROUPEMENT DES LAITS SELON LES CRITERES DES CAHIERS DE CHARGES

Afin de développer des indicateurs pour authentifier le respect des différents critères d'alimentation des cahiers des charges des deux fromages AOP, des seuils ont été définis pour chaque aliment et ont été utilisés pour classer les échantillons de laits en différents groupes. Compte tenu du fait que les critères des cahiers de charges sont exprimés en % de la ration de base par saison (concentrés exclus) et que les quantités maximales autorisées de concentrés sont précisées sur une base annuelle, une conversion en % de matière sèche (MS) de la ration totale a été établie pour chaque type d'aliment. Pour cela la quantité de concentrés maximale sur l'année (rapportée en MS) a été divisée par une lactation de durée standard (305 jours) et rapportée à l'ingestion moyenne en kg de MS, renseignée dans les enquêtes. Pour chaque aliment composant la ration, les laits ont ensuite été classés en deux groupes selon le seuil imposé par les cahiers des charges (« > à » et « < à ») (Tableau 1). L'effectif des échantillons de

ANALYSES STATISTIQUES

La base de données a été séparée pour l'étalonnage (n = 6107) d'une part, et la validation (n = 1500) d'autre part, en utilisant l'algorithme Kennard-Stone (1969). Des modèles de régression PLS (partial least squares) et des analyses discriminantes ont été utilisés pour prédire le pourcentage de MS des différents aliments dans la ration et pour authentifier les critères d'alimentation des cahiers des charges des deux AOP, respectivement. Plusieurs prétraitements mathématiques ont été testés sur les spectres MIR, mais les meilleurs performances de calibration et validation ont été obtenues en utilisant les spectres bruts des laits dans les intervalles de longueurs d'onde suivants : 2989-2561 cm -1 , 1809-1712 cm -1 et 1600-926 cm -1 . Nous avons évalué les modèles de prédictions des proportions d'aliments dans la ration, par le coefficient de détermination en validation externe (R 2 V) et l'erreur standard de prédiction (SEP). Les modèles de discrimination ont été évalués en calculant la sensibilité, la spécificité, la précision et l'exactitude. La sensibilité et la spécificité expriment le taux d'erreur au sein d'un groupe à discriminer (l'adhésion ou non à un critère, respectivement), tandis que la précision exprime la capacité du modèle à détecter les échantillons, en respectant un critère sur l'ensemble des échantillons, et l'exactitude exprime la fiabilité générale du modèle.

RESULTATS

VARIABILITE DES CONDITIONS DE PRODUCTION

La composition de la ration était très variable, allant de rations 100% pâturage, à des rations à base d'ensilage de maïs (Figure 1). La grande variabilité observée pour la taille du troupeau ( 4

PREDICTION DE LA PROPORTION DES ALIMENTS CONSTITUANT LA RATION

La proportion d'herbe pâturée dans la ration, exprimée en % de MS a été prédite avec un modèle ayant un coefficient de détermination en validation externe (R 2 V) de 0,81 et une erreur standard de prédiction (SEP) de 11,7% (Tableau 5). Les proportions de pâturage + foin, d'ensilage de maïs, d'herbe conservée, de fourrages fermentés et d'herbe totale ont été prédites par des modèles ayant des R 2 V > 0,61 et des SEP < 14,8%. Les coefficients de détermination des modèles pour la prédiction de la proportion d'herbe fermentée et d'ensilage dans la ration sont inférieurs à 0,4.

AUTHENTIFICATION DES CRITERES ALIMENTAIRES DES CAHIERS DES CHARGES

Les modèles proposés pour certifier le respect des critères des cahiers des charges relatifs à la proportion de pâturage dans la ration ont été de bonne qualité (Tableau 6). Pour les critères « présence de pâturage » et « proportion de pâturage ≥ 50% et ≥ 57% » les proportions de laits bien classés et la spécificité des modèles sont ≥ 90%. Leur sensibilité et leur précision sont ≥ 80%. Les modèles discriminants pour les critères « proportion de pâturage + foin ≥ 72% », « herbe totale (fraiche + conservée) ≥ 50% », « pâturage + foin ≥ 25% », « absence d'herbe fermentée », « absence d'ensilage de maïs » et « ensilage de maïs ≤ 30% » sont de moins bonne qualité. La proportion d'individus bien classés est ≥ 80%, mais soit la sensibilité, soit la précision sont < à 80%. Les modèles obtenus n'ont pas permis de discriminer correctement les exploitations respectant le critère « concentrés < 28% ». Les modèles proposés pour tester le respect de l'ensemble des critères du cahier des charges de chacune des AOP ont une exactitude, une spécificité, une sensibilité et une précision > 90%.

DISCUSSION

La qualité des modèles obtenus pour prédire la proportion des fourrages dans la ration est équivalente à celle des modèles utilisés en routine pour prédire la composition du lait (en minéraux ou en acides gras par exemple) ou son aptitude fromagère (paramètres de coagulation, rendements fromagers). Ces modèles ont été considérés comme fiables pour une application en sélection génétique ou pour identifier des laits ayant des caractéristiques spécifiques (De Marchi et al., 2014 ;Coppa et al., 2017). Les erreurs de prédiction de ces modèles sont comprises entre 11 et 15%. Lorsque l'on compare les SEP des modèles ici présentés à ceux de constituants déterminés analytiquement, l'incertitude de la méthode de référence doit être prise en compte. En effet, les données issues des enquêtes (considérées ici comme méthode de référence) peuvent être parfois approximatives. Par exemple, les concentrés ne sont pas toujours pesés, le foin est donné en balles de poids variable, la teneur en MS de l'ensilage peut varier entre différentes séries de balles enrubannées ou d'une coupe à l'autre au sein d'un même silo, etc. Les erreurs d'estimation de la consommation d'herbe au pâturage sont encore plus importantes. Une erreur d'estimation de 10 à 15% pour les différents constituants de la ration peut être considérée comme intrinsèque aux données de référence et cohérente avec le SEP de nos modèles. Les principales longueurs d'onde associées dans nos modèles aux prédictions et discriminations des types de rations étaient: i) celles comprises entre 900 et 1 500 cm -1, qui sont liées à les liaisons C-H et C-O; ii) celles comprises entre 1550 et 1570 cm -1, qui sont liées aux protéines iii) et enfin celles de 2 855 à 2 928 cm -1 et de 1 805 à 1 736 cm -1, qui sont liées à l'absorption des lipides. Ces résultats semblent confirmer que les modèles de prédiction et de discrimination des types de rations s'appuient majoritairement sur les différences de profil en acides gras (AG) du lait induites par la composition des rations (Coppa et al., 2017). Les meilleurs modèles ont d'ailleurs été obtenus pour prédire ou discriminer la proportion d'herbe pâturée dans la ration. Cette dernière est connue pour être le facteur de variation le plus important du profil en acides gras (AG) du lait (Coppa et al., 2019;Prache et al., 2020). Ces résultats confirment l'empreinte unique que le pâturage est capable de conférer aux produits laitiers. La sensibilité et la précision des modèles discriminant les différentes proportions de pâturage dans l'alimentation des vaches diminuent lorsque le seuil d'herbe pâturée augmente de 0 à 50 puis 57% de la ration. Ce phénomène peut s'expliquer par la fait que, en considérant un taux d'erreur constant dans les données de référence (discuté précédemment), la probabilité d'une estimation incorrecte sur les données de référence apparaît plus élevée dans le groupe ayant la plus faible amplitude de variation de la proportion d'herbe dans la ration. De plus, les erreurs de discrimination les plus élevées ont été observées pour le petit groupe. Enfin, les faibles valeurs obtenues pour la sensibilité (comme dans les modèles « absence de fourrage fermenté » et « absence d'herbe fermentée ») ou de spécificité (comme dans le modèle « concentré <28% ») ne sont pas surprenants lorsque l'on essaye de discriminer les aliments de la ration considérés un par un. En effet, ces critères offrent in fine de nombreuses possibilités, qui font que des rations complexes et variées coexistent dans les groupes respectant ou non, le critère du cahier des charges ce qui rend la discrimination plus complexe. Les résultats de discrimination des modèles qui regroupent la totalité des critères (tous respectés / aucun respecté) sont très performants pour les deux fromages AOP. Ils sont meilleurs que les modèles discriminant les critères considérés un par un. Ce résultat met l'accent sur l'importance de considérer la ration dans son ensemble pour caractériser les laits correspondants. Plus les rations permises sont simples (avec un nombre restreint d'aliments autorisés) plus les performances de discrimination s'améliorent, comme le montrent les meilleurs résultats obtenus pour le Laguiole comparativement au Cantal. Même si les résultats des modèles obtenus sont prometteurs, il est nécessaire de rappeler qu'il s'agit là d'indicateurs et non pas de mesures. Une interprétation très prudente des résultats est donc toujours requise, notamment si l'on ne considère qu'un échantillon. En revanche, l'incertitude des modèles pourrait être en partie compensée par des mesures sur un grand nombre d'échantillons. Cela pourrait être le cas lors d'une utilisation en routine qui permettrait de prédire la composition de la ration lors de chaque prélèvement de lait de tank réalisé pour le payement du lait (> 3 échantillons par semaine /ferme). Au-delà de l'incertitude de la mesure de référence et de l'erreur du modèle, il est nécessaire de rappeler également que les critères et les seuils ont été calculés sur une moyenne annuelle. Cette simplification oblige à ne pas considérer une prédiction sur un seul échantillon de lait pour vérifier ou non le respect du cahier de charge, mais plutôt de se focaliser sur un bilan sur une durée plus longue (par exemple à l'échelle de la saison pour le pâturage ou sur l'année pour les concentrés). Ce principe est d'autant plus important en considérant les variations saisonnières de la composition de la ration selon la disponibilité des fourrages. Enfin, les indicateurs développés avec les modèles décrits n'ont pas pour vocation de se substituer aux contrôles actuellement mis en oeuvre pour vérifier les promesses faites aux consommateurs par les cahiers des charges. Ils constituent plutôt un outil pour mieux orienter et cibler les contrôles. Ils permettront également de consolider le système d'authentification et de contrôle, en renforçant l'image et la crédibilité des filières vis-à-vis des consommateurs.

CONCLUSION

Les modèles de prédiction de la proportion des aliments dans la ration, tout comme les modèles de discrimination, peuvent permettre de fournir via les analyses de routine réalisées sur les laits de tank, des indicateurs sur la composition de la ration des troupeaux laitiers.

  Prédiction de la composition de la ration des vaches et authentification des pratiques des cahiers des charges des fromages AOP par spectroscopie dans le moyen infrarouge sur des échantillons de lait de tank COPPA M. (1), MARTIN B. (1), HULIN S. (2), GUILLEMIN J. (3), GAUZENTES J.V. (4), PECOU A. (5), ANDUEZA D. (1)

  Protected Designation of Origin cheese using mid-infrared spectroscopy on bulk milk COPPA M. (1), MARTIN B. (1), HULIN S. (2), GUILLEMIN J. (3), GAUZENTES J.V. (4), PECOU A. (5), ANDUEZA D. (1) (1) Université Clermont Auvergne, INRAE, VetAgro Sup, UMR 1213 Herbivores, F-63122 Saint-Genès-Champanelle, France

  à 192 vaches traites) et le niveau de production laitière (4,2 à 40,0 kg/vache/J) témoigne d'une grande diversité de types d'élevage qui s'échelonnent le long d'un gradient qui va d'exploitations utilisant des doubletroupeaux (lait et viande) de petite taille avec des races locales assez fréquentes au sein du Massif central, aux grands troupeaux de races laitières spécialisées à haut rendement caractéristiques d'exploitations laitières plus intensives. Les parts moyennes de pâturage, de foin, d'ensilage d'herbe, d'ensilage de maïs et de concentrés étaient respectivement de 18, 17, 12, 19 et 25% de l'apport quotidien en MS. Ces valeurs moyennes (similaires pour le groupe de calibration et de validation) masquent une variabilité très importante en cohérence avec la disponibilité fourragère et l'adaptation saisonnière du régime alimentaire des troupeaux.

Figure 1

 1 Figure 1 Variation du % des aliments dans la ration des troupeaux. Aliment (%MS/ ration totale) Validation SEP R 2 V Foin 13,3 0,42 Ensilage d'herbe 15,1 0,26 Ensilage de maïs 12,2 0,61 Pâturage 11,7 0,81 Concentrés 6,2 0,26 Herbe fermentée 14,6 0,37 Herbe conservée 14,8 0,61 Fourrages fermentés 14,0 0,67 Herbe totale 12,3 0,68 Tableau 5 Résultats des modèles de prédiction du % des aliments dans la ration.

  2017. J. Dairy Sci., 100 ,8705-8721. De Marchi, M., Toffanin V., Cassandro M., Penasa M.. 2014. J. Dairy Sci., 97, 1171-1186. Kennard R.W., Stone L.A., 1969. Technometrics, 11:137-148. Prache S., Martin B., Coppa M., 2020. Animal. 14:854-863. Valenti, B., Martin B., Andueza D., Leroux C., Labonne C., Lahalle F., Larroque H., Brunschwig P., Lecomte C., Brochard M., Ferlay A., 2013. Int. Dairy J. 32, 26-32.

Fromage

  chaque groupe pour les différents critères des cahiers des charges est reporté dans le Tableau 2 pour le fromage AOP Cantal et dans le Tableau 3 pour le fromage AOP Laguiole. Deux autres groupes avec, pour l'un, les échantillons respectant l'ensemble des critères de chaque cahier des charges en même temps, et pour l'autre, les échantillons n'en respectant aucun, ont également été créés. Valeurs seuils pour chaque type d'aliment, exprimée en % de MS sur la ration totale, respectant les cahiers des charges des AOP Cantal et Laguiole. Effectifs des différents groupes pour tester les critères du cahier des charges de l'AOP Laguiole.

	Aliment		Fromage AOP
	(% de MS / ration totale)	Cantal	Laguiole
	Pâturage		> 50%	> 57%
	Concentrés		< 28%
	Pâturage + foin		> 25%	> 72%
	Ensilage de maïs		< 30%	0
	Herbe totale		> 50%	> 72%
	Herbe fermentée		-	0
	Fourrages fermentés		-	0
	Tableau 1 Critères	Groupe	N	N
	(% de MS / ration totale)		calibration	validation
	Présence de Pâturage	Oui Non	2531 3577	620 880
	Pâturage ≥ 50%	≥ 50 < 50	1029 5078	256 1255
	Concentrés < 28%	< 28 ≥ 28	4119 1989	1009 491
	Pâturage + foin ≥ 25%	≥ 25 < 25	3211 2897	799 701
	Ensilage de maïs < 30%	< 30 ≥ 30	4091 2016	1021 479
	Herbe totale ≥ 50%	≥ 50 <50	3614 2493	917 583
	Respect simultané de	Tous	1905	650
	tous les critères	Aucun	1683	482
	Tableau 2 Effectifs des différents groupes pour tester les
	critères du cahier des charges pour l'AOP Cantal.
	Critères	Groupe	N	N
	(% de MS / ration totale)		calibration	validation
	Pâturage ≥ 57%	≥ 57 < 57	815 5292	197 1303
	Pâturage + foin ≥ 72%	≥ 72 < 72	1021 5086	234 1266
	Absence de fourrages	Oui	1303	308
	fermentés	Non	4804	1192
	Absence d'herbe	Oui	2005	484
	fermentée	Non	4102	1016
	Absence d'ensilage de	Oui	2577	680
	maïs	Non	3531	820
	Respect simultané de	Tous	488	134
	tous les critères	Aucun	1666	477
	Tableau 3

  Ces indicateurs, obtenus à faible coût, seront utiles aux éleveurs, aux filières et aux consommateurs pour vérifier et consolider les promesses faites par les filières en terme d'alimentation des animaux via leurs cahiers de charges. Ce projet « Alimir » a été financé par le CNIEL. Les auteurs remercient également le Comité Interprofessionnel des fromages du Cantal (CIF) et le syndicat du fromage de Laguiole, tous deux fortement impliqués dans le projet. Enfin, les auteurs remercient Y. Goudron et F Picard de l'INRAE pour leur appui technique.

  Tableau 6 Résultats de validation des modèles de discrimination pour authentifier les critères alimentaires indiqués dans les cahiers des charges des fromages AOP Cantal et Laguiole.

	AOP	Critères	Sensibilité (%) Spécificité (%)	Précision (%) Exactitude (%)
		Présence de Pâturage	87,6	92,4	89,0	90,4
		Pâturage ≥ 50%	78,5	96,7	82,8	93,7
		Concentrés < 28%	88,8	34,2	73,6	71,0
	Cantal	Pâturage + foin ≥ 25%	82,5	84,7	86,0	83,5
		Ensilage de maïs < 30%	88,3	74,7	87,8	83,9
		Herbe totale ≥ 50%	87,9	84,3	89,2	86,5
		Respect simultané de tous les critères	91,1	89,2	92,6	90,3
		Pâturage ≥ 57%	66,5	97,6	81,1	93,5
		Pâturage + foin ≥ 72%	44,5	97,8	80,3	89,1
	1 Laguiole	Absence de fourrages fermentés Absence d'herbe fermentée	54,4 61,1	96,1 89,7	79,0 74,3	87,3 80,3
		Absence d'ensilage de maïs	80,0	85,9	80,9	83,4
		Respect simultané de tous les critères	100,0	99,4	97,8	99,5

1 le modèle pour le critère des concentrés (< 28 %) est commun aux 2 fromages AOP.

Renc. Rech. Ruminants, 2020, 25


Marion Ferreira
email: marion-ferreira@hotmail.fr

Raphaël Borie

Bruno Crestani

Pierre Rigaud

Lidwine Wemeau

Dominique Israel-Biet

Sylvie Leroy

Sébastien Quétant

Laurent Plantier

Jean-Charles Dalphin

Vincent Cottin

Sylvain Marchand-Adam

Orphalung Network

Efficacy and safety of rituximab in patients with chronic hypersensitivity pneumonitis (cHP): A retrospective, multicentric, observational study

Keywords: Rituximab, hypersensitivity pneumonitis hypersensitivity pneumonitis, rituximab, forced vital capacity

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

INTRODUCTION:

Hypersensitivity pneumonitis (HP) results from repeated exposure to a variety of inhaled particles. In previously sensitized individuals, these antigens elicit an exaggerated immunological response within the small airways and pulmonary parenchyma [START_REF] Selman | Hypersensitivity pneumonitis: insights in diagnosis and pathobiology[END_REF].

In contrast to idiopathic pulmonary fibrosis (IPF), there are no defined criteria for chronic HP (cHP) diagnosis, which is established by bringing together clinical, radiological and pathological information at multidisciplinary team meetings. To determine probability of cHP, Morisset et al proposed an algorithm based on compatible symptomatology, exposure to antigens known to be pathogenic or presence of serum precipitins, typical computerized tomography (HRCT) features, and histopathological characteristics [START_REF] Morisset | HP Delphi Collaborators, Identification of Diagnostic Criteria for Chronic Hypersensitivity Pneumonitis: An International Modified Delphi Survey[END_REF].

Vasakova et al distinguished between acute forms with symptoms lasting less than 6 months, and chronic forms. These chronic forms can be associated with fibrosis shown on the HRCT as well as by histological analysis. Chronic forms can progress to an imaging pattern of pulmonary fibrosis and show histological features similar to those of non-specific interstitial pneumonitis (NSIP) or usual interstitial pneumonitis (UIP) [START_REF] Vasakova | Hypersensitivity Pneumonitis: Perspectives in Diagnosis and Management[END_REF]. Chronic HP has a poor prognosis; in a longitudinal study of 160 cHP cases, the survival rate at 5 years was 31% [START_REF] Ojanguren | Long-term outcomes in chronic hypersensitivity pneumonitis[END_REF].

Pathophysiology is not fully understood, although type III and type IV hypersensitivity to inhaled antigen are suspected. The coexistence of genetic and environmental susceptibility factors can cause an exaggerated immune response, causing inflammation in the lungs [START_REF] Selman | Hypersensitivity pneumonitis: insights in diagnosis and pathobiology[END_REF]. Inflammatory mediators secreted by activated lymphocytes and macrophages play a role in the local inflammatory state. In addition, B cells secrete immunoglobulins G (IgG) specific to the antigens underlying HP. Fibrocytes and interleukin-17 are involved in the fibrotic appearance of chronic HP [START_REF] Lacasse | Recent advances in hypersensitivity pneumonitis[END_REF].

Management of HP is primarily based on antigen avoidance [START_REF] Zacharisen | The long-term outcome in acute, subacute, and chronic forms of pigeon breeder's disease hypersensitivity pneumonitis[END_REF], even if, fibrosing forms of the disease can continue to progress. There is no pharmacological treatment for cHP and only lung transplantation can improve the survival rate of patients with impaired respiratory function [START_REF] Kern | Lung transplantation for hypersensitivity pneumonitis[END_REF]. Although the mechanisms of pulmonary inflammation and fibrosis in cHP are complex, T and B lymphocytes probably play a central role [START_REF] Lacasse | Recent advances in hypersensitivity pneumonitis[END_REF]. This indicates the use of anti-inflammatory or immunosuppressive drugs such as systemic corticosteroids, but with no influence on long-term survival [START_REF] Agache | Management of hypersensivity pneumonitis[END_REF] [START_REF] Kokkarinen | Effect of corticosteroid treatment on the recovery of pulmonary function in farmer's lung[END_REF]. Several retrospective studies evaluating two main lines of therapeutic options: on the one hand immunoregulatory molecules (corticosteroids, azathioprine and mycophenolate mofetil) and in the other hand anti-fibrotic molecules have been published. The first reported the use of pirfenidone [START_REF] Behr | Exploring efficacy and safety of oral Pirfenidone for progressive, non-IPF lung fibrosis (RELIEF) -a randomized, double-blind, placebo-controlled, parallel group, multi-center, phase II trial[END_REF]which was not shown to have a significant effect on forced vital capacity (FVC) decline. Introduction of mycophenolate mofetil and azathioprine was associated with improved gas exchange and in reduction of dose in those who were in treatment with prednisone [START_REF] Morisset | Use of Mycophenolate Mofetil or Azathioprine for the Management of Chronic Hypersensitivity Pneumonitis[END_REF].. Two another retrospective studies found less treatment related-adverse events compared to those treated with prednisone alone without a significant effect on disease progression [START_REF] Adegunsoye | Outcomes of immunosuppressive therapy in chronic hypersensitivity pneumonitis[END_REF] or an improvement in carbon monoxide diffusing capacity (DLCO) and reduction in dose of corticosteroids with mycophenolate mofetil or azathioprine [START_REF] Fiddler | Use of mycophenolate mofetil and azathioprine for the treatment of chronic hypersensitivity pneumonitis-A single-centre experience[END_REF];. More recently, a prospective, randomized, double-blind, INBUILD study found that the annual decline in FVC is reduced in patients with active fibrotic interstitial lung disease (ILD) treated with nintedanib; a quarter of these patients had cHP [START_REF] Flaherty | INBUILD Trial Investigators, Nintedanib in Progressive Fibrosing Interstitial Lung Diseases[END_REF].

Another treatment option for patients with cHP is rituximab [START_REF] Morell | Addition of Rituximab to Oral Corticosteroids in the Treatment of Chronic Hypersensitivity Pneumonitis[END_REF], an anti-CD20 monoclonal antibody depleting B lymphocytes which has been shown to be beneficial in ILD associated with connective tissue disease [START_REF] Keir | Severe interstitial lung disease in connective tissue disease: rituximab as rescue therapy[END_REF]. A patient with histologically proven HP, who did not respond to corticosteroids, showed improved FVC after taking rituximab [START_REF] Lota | Novel use of rituximab in hypersensitivity pneumonitis refractory to conventional treatment[END_REF]. Two other clinical case reports published in 2019 showed similar results [START_REF] Morell | Addition of Rituximab to Oral Corticosteroids in the Treatment of Chronic Hypersensitivity Pneumonitis[END_REF] [START_REF] Tamm | Rituximab for Salvage Therapy of Refractory Hypersensitivity Pneumonitis[END_REF]. A retrospective study of 6 patients with cHP found that lung function stabilized or improved in 3 and continued to deteriorate in the other 3, after treatment with rituximab [START_REF] Keir | Rituximab in severe, treatmentrefractory interstitial lung disease[END_REF]. To date, there is no recommended treatment for patients whose respiratory function continues to deteriorate in spite of antigen avoidance.

We hypothesized that rituximab could benefit patients with cHP who have not responded to standard treatment including antigen avoidance and corticosteroid therapy. Our aim was to study changes in FVC in 20 cHP cases after 6 months of treatment.

METHODS:

This retrospective and observational study was conducted in the pulmonology departments of seven French University Hospitals. All patients treated with rituximab between January 2014 and June 2019 and with a diagnosis of cHP validated during a multidisciplinary team meetings with criteria adapted from Morisset et al. [START_REF] Vasakova | Hypersensitivity Pneumonitis: Perspectives in Diagnosis and Management[END_REF] (Table 1) were screened for our analysis. We included patients with no improvement in lung function (deteriorationin their FVC) after a minimum of 3 months treatment including antigen avoidance and who had received at least one injection of rituximab. There were no exclusion criteria.

Primary endpoint was change in FVC (% of the predicted value) 6 months after initiation of rituximab. Secondary endpoints included change in percentage of the predicted value of FVC at 12 months and change in the percentage of the predicted value of DLCO at 6 months. We also studied changes in corticosteroids dose at 6 months and any side effects, in particular infection, in the 12 months following onset of treatment with rituximab.

Local research ethics committee approval was in place for retrospective review of cHP patients.

Statistical analysis

Characteristics of the participants were identified with quantitative (median;

[minimum-maximum]) and qualitative (frequency) variables. For analysis of the primary endpoint, given the small size of the study population, non-parametric tests were used. The Wilcoxon test was used to analyze changes in FVC and DLCO before and after treatment with rituximab.

To compare characteristics of responders and non-responders to rituximab (positive or strictly negative change), and to identify predictors of response to treatment, a Mann Whitney test was used for the unpaired quantitative variables and Chi2 test for qualitative variables.

The sample was too small to perform multivariate statistical analyses.

Statistical analyzes and graphs were performed using Excel software and GraphPadPrism 6 software. For all analyzes, a p <0.05 was considered significant.

RESULTS:

Patients

The sample comprised 20 patients who had been treated with rituximab between January 8, 2014 and June 14, 2019. Median duration of follow-up after the first administration was 18 months [6; 50 months]. Forty percent (10/20) of patients had a history smoking; all were former smokers, with a median cumulative smoking rate of 30 pack-years [5; 60 pack-years]. One of the 20 patients (5%) had no history of antigen exposure. For the others, exposure was domestic in 85% (17/20) of cases and occupational in 10% (2/20). The most frequently found precipitins were avian (50%) or household molds (10%). Sixty-five percent (13/20) of patients had had a surgical lung biopsy and histological analysis supporting the diagnosis of cHP. All chest CT were suggestive of cHP; majority of patients had HRCT signs of fibrosis : 90% with traction bronchiectasis, 75% with intra-lobular reticulations, 65% with lobular distortions and 45% with honeycombing. Thus, the probability of diagnosis, according to criteria adapted from Morisset et al. [START_REF] Vasakova | Hypersensitivity Pneumonitis: Perspectives in Diagnosis and Management[END_REF], was definite for 55% of patients (11/20) and very likely for the remaining 45% (9/20) (Table 1). The median number of infusions received was 3 [2; 10] with a dosage of 500 or 1000 mg and an interval of 6 months between doses (Table S2). Characteristics of the study population are presented in Table 2.

Six months before the introduction of rituximab (M-6), 20 patients had impaired respiratory function with median FVC of 65% [44; 112%] and median DLCO of 47% [21; 68%] in the 8 patients whom DLCO was avaible at the initiation of rituximab (M0) and M+6 too. All patients received rituximab because their respiratory function had deteriorated significantly, with a median FVC of 59% [39; 102%] (p =0.0001) at M0. One patient who presented 112% of FVC at M-6 and 102% at M0 was treated with rituximab because the dissociation of the DLCO value which appeared low to begin with, because he had received none clinical benefit from the corticosteroid therapy and because he had major fibrosis lesions on his HRCT scan. All patients had previously received systemic corticosteroids, with a median dose of 18 mg [10; 60 mg] at M0 (Table 2).

Evolution of lung function and corticosteroids

We analyzed changes in FVC 6 months after the start of treatment (M+6Between M0 and M+6, FVC remained stable (59% [39; 102%] versus 61% [38; 99%]; p=0.28). All patients showed a decrease in FVC between M-6 and M0, and in seven cases (35%), FVC improved or remained stable at M+6 (Figure 1). Although FVC continued to decline after administration of rituximab, the rate of decline was significantly less between M0 and M+6 (-3% [-15; + 19%]) than between M-6 and M0 (-8% [-21;0%]); p=0.0002). At M+12, FVC data were available for 13 of the 20 patients, who showed a significant decrease between M-6 (62% [51;80%]) and M0 (55% [39; 74%]; p=0,03), with no significant change between M0 and M+12 (52% ([31; 74%]; p=0.10). Twelve of the 20 patients (60%) had a rituximab administration between M+6 and M+12. The decline over the 12-month period between M0 and M+12 (-7% [-15; + 10%]), was significantly less than over the 6-month period between M-6 and M0 (-8% [-21; 0%]; p=0.0017).

Data for DLCO at initiation of rituximab were only available for 8 of the 20 patients; median DLCO was 47% [21; 68%] 6 months before the first administration of rituximab, and 37% [16; 73%] at onset. Between M-6 and M0, DLCO showed a downward trend. At M+6, the median DLCO remained stable (41% [15; 70%]; p = 0.29) (Figure 2), and improved in five of the 8 patients (63%).

Median daily dose of corticosteroid between M-6 and M0 remained stable (15mg/d of prednisone [0; 40mg] and 18mg/d [10; 60mg] respectively; p=0.55), while it decreased significantly between M0 and M+6 (12mg/d [0; 30mg]; p=0.013) (Figure 3). The corticosteroid dose of 11 patients was reduced between M0 and M+6, remained the same for 8 patients and was increased by 10mg to the M-6 dose for one patient.

Safety and tolerability of rituximab

Seven patients (35%) had developed an infection within 12 months of the first injection of rituximab; 6 had respiratory infections with no microbiological identification, and no Pneumocystis infection and one had a urinary tract infection caused by Escherichia coli.

Of the 6 patients with viral or bacterial respiratory infection, only one had no co-trimoxazole prophylaxis. One patient (5%) died from his respiratory infection.

Responder analysis

We compared 2 groups of patients: "responders" (n=7), whose FVC at M+6 showed a positive change; and "non-responders" (n=13), whose FVC showed a strictly negative change compared to M0.

Monovariate analysis revealed that no parameter was significantly associated with improvement in FVC at M+6, only the absence of a UIP or NSIP pattern on histology analysis tended to be approach statistical significance (p=0.06) (Table S1).

DISCUSSION:

To our knowledge, this is the largest study suggesting a possible beneficial effect of rituximab in a cohort of patients with cHP whose condition has not improved despite antigen avoidance and corticosteroid therapy. The data support the hypothesis that rituximab could limit FVC decline. For seven (30%) of the 23 patients in this study, FVC improved or remained stable 6 months after the introduction of rituximab. When a histological analysis was available, the absence of UIP or NSIP could be associated with improved FVC.

Our study population was generally comparable to that of other cohorts of cHP [12][11][20][4][21] [START_REF] Wälscher | Comorbidities and survival in patients with chronic hypersensitivity pneumonitis[END_REF]. In those studies, 32 to 57% of the patients were male (47% in our study), ages ranged between 61 and 65 years (63 years for our sample), and 34 to 56% had never smoked (52% of our patients). Antigen exposure was avian in 41 to 59% of cases (48% in our study). At the radiological level, honeycombing was found on 37 to 60% of scans (48% in our study) and air trapping was observed in 54 to 82% of cases (61% in our study).

However, some differences between our patients and those cohorts should be noted:

FVC was more severely impaired at M-6 and M0 in our patients than in the other cohorts (63% and 56% vs 73 and 65% at M-6 and M0 respectively). Antigen exposure was not found in 9% of our cases, compared to 11 to 41% in the other cohorts. Finally, the median value of broncho-alveolar lavage (BAL) lymphocytosis was higher in our patients (32% vs 14 to 20%).

Rituximab is a monoclonal antibody that depletes B cells [START_REF] Lota | Novel use of rituximab in hypersensitivity pneumonitis refractory to conventional treatment[END_REF]. After binding of the Fab fragment of rituximab and the CD20 antigen of B lymphocytes, the Fc fragment activates immune effectors responsible for lysis of B lymphocytes [START_REF] Cartron | From the bench to the bedside: ways to improve rituximab efficacy[END_REF]. It was therefore initially used in lymphoproliferative syndromes and is now used in many autoimmune pathologies and its efficacy has been observed in diffuse interstitial pathologies associated with anti-synthetase syndrome or scleroderma [24][25]. Despite the lack of randomized trials evaluating rituximab in cHP, many centers have used it in patients who have not responded to antigen avoidance and/or corticosteroid therapy, based on its efficacy in the treatment of severe ILD associated with connective tissue disease [START_REF] Keir | Severe interstitial lung disease in connective tissue disease: rituximab as rescue therapy[END_REF]. The rationale for using this molecule is that it allows a systemic deactivation of the immune system in order to prevent the progression of fibrosis, stabilizing and possibly improving the condition of patients with pulmonary disease from a clinical, functional and radiological point of view [START_REF] Koo | Treatment of connective tissue disease-associated interstitial lung disease: the pulmonologist's point of view[END_REF].

There are many hypotheses regarding the mechanisms whereby rituximab stabilizes or improves the lung function of patients with ILD with autoimmune features. Rituximab induces rapid depletion of CD20 + B lymphocytes present in peripheral circulation; although the role of these lymphocytes in the production of autoantibodies is well documented, the action of rituximab in ILD is probably only partially explained by the inhibition of this function. Clinical observations of patients with idiopathic thrombocytopenic purpura or graftversus-host reaction have suggested that rituximab may "normalize" self-reactive T cells [START_REF] Stasi | Response to B-cell depleting therapy with rituximab reverts the abnormalities of T-cell subsets in patients with idiopathic thrombocytopenic purpura[END_REF].

B cell repopulation following introduction of rituximab comprises antigenically undetermined B cells, suggesting a resetting of the immune system, which may contribute to therapeutic effect [START_REF] Keir | Rituximab in severe, treatmentrefractory interstitial lung disease[END_REF].

Finally, use of rituximab seems to be relatively safe: we documented 30% of infections (7/23 patients), most of them respiratory with only one reported death, attributable to a pulmonary infection. In the only retrospective study that tested rituximab in 50 patients with ILD, the respiratory infection rate was 22% without death directly related to rituximab toxicity or an infection secondary to its use [START_REF] Keir | Severe interstitial lung disease in connective tissue disease: rituximab as rescue therapy[END_REF]. If we plan to treat cHP patients with longterm rituximab, we must consider potential risks associated with the treatment, and in particular in comparison with prolonged corticosteroid therapy or other possible treatments such as mycophenolate mofetil or azathioprine. In the supplementary date, Adegunsoye et al detail the side effects of these molecules: in the 24 patients treated with azathioprine, 4 patients presented grade II upper respiratory infections, 2 patients recurrent grade III urinary tract infections; for the 28 patients under mycophenolate mofetil, there were 4 grade IV respiratory toxicities without specifying whether they were infections and 3 recurrent urinary tract infections [START_REF] Adegunsoye | Outcomes of immunosuppressive therapy in chronic hypersensitivity pneumonitis[END_REF]. With rituximab, there is a risk of hepatitis B reactive and there are reported cases of hepatitis C reactive, CMV infection, VZV infection, tuberculosis and fungi infections [START_REF] Malpica | Preventing infectious complications when treating non-malignant immune-mediated hematologic disorders[END_REF].In addition, long term hypogammaglobulinemia has been observed after repeated rituximab courses [START_REF] Cooper | Repeated courses of rituximab for autoimmune cytopenias may precipitate profound hypogammaglobulinaemia requiring replacement intravenous immunoglobulin[END_REF]. Vaccine recommendations also exist for measures associated with these molecules [START_REF] Malpica | Preventing infectious complications when treating non-malignant immune-mediated hematologic disorders[END_REF].

This retrospective study, without a control group has certain limitations. First, missing data did not allow to calculate monthly declines in FVC or DLCO. In addition, the apparent change in the decline in FVC can be due to the effect of treatment but alternatively could in part be explained in part by the natural course of FVC, which is not linear. We have also to criticize that even if the change in FVC decline appears statistically significant, absolute values are little different and are not necessarily associated with clinical benefit. There is a large inter-individual variability in FVC variation at M+6, reflecting a variety of possible responses to treatment. Subgroup analysis by presence of serum precipitins or of the level of immunoglobulins G could not be performed. Finally, heterogeneity in the number of rituximab infusions received by our patients limits the comparison. Bellan et al. stressed the importance of repeating courses of treatment with rituximab in order to be able to observe its benefits [START_REF] Bellan | Targeting CD20 in the treatment of interstitial lung diseases related to connective tissue diseases: A systematic review[END_REF]. A larger study population could have better identified some predictors of response.

ACKNOWLEDGEMENTS:

V. Cottin is a member of ERN-LUNG.

FORMATTING OF FUNDING SOURCES:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

DECLARATION OF INTEREST

We have no conflicts of interest to disclose.

REFERENCES:

Figure 1 :Figure 2 :

 12 Figure 1: relative change in FVC (% of predicted value), 6 months before and after the introduction of rituximab (n = 20The median value is represented by the bold line. ** and ***: p <0.01 and <0.001, respectively.

Figure 3 :

 3 Figure 3: Difference in the dose of corticosteroids 6 months before and after the introduction of rituximab (n = 20). The median values are represented by the bold lines. * and **: p <0.05 and <0.01 respectively.

  Thus, there are recommendations for hepatitis B vaccination in patients treated with rituximab and hepatitis B and C screening should be done before rituximab onset; other vaccinations are recommended: Streptococcus pneumonia, Haemophilus influenza and Neisseria meningitidis[START_REF] Malpica | Preventing infectious complications when treating non-malignant immune-mediated hematologic disorders[END_REF].The other treatments that can be used in cHP are not without risk: corticosteroids expose the risk of bacterial (tuberculosis), viral

(herpes viruses), fungal (Pneumocystis jirovecii) and parasitic infections; Aza and MMF can be complicated by viral (CMV, VZV) and fungal (Pneumocystis jirovecii) infections.

TABLES and FIGURES:

Table 1: diagnostic criteria for chronic hypersensitivity pneumonitis (adapted from Morisset [START_REF] Vasakova | Hypersensitivity Pneumonitis: Perspectives in Diagnosis and Management[END_REF]). The diagnosis is "very likely" when the probability is greater than 70%; the diagnosis is "likely" when the probability is greater than 50%.* Images compatible with thoracic CT scan: diffuse centrilobular micronodulation, diffuse ground glass, preserved lobules (mosaic) and expiratory trapping, presence of thin-walled cysts, traction bronchiectasis, intralobular reticulations, honeycombing, emphysema.** Suggestive histology: peribronchiolar lymphocytic infiltration, malformed granulomas, giant cells, peribronchiolar fibrosis

SUPPLEMENTARY INFORMATION Table S1-Determinants of variation in FVC (monovariate analysis). n = 20 in responders (variation in FVC at M + 6 greater than or equal to 0%) vs non-responders (variation in

FVC at M+6 less than 0%)