MD Karim Benali
email: k.benali@chru-nancy.fr

MD Louis Rigal

BSc Antoine Simon

PhD Julien Bellec

BSc Pierre Jaïs

MD, PhD; Tsukasa Kamakura

PhD Clifford G Robinson

MD Phillip Cuculich

MD Renaud De Crevoisier

PhD Raphaël P Martins

Correlation between radiation dose and myocardial remodeling after stereotactic radiation therapy for ventricular tachycardia: First assessment of dose-effect relationship in human

Keywords: Ventricular tachycardia, Radiation therapy, Arrhythmias, Cardiac SBRT, Radiation dose, Antiarrhythmic effects

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

Cardiac stereotactic body radiation therapy (SBRT) is an emerging treatment for refractory ventricular tachycardias (VT) resistant to medication and/or catheter ablation (CA) [START_REF] Robinson | Phase I/II Trial of Electrophysiology-Guided Noninvasive Cardiac Radioablation for Ventricular Tachycardia[END_REF]. Data regarding myocardial remodeling resulting from cardiac SBRT in humans are scarce. We aimed to describe the correlation between radiation dose received and the resulting electrophysiological effect.

A 62-years-old man with non-ischemic cardiomyopathy was referred for cardiac SBRT for refractory VT after unsuccessful CA procedures, most likely due to an intramural location of the substrate. Using Versa HD linear accelerator (Elekta, Crawley, UK), a single radiation dose of 25 Gy was delivered to the basal inferolateral left ventricle (LV), over a planning target volume (PTV) of 85 cc (Figure 1A). Eight months later, the patient presented with a new VT different from the one initially targeted, requiring redo CA. Both procedures were performed using CARTO® 3D system (Biosense Webster Inc., California, USA). High density voltage maps (Pentaray®) were performed in sinus rhythm (Figure 1B). Dense scar, low voltage areas and normal myocardium were defined as bipolar voltage < 0.5 mV, 0.5-1.5 mV and > 1.5mV, respectively. Pre-and postradioablation electroanatomical data were registered to the cardiac computed tomography (CT) using automatic registration (Coherent Point Drift probabilistic algorithm), manually corrected and validated by two cardiologists (RPM, KB). Dose distribution was registered to the cardiac CT using automatic rigid registration based on intensity correlation with the corresponding thoracic CT, manually corrected by a cardiologist (RPM) and a radio-oncologist (RDC). Each pre-SBRT measurement with bipolar voltage > 0.5mV was linked to the nearest post-SBRT point. Pre-SBRT measurements with no corresponding post-SBRT measurements closer than 3mm were not considered. The study was approved by local ethical committee.

Based on this matching, 631 pairs of voltage measurements were analyzed. Regarding radiation dose, 82 (13.0%), 116 (18.4%), 106 (16.8%), 87 (13.8%), 84 (13.3%) and 156 (24.7%) voltage pairs received a dose of 0-5 Gy, 5-10 Gy, 10-15 Gy, 15-20 Gy, 20-25 Gy and above 25 Gy, respectively. LV myocardium receiving less than 15 Gy had no significant change in mean bipolar voltage between pre-and post-SBRT. Conversely, LV myocardium in the 15-20 Gy, 20-25 Gy and >25 Gy dose ranges had a significant change in mean bipolar voltage, decreasing from 3.9±3.3mV to 0.4±0.5mV (p<0.001), 1.1±0.7mV to 0.3±0.2mV (p<0.001), and 1.0±0.6mV to 0.2±0.3mV (p<0.001), respectively (Figure 1C).

Cardiac SBRT is a promising technique for the treatment of refractory VTs. Pre-clinical studies have specified the dose-effect relationship leading to the current radiation dose of 25 Gy(2). However, there is no data regarding myocardial remodeling resulting from SBRT in humans, particularly the dose-effect relationship in real-world SBRT setting. Amino et al previously reported in rabbit hearts the same threshold of 15 Gy for reducing ventricular arrhythmias vulnerability(3). Zhang's paper recently suggested that cardiac SBRT would promote an increase in conduction velocities through cellular reprogramming, potentially explaining the observed acute effects of SBRT, even for low radiation doses. Our work suggests the existence of a delayed radioablative effect with reduced bipolar voltage and dense scar formation in myocardial regions receiving a radiation dose ≥15 Gy. These two electrophysiological mechanisms most likely coexist after cardiac SBRT, at different degrees depending on the delay from treatment delivery.

In conclusion, this case of remapping 8 months after SBRT shows a reduction in bipolar voltages for myocardium exposed to radiation doses ≥15 Gy. This result and recent works suggest that for doses below 25 Gy, two anti-arrhythmic effects of SBRT may coexist: an early effect explained by electrical cell reprogramming and a delayed ablative effect explained by increased scar formation. However, there is no data regarding the durability of these low-dose effects on the control of ventricular arrhythmias. Although further studies are needed to confirm these results, these data may be of interest to optimize future cardiac SBRT treatment plans, particularly in complex cases where organs at risk are close to the PTV.

Figure 1 :

 1 Figure 1: Panel A -Cardiac SBRT target volume and dose distribution. Panel B-

funding for this work.


Odile B Harrison
email: odile.harrison@zoo.ox.ac.uk

Nicholas J Evans

Jessica M Blair

Holly S Grimes

Colin R Tinsley

Xavier Nassif

Paula Kriz

Roisin Ure

Steve J Gray

Jeremy P Derrick

Martin C J Maiden

Dr Ian M Feavers
email: ifeavers@nibsc.ac.uk

Epidemiological evidence for the role of the haemoglobin receptor, HmbR, in meningococcal virulence

Keywords: haemoglobin receptor, meningococcal disease, virulence, pathogenicity island

The distribution of the haemoglobin receptor gene (hmbR) was investigated in disease and carried Neisseria meningitidis isolates revealing that the gene occurred at a significantly higher frequency in disease isolates compared to those obtained from carriage. Where hmbR was absent, the locus was occupied by the cassettes exl2 or exl3, or with a "pseudo hmbR" gene designated exl4. The hmbR locus in published N. meningitidis genomes, as well as N. gonorrhoeae and N. lactamica ST-640, exhibited characteristics of a pathogenicity island. These data are consistent with a role for the hmbR gene in meningococcal disease.

INTRODUCTION

The documented absence of the haemoglobin receptor gene among a number of Neisseria meningitidis isolates led to the discovery that the gene was located on a genetic island containing multiple cassettes including the putative proteins Exl2, Exl3 followed by other unidentified open reading frames (ORF) [START_REF] Richardson | HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation[END_REF][START_REF] Kahler | exl, an exchangeable genetic island in Neisseria meningitidis[END_REF]. Lack of the hmbR gene among some meningococcal isolates, combined with the association of the gene with mobile genetic elements, indicates that it may form part of a DNA virulence island recently acquired by meningococci [START_REF] Hacker | Pathogenicity islands and the evolution of microbes[END_REF][START_REF] Gal-Mor | Pathogenicity islands: a molecular toolbox for bacterial virulence[END_REF].

Here the distribution of the hmbR gene among over 700 disease and carriage-associated meningococci from three separate isolate collections was investigated, revealing a statistically significant association between disease and the presence of the hmbR gene. In silico analysis of the hmbR genetic locus among the genomes belonging to N. meningitidis isolates Z2491, FAM18, MC58, NMC053442, N. gonorrhoeae FA1090 and N. lactamica ST-640 indicated that the gene was located on a putative pathogenicity island. DNA arrays comparing the genomes of isolates belonging to the ST-41/44 clonal complex showed a higher likelihood of the hmbR gene being found among isolates from cases of invasive disease.

METHODS

Meningococcal isolates

Three isolate collections were examined. The first comprised 96 mainly disease-associated isolates assembled globally from 1937 to 1996 and described by Maiden et al [START_REF] Maiden | Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms[END_REF]. The second contained 203 carried and invasive isolates forming part of a collection assembled in 1993 in the Czech Republic [START_REF] Jolley | Carried meningococci in the Czech Republic: a diverse recombining population[END_REF]. The third collection included 462 isolates obtained in England and Wales between 1999 and 2000, of which 265 were carried and 197 were disease-associated. These isolates were from the Meningococcal Reference Unit and the UK carriage study.

PCR amplification and nucleotide sequence determination

The complete hmbR gene was amplified using primers PN1 5′-CCGGAAGGAATGATGCCGCACAGG-3′ and NE3 5′-TTAAAACTTCCATTCCAGCG-3′. Isolates hmbR negative were further screened by PCR using primers hemO 5′-GAATGATGCCGCAGAG-3′ and col2 5′-GCATTCAGGTCAAAATCC-3′, which are located in the hemO and col genes on either side of the genetic island thus amplifying the whole region. Following this procedure, amplicons were sequenced using the primers hemO, exl2-R 5′-CCGTTCAGGGATTTACCGGC-3′, exl3-F 5′-GCCTGCAAATACGCCGAAGCC-3′, exl3-R 5′-GCCTGCAAATACGCCGAAGCCG-3′, exl3-seqF 5′-GGCGGAGTGCAAAATGATGC-3′ and exl3-seqR 5′-GCCATCTTTTAATTTAGCCGC-3′ revealing the exchangeable cassette replacing the hmbR gene. Nucleotide accession numbers can be found in the Appendix A1.

Whole genome comparisons of the ST41/44 clonal complex meningococci by DNA array

Techniques and data analysis were as described previously [START_REF] Bille | A chromosomally integrated bacteriophage in invasive meningococci[END_REF][START_REF] Perrin | Comparative genomics identifies the genetic islands that distinguish Neisseria meningitidis, the agent of cerebrospinal meningitis, from other Neisseria species[END_REF] with data deposited in the Array express database www.ebi.ac.uk/arrayexpress/query/entry with accession number E-MEXP-343.

Statistical analyses

These were conducted using Intercooled STATA version 10 software for windows. Multivariable logistic regression models were used to estimate the strength of association between hmbR status and whether the isolate originated from a healthy carrier or a clinical case.

RESULTS

Distribution of the hmbR gene and association with disease

A total of 761 N. meningitidis isolates were screened for the presence of the hmbR gene. A minority of disease isolates lacking the gene were present with 11 (14%), 1 (7%) and 5 (3%) hmbR negative isolates present in all three collections respectively, indicative of a significant association of the gene with virulence (Table 1). The odds ratio for the association of the hmbR gene with all disease isolates and for each isolate collection provided further evidence for the predisposition of disease isolates to contain the hmbR gene. The over-representation of the gene among disease N. meningitidis isolates was apparent with the hyper-invasive lineages ST-4, ST-5, ST-8, ST-11, ST-18 and ST-32 exclusively hmbR positive (Appendix A2).

Association of exl2 and exl3 with carried clonal complexes

Isolates lacking the hmbR gene were found among clonal complexes ST-1, ST-22, ST-23, ST-41/44, ST-60, ST-106, ST-198, ST-549 in addition to many isolates currently unassigned to a clonal complex (Appendix A2). The exl2 exchangeable cassette was principally associated with serogroup Y isolates of the ST-23 clonal complex (59%).The exl3 genetic island was present in clonal complexes ST-1, ST-106, ST-549, ST-22 and ST-60.

Characterisation of a "pseudo hmbR" gene designated exl4

A total of 28 isolates from the clonal complexes ST-41/44 and ST-198 lacked both exl genetic islands and the hmbR gene (Appendix A2). BLAST searches of the sequence replacing exl2, exl3 and hmbR in these isolates revealed 97% sequence identity with a "pseudo hmbR gene" identified in the sequenced genome of isolate NMC053442, a Chinese ST-4821 serogroup C isolate [START_REF] Peng | Characterization of ST-4821 complex, a unique Neisseria meningitidis clone[END_REF]. Further analysis of the 2, 477 bp long "pseudo hmbR gene" belonging to isolate NMC053442 revealed 62% sequence identity over a length of 977 bp fragments with published hmbR sequences indicating that this may not be an hmbR gene but may instead contain fragments of the hmbR gene. Furthermore, the "pseudo hmbR gene" identified in the ST-41/44 and ST-198 isolates, as well as isolate NMC053442, was found to contain a polydeoxyguanosine phosphate tract occurring at 663 bp, much earlier than that of published hmbR genes, which are known to occur at 1159 bp. Consequently, this novel ORF replacing hmbR was designated exl4.

Clonal Complex ST-41/44 and hmbR

DNA arrays were used to compare the genomes of isolates belonging to recognised invasive sequence types (e.g. ST-41) with isolates belonging to sequence types not associated with disease (e.g. ST-44, ST-110 and ST-111). Within the ST-41/44 clonal complex, the association of hmbR with disease N. meningitidis isolates was significant (P< 0.0001) with 65 (98%) of the 66 disease-associated isolates containing the hmbR gene compared to 41 (69%) out of 59 carriage isolates. Genome comparisons between ST-41 and ST-44 isolates did not show any other major differences.

Genetic arrangement of the hmbR and exl loci in Neisseria

In silico analysis of the hmbR locus among the sequenced genomes from N. meningitidis, N. gonorrhoeae and N. lactamica identified seven different cassettes (A-G). Region I contained hemO, a gene involved in the catabolism of heme. Region II included the collagenase gene homologue, col, bordered by a 27 bp inverted repeat. Region III contained the hmbR gene, or the exchangeable islands exl2, exl3 and exl4 (Figure 1). The previously documented hmbR locus belonging to N. meningitidis GA0929 containing the exl2 exchangeable cassette was also included in the alignment [START_REF] Kahler | exl, an exchangeable genetic island in Neisseria meningitidis[END_REF]. Multiple unidentified ORFs, Correia elements [START_REF] Correia | A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA[END_REF] and inverted repeats were identified -all key characteristics of pathogenicity islands.

DISCUSSION

Neisseria meningitidis remains a major cause of meningitis and septicaemia worldwide, however despite its pathogenic potential, N. meningitidis is also a common human commensal [START_REF] Yazdankhah | Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway[END_REF][START_REF] Cartwright | The Stonehouse survey: nasopharyngeal carriage of meningococci and Neisseria lactamica[END_REF]. The capsular polysaccharide, type IV pili and iron acquisition systems are all necessary pathogenesis determinants, but while each of these factors is required for invasive disease it is not sufficient, with the likelihood that many other genetic factors contribute to the ability of a meningococcus to cause invasive disease. The search for new virulence factors in this species is hampered by the lack of a suitable animal model representative of human disease. An alternative strategy is to employ a molecular epidemiological approach to establish a statistical association of a gene with disease. Here, the distribution of the haemoglobin receptor gene was examined in over 700 meningococci isolated from cases of disease and asymptomatic carriage.

The hmbR gene was first identified in 1995 and the importance of haemoglobin utilisation during invasion was demonstrated when an N. meningitidis hmbR mutant was attenuated in an infant rat model of meningococcal infection [START_REF] Stojiljkovic | The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence[END_REF]. Data presented in this study described a statistically significant association of the hmbR gene with disease N. meningitidis isolates, providing epidemiological evidence for the importance of the hmbR gene in meningococcal virulence (Table 1). Combined with the documented low diversity of the gene [START_REF] Stojiljkovic | HmbR outer membrane receptors of pathogenic Neisseria_spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation[END_REF], these data indicate a potential role of HmbR in future vaccines.

The absence of hmbR among N. meningitidis isolates has been reported [START_REF] Richardson | HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation[END_REF] and, due to the prevalence of the hpuAB complex among those isolates, it was suggested that hmbR may have been a recent acquisition by meningococci. Examination of the genetic island among the genomes from N. meningitidis FAM18, Z2491, MC58 and NMC053442, N. gonorrhoeae FA1090 and N. lactamica ST-640 revealed that the region contained many inverted repeats, a feature of pathogenicity islands (PAIs) (Figure 1) [START_REF] Gal-Mor | Pathogenicity islands: a molecular toolbox for bacterial virulence[END_REF]. Further PAI characteristics included an overall G+C content of 47% below the mean 51% of the Neisseria genome content, the presence of cryptic ORFs, the insertion sequence IS1060 in N. gonorrhoeae FA1090 as well as Correia elements among N. meningitidis isolates [START_REF] Hacker | Pathogenicity islands and the evolution of microbes[END_REF]. Finally, the hmbR locus was not composed of a homogeneous piece of DNA but was instead made up of several mosaic-like ORFs, another key PAI feature [START_REF] Hacker | Pathogenicity islands and the evolution of microbes[END_REF][START_REF] Gal-Mor | Pathogenicity islands: a molecular toolbox for bacterial virulence[END_REF][START_REF] Hacker | Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes[END_REF].

In the present study, a minority of 17 (5%) of the 314 disease N. meningitidis isolates from all three isolate collections lacked the hmbR gene indicative of the over-representation of the gene among disease isolates (Table 1). Most cases of meningococcal disease are caused by only a few clonal complexes of related sequence types (STs) referred to as hyper-invasive lineages [START_REF] Yazdankhah | Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway[END_REF]. The association of the hmbR gene with disease was apparent in isolates belonging to such lineages including ST-4, ST-5, ST-8, ST-11, ST-18 and ST-32 where, without exception, all isolates contained the gene (Appendix A2).

The importance of the hmbR gene in disease isolates was consistent among meningococci of the ST-41/44 clonal complex, members of which have been the prevalent cause of disease in parts of Northern Europe during the past decade and responsible for epidemic disease in New Zealand [START_REF] Dyet | New Zealand's epidemic of meningococcal disease described using molecular analysis: implications for vaccine delivery[END_REF]. This complex of closely related clones is unusual in that it is centred on two sequence types, ST-41 and ST-44, rather than possessing a single central ST [START_REF] Maiden | Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms[END_REF]. ST-41 related meningococci are in general isolated from cases of invasive disease, whereas ST-44 related clones are typically associated with carriage [START_REF] Yazdankhah | Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway[END_REF]. In the current study, all of the ST-41 related isolates possessed an hmbR gene. Conversely, all of those isolates lacking the gene were among ST-44 related meningococci. Using this DNA array, no other major differences were observed between the ST-41 and ST-44 related meningococci.

Alternative cassettes for the HmbR locus were the putative lipoproteins Exl2 and Exl3 [START_REF] Kahler | exl, an exchangeable genetic island in Neisseria meningitidis[END_REF]. The function of both Exl2 and Exl3 is unknown, however they both present 69% sequence identity with the TbpB-like protein, GNA2132, as well as TbpB, LbpB and HpuAconsistent with a putative role in iron uptake or metabolism [START_REF] Kahler | exl, an exchangeable genetic island in Neisseria meningitidis[END_REF]. In agreement with Kahler et al, the genetic island, exl2, was predominantly found among meningococci with a serogroup Y polysaccharide capsule belonging to the ET-508/ST-23 clonal complex (Appendix A2). The predominance of exl2 in meningococci from the ST-23 clonal complex may be a result of clonal expansion. Clonal complexes are often associated with particular phenotypic characteristics such as serogroup or subcapsular antigens [START_REF] Urwin | Distribution of surface protein variants among hyperinvasive meningococci: implications for vaccine design[END_REF].

The exl3 genetic island was predominantly found among meningococci belonging to the lineages ST-22, ST-106, ST-549 and ST-60 (Appendix A2). However, in agreement with previous analysis, exl3 was also found among isolates in the ST-1 clonal complex which, in contrast, primarily contained disease-causing serogroup A meningococci [START_REF] Kahler | exl, an exchangeable genetic island in Neisseria meningitidis[END_REF]. The absence of the hmbR gene among serogroup A meningococci has been documented, however all of the isolates contained the hpuB gene part of the HpuAB bipartite receptor for Hb and haptohaemoglobin acquisition indicating that this was the main haemoglobin receptor for serogroup A meningococci [START_REF] Richardson | HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation[END_REF]. In the present study, all of the serogroup A lineage ST-5 (N=13) and ST-7 (N=2) meningococci contained hmbR genes, suggesting that replacement of the hmbR gene with the exl3 locus was limited to serogroup A clonal complex ST-1 meningococci.

A total of 19 clonal complex ST-41/44 and nine ST-198 isolates contained neither the hmbR gene nor either of the exl genetic islands. Instead the locus was replaced with an ORF designated exl4 sharing 97 % identity with the "pseudo hmbR" gene from the recently sequenced genome of ST-4821 isolate NMC053442 [START_REF] Peng | Characterization of ST-4821 complex, a unique Neisseria meningitidis clone[END_REF]. The "pseudo hmbR" gene belonging to isolate NMC053442 was highly divergent from other hmbR genes with BLAST searches revealing 62% identity in a 977 bp fragment of the hmbR gene. Previous studies have documented the relatively high conservation of hmbR among N. meningitidis isolates raising the question of whether the gene identified in the Chinese isolate NMC053442 may be another exl island containing a hmbR fragment [START_REF] Stojiljkovic | HmbR outer membrane receptors of pathogenic Neisseria_spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation[END_REF].

In conclusion, an epidemiological survey of the hmbR gene among both disease and carriage associated N. meningitidis isolates revealed a statistically significant association of the hmbR gene with virulent N. meningitidis isolates consistent with a role for hmbR during pathogenesis. This study highlights the power of molecular epidemiological studies in determining novel virulence determinants.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Figure 1 .

 1 Figure 1. Genetic arrangement of the hmbR locus among Neisseria A. N. gonorrhoeae FA1090 [accession number AE004969]; B. N. meningitidis FAM18 [accession number AM421808]; C. N. meningitidis MC58 [accession number AE002098]; D. N. meningitidis Z2491 [accession number AL157959]; E. N. meningitidis NMC053442 [accession number CP000381]; F. N. lactamica ST-640 and G. N. meningitidis isolate GA0929 [accession number AF319532]. Annotation was derived from the published genome data available at TIGR, the Sanger institute and data published by Kahler et al [2]. Permission for use of the N. lactamica genome data was kindly given by Julian Parkhill at the Sanger Institute and genome data is available at http://www.sanger.ac.uk/Projects/ N_lactamica//. Each ORF is represented by an arrow the direction of which indicating the theoretical direction of transcription. Thin black vertical bars indicate Correia elements, hatched vertical bars represent inverted repeats. Open horizontal blocks denote highly conserved 174 bp regions located at the 5′ end of the hmbR and exl4 ORFs. Black horizontal blocks represent 112 bp conserved regions located immediately after the hmbR or exl4 ORFs. Both conserved regions were not found in N. lactamica or N. meningitidis GA0929.

J Infect Dis. Author manuscript; available in PMC 2009 August 24.

Acknowledgments

This work was funded by the National Institute for Biological Standards and Control (NIBSC), the Wellcome Trust and the University of Manchester.


V Munoz

B Vales

M Perrin

M L Pastor

H Welemane
email: helene.welemane@enit.fr

A Cantarel
email: arthur.cantarel@iut-tarbes.fr

M Karama

Coupling infrared thermography and acoustic emission for damage study in CFRP composites

Improvement of the design and reliability of aeronautical composite structures requires an identification and characterization of their damage evolution. By providing surface thermal fields, infrared thermography has allowed considerable progress in the detection of degradation phenomena. On the other hand, acoustic emission is employed to record the transient waves resulting from released energy during a damage process. This study intends to combine simultaneously these nondestructive methods to investigate the damage behaviour of carbon-fibre composites under tensile load. Experimental results show some correlations between thermal and acoustic events induced by the load according to the damage development.

Introduction

The use of composites in the aerospace industry has increased dramatically since the 1970s. The primary benefits of composite structures are the reduction of weight and the simplification of assembly. Composite materials such as carbon fibre-reinforced composites are very used for many structural applications [START_REF] Soutis | Fibre reinforced composites in aircraft construction[END_REF]. When they are in service, different types of mechanical and thermal loads are applied to these structures, resulting delamination or disbonding for high level of solicitations. Defects such as random inclusions or undesirable material have also been found during the manufacture process of composite materials [START_REF] Goidescu | Damage investigation in CFRP composites using full-field measurement techniques: combination of digital image stereo-correlation, infrared thermography and X-ray tomography[END_REF]. These damages or defects may decrease even more the composite structures reliability lading consecutively to the introduction of high safety factors.

To optimize the design of composite structures, it is then required to identify and characterize as precisely as possible the damage evolution of these materials. The identified information will then allow predicting the behaviour of the structure by the use of adjusted models.

Nondestructive experimental characterizations show nowadays a major advantage for studying the damage process. In the aeronautic industry, there exists various nondestructive testing (NDT) used either for health monitoring of the aerostructure [START_REF] Staszewski | Health monitoring of aerospace composite structures -Active and passive approach[END_REF][START_REF] Garnier | Fatigue behavior of impacted composite structures[END_REF] or for damage mechanisms monitoring [START_REF] Naderi | Dissipated thermal energy and damage evolution of Glass/Epoxy using infrared thermography and acoustic emission[END_REF]. Some results have showed that infrared thermography and acoustic emission are qualified techniques for these studies, either for monotonic or fatigue tensile mechanical loads [START_REF] Kordatos | Rapid evaluation of the fatigue limit in composites using infraredlock-in thermography and acoustic emission[END_REF].

Infrared thermography (IT) is a non-destructive, non-intrusive, non-contact technique that provides mapping of thermal patterns (or thermograms) on the surface of objects. In the mechanical field, the measured radiation can result from two kinds of sources: an external excitation (halogen lamps, xenon flash, etc.) or from the deformation of the material itself due to the thermoelastic couplings and the dissipations. This method has been used for monitoring damages under stress in monotonic loading [START_REF] Goidescu | Damage investigation in CFRP composites using full-field measurement techniques: combination of digital image stereo-correlation, infrared thermography and X-ray tomography[END_REF] or fatigue [START_REF] Pastor | Applying infrared thermography to study the heating of 2024-T3 aluminium specimens under fatigue loading[END_REF].

Acoustic emission (AE) has been developed to acquire in situ information during a mechanical load of a structure. The energy released during a damage process is detected by piezoelectric sensors in form of transient elastic waves. About 15 waveform parameters (amplitude, energy, counts…) can be studied from the acquired waves. These information can be then used to correlate the acoustic emission activity with the damage evolution of the materials. Some works have showed that damage in composite materials such as matrix cracking, fibre cracking or interfacial debonding can be identified by the use of acoustic emission [START_REF] Loutas | Healt monitoring of carbon/carbon, woven reinforced composites. Damage assessment by using advanced signal processing techniques. Part I: Acoustic emission monitoring and damage mechanisms evolution[END_REF][START_REF] Kempf | Acoustic emission analysis for characterisation of damage mechanisms in fibre reinforced thermosetting polyurethane and epoxy[END_REF]. Some other works have showed that these both techniques (IT and AE) can be correlated for damage identification and characterization. By combining both methods, it is possible to acquire in situ information about the fatigue damage in metallic materials, ceramic or glass composites [START_REF] Naderi | Dissipated thermal energy and damage evolution of Glass/Epoxy using infrared thermography and acoustic emission[END_REF][START_REF] Kordatos | Rapid evaluation of the fatigue limit in composites using infraredlock-in thermography and acoustic emission[END_REF][START_REF] Kordatos | Monitoring mechanical damage in structural materials using complementary NDE techniques based on thermography and acoustic emission[END_REF]. Nevertheless, the coupling of surface temperature (thermograms acquired by IT) with AE parameters can be difficult in the case of monotonic load of composite materials due to low temperature variations [START_REF] Pastor | Applying infrared thermography to study the heating of 2024-T3 aluminium specimens under fatigue loading[END_REF]. The temperature itself could reveal some important dissipative phenomena [START_REF] Goidescu | Damage investigation in CFRP composites using full-field measurement techniques: combination of digital image stereo-correlation, infrared thermography and X-ray tomography[END_REF] but yet it should not be considered as a relevant indicator since it does not take into account the external exchanges with the outside for example [START_REF] Chrysochoos | Infrared thermography, a potential tool for analysing the material behaviour[END_REF]. That is why the damage analysis with IT should be studied through the heat source and not only through the surface temperature variations.

The aim of this paper is to combine infrared thermography with AE parameters for damage identification in carbon-epoxy unidirectional laminates subjected to axis and off-axis tensile loads. In the case of IT, analyses are done by determining the heat source fields from the temperature fields by taking into account the anisotropic material symmetry [START_REF] Pastor | Applying infrared thermography to study the heating of 2024-T3 aluminium specimens under fatigue loading[END_REF]. In the case of AE, Unsupervised Pattern Recognition Algorithms are used to identify the damage mechanisms.

Specimen and experimental setup

Specimen

A carbon fibre reinforced laminate is considered for this study. The composite is made of 14 unidirectional plies of prepreg Hexply M10R/38%/UD150/CHS leading to a 2 mm thickness composite with fibre volume fV of 62%. The reference of the fibre is TORAY T700S. Sample fabrication is carried out using the manual lay up technique. Then the sample is cured at 125°C during 90 minutes at a pressure of 2 bars. This sample is representative of a transversely isotropic composite. To obtain the tensile samples, the cured plate is cut according to the standard ISO 527-5 [START_REF]Plastics -Determination of tensile properties -Part 5: Test conditions for unidirectional fibre-reinforced plastic composites[END_REF]. Two composite directions are obtained after cutting the plate (0° and 90°). Figure 1 shows the axis fibre direction (direction x3) for each category of specimen.

Fig. 1. Fibre direction for each kind of specimen

The thermophysical properties are given in table 1. ρ is the density measured experimentally; C is the specific heat provided by the data software Granta CES selector; α is the coefficient of thermal expansion obtained by [START_REF] Gay | Matériaux composites[END_REF]; kLf (respectively kTf) are the heat conduction coefficient of the fibre in the longitudinal (respectively transversal) direction given by fibre manufacturer (Toray Carbon Fibers Europe); km is the heat conduction coefficient of the matrix (epoxy) indicated in the software Granta CES selector; time decrement τ is experimentally evaluated by warming the specimen and then by waiting to the return to initial temperature.

Table 1. Thermal properties of the carbon/epoxy laminated composite obtained from different references

Properties ρ C τ αL αT kLf kTf km Units [kg m -3 ] [J kg -1 °C-1 ] [s] [C -1 ] [C -1 ] [W m -1 C -1 ] [W m -1 C -1 ] [W m -1 C -1 ]

Experimental setup

The tensile tests are made at ambient temperature. These tests are performed with an electromechanical testing machine INSTRON 5500. 2 tests are carried out according to the fibre direction, 0° (axial load along the direction x3) and 90° (axial load along direction x2) (figure1). To check the reproducibility of the response, three specimens are considered for each loading direction.

The thermal acquisition is done with a FLIR Titanium SC7000 retrofitted camera. Automatic calibration, control of the camera and data recording are done by Altair software. Acquisition frequency is 150 Hz. So as to limit surrounding influences, a data processing after the acquisition phase has provided the temperature variations fields ΔT = T -T0 over the sample, with T the temperature field at time t and T0 the temperature field in the initial state t = 0. Such relative analysis avoids the most disturbing effects, including environmental ones, and highlights the thermal events induced in the material.

The acoustic emission activity is recorded during the tensile test using AEwin for SAMOS software. The data acquisition system is composed by a PCI8 board in which two channels are used. The AE monitoring is conducted using two piezoelectric sensors mounted on one side of the specimen. The sensors are attached using mechanical support while supplying the necessary acoustic coupling agent. The WD sensors are wideband with frequency band-width between 100 kHz and 1 MHz. In view of environmental noise in the laboratory, the acquisition threshold is set to 32-35 dB. The acquired signals are amplified by 40 dB. Two sensors have been positioned at the top and bottom of the specimen in order to derive the location of the AE events. The full experimental setup is shown in figure 2.

Fig. 2. Experimental setup

Heat source identification

As explained in the introduction section, the purpose of the present study is to correlate AE information with the heat source obtained by infrared thermography. Heat source st produced by the material is composed of the mechanical dissipation and of the thermoelastic coupling (temperature and strain). The heat source assessment is based upon the following assumptions: the temperature variation has no influence on the microstructure state, internal coupling sources are neglected, external heat rext does not depend on the time, coefficients k, ρ and C remain constant during the test and heat sources are assumed homogeneous through the thickness (low thickness compared to the sample's length and width). Under these assumptions, the heat diffusion equation can be written as follows (Eq. ( 1), [START_REF] Chrysochoos | Infrared thermography, a potential tool for analysing the material behaviour[END_REF]).

( ) ( ⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ ) (1)
Regarding the heat sources determination, one of the main contribution of this work is to take into account the anisotropic symmetry of the material. Existing works often deal with isotropic materials (especially metallic, [START_REF] Pastor | Applying infrared thermography to study the heating of 2024-T3 aluminium specimens under fatigue loading[END_REF][START_REF] Kaganova | On calculation of effective conductivity of inhomogeneous metals[END_REF]) in which the thermal conductivity k remains a scalar value. In our case, 2D orthotropy is considered. It means that the second order conductivity tensor exhibits two eigenvalues related to fibre and transversal directions of the material. In view of the lack of experimental data, this work relies on a homogenization approach to address such issue. In agreement with the material morphology, the Mori-Tanaka approach [START_REF] Phan | Etude expérimentale et numérique de la conductivité thermique d'un composite chaux-chanvre[END_REF][START_REF] Gruescu | Effective thermal conductivity of partially saturated porous rocks[END_REF] is used to derive the effective thermal conductivity of the material by assuming fibres as infinite length cylinders inclusions [START_REF] Do | Application des approches d'homogénéisation à l'étude des propriétés themo-hydro-mécaniques des roches[END_REF]. Expressions of thermal conductivities related to fibre direction and to the transversal direction are detailed in Eqs. ( 2) and ( 3) and table 2 gives the numerical results in the present case.

( )

(2) [ ( ) ( ) ( ) ( ) ] (3)

Table 2. Effective conductivities of the composite according to the fibre direction

Conductivities [W m -1 C -1 ] [W m -1 C -1 ]
Values 5.98 0.87

To determine the heat source, a spatial-median filter is first applied to the recorded thermal fields due to high noise in the recorded temperature. That operation takes the temperature of the pixel and replaces it by the median value of all the pixels in its spatial surrounding [START_REF] Vales | Caractérisation thermo-mécanique de l'endommagement des composites stratifiés par Thermographie Infrarouge[END_REF]. This operation is carried out for each thermal frame in the analyzed game of frames.

To solve Eq. ( 1), the classical left derivation is applied for temporal term ( ) and for the spatial conduction laplacian term ( ( ⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ )) central derivation is used. This type of derivation is commonly used in signal processing field [START_REF] Vales | Caractérisation thermo-mécanique de l'endommagement des composites stratifiés par Thermographie Infrarouge[END_REF]. Filtering on the derived terms is needless due to well reduction of noise of the thermal fields with the spatialmedian filter.

Pattern recognition method of AE

Noesis Software is used for post processing of the recorded acoustic emission signals. Pattern recognition techniques are applied by the use of k-means algorithm for acoustic emission signal classification [START_REF]Advanced Acoustic Emission Data Analysis Pattern Recognition & Neural Networks Software for Acoustic Emission Applications[END_REF]. The aim is to identify the damage mechanisms for each fibre direction. For instance, some works [START_REF] Kempf | Acoustic emission analysis for characterisation of damage mechanisms in fibre reinforced thermosetting polyurethane and epoxy[END_REF][START_REF] Kostopoulos | Fracture behavior and damage mechanisms identification of SiC/glass ceramic composites using AE monotoring[END_REF] have showed that fibre breakings, interphase failures and matrix crackings can be identified separately by this way.

K-means is a simple iterative algorithm, aims at minimizing the square error for a given number of clusters. The algorithm, starting with the initial clusters specified, assigns the remaining points to one of the predefined clusters by nearest neighbour classification. The cluster centers are updated and the process continues until none of the patterns changes class membership [START_REF]Advanced Acoustic Emission Data Analysis Pattern Recognition & Neural Networks Software for Acoustic Emission Applications[END_REF].

The classification procedure is investigated with several different features and number of classes. The features taken into consideration are chosen according to their degree of correlation to each other and can be different for each loading direction (that is for each damage process involved).

Results

For 0° specimens, AE analysis is based on rise time, energy, duration and amplitude. Those four parameters are correlated to identify 3 classes, each class corresponding to one specific damage mechanism.

For 90° specimens, rise time, duration and amplitude are the features chosen to converge into 2 classes. Fibre breaking is not a damage mechanism for 90° specimens.

With the purpose of correlating acoustic emission to heat sources, two assumptions are taken into account. The liberation of energy due to a specific damage phenomenon produces an acoustic emission and a heat source at the same time. The second assumption is that the acoustic emission with the most high amplitude and energy is related to a high heat damage source.

Correlation results for 0° tensile test

Figure 3 shows on the amplitude versus time graph the classes of AE data obtained after the application of the pattern recognition method. Three groups are clearly distinguished. The red group with the lowers amplitudes corresponds to the matrix cracking, the green group refers to the debonding and interphase failure and the last blue group corresponds to the fibre breaking. We can see that the fibre breaking generates AE at high amplitudes in regards to the others damage mechanisms.

Fig. 3. Cluster results of acoustic emission for 0° tensile test

Correlations between AE and TI measurements have been done between several heat sources and acoustic events of high amplitude and high energy. As an illustration of the analysis, the procedure and results are explained here on the case of the one specific AE event selected on figure 3. Figure 4 shows the thermogram (figure 4a), the heat source (figure 4b) and the located amplitude (figure 4c) for this damage that appeared at 128.428 seconds after the beginning of the tensile test. According to the figure 3, this damage (at this specific time) corresponds to a fibre breaking. We can see that the amplitude of the analyzed acoustic event is around 79 dB (figure 4c). To correlate that specific AE to the heat source, it is needed to analyse the maximum of heat source at a specific timespan. We have considered an interval of 1 s around the AE event (128.428 0.5 s) which includes 150 thermal frames. During this timespan the maximal heat source is obtained at 128.407 s. (error of 0.02%) for the pixel highlighted in figure 4b. Figure 5 shows that for such analyzed pixel, the heat source gets indeed its maximal value at the AE event time.

Fig. 4. Correlation between heat source and acoustic emission amplitude for the 0° specimen

Figure 4c provides the location of AE events during the test. Events take place all along the specimen length, which stands in agreement with the post-mortem observation shown in figure 4d and its heat source (breaking time) (figure 4e). We can clearly see that the final breaking of 0° specimens results from macroscopic debonding in the fibre direction.

Regarding the specific event highlighted in figures 3 and 4b, we can see that the location of the heat source and acoustic event stand in the same zone of the specimen (lower part) near the sensor number 2 (indicated by the green number 2 in figure 4c). The error of the AE location in regards to the heat source is 25%. However, it is important to note here that the location in AE is done by measuring the velocity of the wave for each specimen in the initial state. The slight difference in location may thus be explained by velocity change induced by the damage and load applied.

Correlation results for 90° tensile test

The same analyzes were carried out for 90° specimens. In this case, not the amplitude but the acoustic energy is taken in consideration for the analysis.

Figure 6 shows the classes of AE for the 90° specimen. As explained before, only two groups are needed according to the damage mechanisms for this fibre direction: the red group represents the matrix cracking and the green group refers to debonding. The AE corresponding to the matrix cracking have the lowest energy and amplitude. In this case the analyzed AE corresponds to a debonding (selected AE event in figure 6). This allows observing that debonding or delamination also create heat sources as important as the fibre breaking.

Fig. 6. Cluster results of acoustic emission for 90° tensile test

In the same way, a specific timespan is selected for the analysis. Figure 7 shows the thermogram (figure 7a), the heat source (figure 7b) and the located acoustic energy (figure 7c) for the selected damage at 42.220 seconds after beginning the tensile test. As explained above and according to figure 6, this damage (at this specific time) corresponds to an internal debonding. The acoustic energy for this specific damage is 38 aJ (figure 7c) which is high in regards to the others AE. For the analysis we have considered an interval of 1 s (42.220 0.5 s) which leads to 148 thermal frames to be analyzed. During this timespan the maximal heat source is founded at 42.216 s. (error of 0.01%) (Figure 7b). Again, figure 8 shows the correspondence between AE event and maximal value of the heat source for the analyzed pixel. Some high heat sources can be seen some milliseconds before and after the analyzed instant but should be considered as parasite events generated by some thermal noise since they do not correspond to any AE.

Fig. 7. Correlation between heat source and acoustic emission energy for the 90° specimen

In this case, the location of the heat source and the acoustic event are also in the same zone of the sample (near sensor number 1) with an error of 21%. In addition to the changes in velocity, such difference may be related to much more brittle character, and therefore instable of the failure in that case. Indeed, it is shown in figures 7d and 7e the final failure breakdown occurs in the fibre axis, induced by the failure of the weakest point of the epoxy matrix (the breakdown is transversal to the load axis). The full load of the tensile test is supported by the matrix (epoxy) and so the break takes place transversally to the load axis. Fig. 8. Heat source variation 0.5 s before and after the studied instant for the analyzed pixel (90° specimen)

Conclusions

In this study, combined analysis of carbon-epoxy composite subjected to axis and off-axis tensile tests is done by means of acoustic emission and infrared thermography measurements. Heat sources are obtained from the processing of the thermal fields and by using homogenization technique (Mori-Tanaka approach) to derive the effective orthotropy conductivity tensor. Acoustic emission technique is also used as a damage detection technique to identify and locate the damage events during the load.

By the use of Unsupervised Pattern Recognition Algorithms, three damage mechanisms are found in specimens at 0° according to the load axis matrix cracking, debonding and fibre breaking). In the case of 90° specimens, two damage mechanisms are found (matrix cracking and debonding).

Disbonding and fibre breaking are damage mechanisms that produce heat sources. It is found that acoustic emissions with high amplitude and high energy can be well correlated to high heat sources produced by these damage mechanisms evolutions. The accuracy of detection in the time is very optimal, while accuracy regarding the location is not precise and could be attributed to velocity change during the test.

Further works need now to be conducted to decompose the total heat source between the mechanical dissipation and the thermoelastic coupling. Also, this combined measurement approach should be put in place for the analysis of damage process of composite under dynamic load (fatigue) to confirm the correlation between acoustic and thermal events.

Fig. 5 .

 5 Fig. 5. Heat source variation 0.5 s before and after the studied instant for the analyzed pixel (0° specimen)