Laura Bergamaschi

Maria Ban

Nadia Barizzone

Maurizio Leone

Maria Edvige Fasano

Franca R Guerini

Lucia Corrado

Paola Naldi

Ennia Dametto

Cristina Agliardi

Daniela Ferrante

Marco Salvetti

Rosella Mechelli

Daniela Galimberti

Elio Scarpini

Paola Cavalla

Valeria Bargiggia

Domenico Caputo

Susanna Cordera

Francesco Monaco

Patricia Momigliano-Richiardi

Sandra D ' Alfonso
email: dalfonso@med.unipmn.it

Association of HLA class I markers with Multiple Sclerosis in the Italian and UK population: evidence of two independent protective effects

Keywords: multiple sclerosis, HLA class I markers, haplotypes, genetic association

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

INTRODUCTION

Multiple Sclerosis (MS) is a complex disease characterized by inflammatory foci in the central nervous system (CNS) causing a progressive demyelination of axons and neuronal cell degeneration, resulting in a severe disabling condition.

The pathogenesis of MS is not well understood, but it is well established that both genetic and environmental factors contribute together to the development of the disease.

Association with the human leukocyte antigen (HLA) genes from the major histocompatibility complex on chromosome 6p21.3 was identified almost 40 years ago. It is well established that the HLA-DRB1*1501 allele, tagging the DRB1*1501-DQB1*0602 (also known as DR15) haplotype in the HLA class II region, is the strongest genetic risk factor conferring an OR of approximately 3 in most studied populations. [START_REF] Ballerini | HLA-multiple sclerosis association in continental Italy and correlation with disease prevalence in Europe[END_REF][START_REF] Fogdell | The multiple sclerosis-and narcolepsy-associated HLA class II haplotype includes the DRB5*0101 allele[END_REF][START_REF] Olerup | HLA class II-associated genetic susceptibility in multiple sclerosis: a critical evaluation[END_REF][START_REF] Barcellos | Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis[END_REF] Through careful analysis of this complex region, which is characterized by an extreme polymorphism and extensive linkage disequilibrium, evidence for an independent effect on risk from the class I region has emerged. In 2007, IMSGC presented data from a large study based on UK and North American Caucasians suggesting that this signal might be determined by the HLA Cw*05 allele. The HLA Cw*05 allele was less frequent in patients than controls conferring a protective effect. [START_REF]A second major histocompatibility complex susceptibility locus for multiple sclerosis[END_REF] At the same time, two other groups identified an association between the HLA A*02 allele and MS with a similar OR in two populations (Swedish and Tasmanians) with a different genetic background. This allele, like HLA Cw*05, conferred a protective effect. [START_REF] Brynedal | HLA-A confers an HLA-DRB1 independent influence on the risk of multiple sclerosis[END_REF][START_REF] Burfoot | SNP mapping and candidate gene sequencing in the class I region of the HLA complex: searching for multiple sclerosis susceptibility genes in Tasmanians[END_REF] We recently tested the association of both HLA A*02 and HLA Cw*05 in an Italian cohort of 1273 MS patients and 1075 controls. [START_REF] Bergamaschi | HLA-class I markers and multiple sclerosis susceptibility in the Italian population[END_REF] Our analyses confirmed the protective effect of HLA A*02 whereas HLA Cw*05 did not present any independent association. However, the presence of HLA Cw*05 appeared to significantly enhance the protective effect due to HLA A*02 resulting in a 3 fold decreased OR for the HLA A*02-Cw*05 combination (OR = 0.22, 95% CI: 0.13-0.38) relative to the presence of HLA A*02 alone (OR = 0.69, 95% CI: 0.58-0.83). In the Caucasian population, the HLA A*02-Cw*05 combination is carried by two HLA extended haplotypes, namely HLA A*0201-Cw*0501-B*4402-DRB1*0401 and HLA A*0201-Cw*0501-B*1801-DRB1*1102 (also known as 44.1 and 18.3 haplotypes, respectively, [START_REF] Cattley | Further characterization of MHC haplotypes demonstrates conservation telomeric of HLA-A: update of the 4AOH and 10IHW cell panels[END_REF][START_REF] Alper | The haplotype structure of the human major histocompatibility complex[END_REF][START_REF] Dorak | Conserved extended haplotypes of the major histocompatibility complex: further characterization[END_REF]). These two haplotypes are present in the Italian population with approximately the same frequency (Rendine, unpublished). Thus it is possible that the strong protective effect we detected for the A*02-Cw*05 combination is due to a haplotypic effect, i.e. to the presence of a primarily associated factor carried by one or both the above haplotypes. Interestingly HLA B*4402 was recently identified as an MS protective factor. [START_REF] International | Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases[END_REF][START_REF] Jager | Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci[END_REF][START_REF] Healy | HLA B*44: protective effects in MS susceptibility and MRI outcome measures[END_REF] In order to better understand whether the enhanced protective effect of the HLA A*02-Cw*05 combination is due to a haplotypic effect or a direct interactive role of the two markers as well as test the possible role of B*44, in the present study we extended the analysis in our Italian cohort to the involved HLA-B alleles, namely HLA B*44 (and its subtypes B*4402 and B*4403) and B*18.

The association with MS of the various haplotypic combinations of the A*02-Cw*05 set with or without B*44 or B*18 was also analysed in a UK cohort fully typed for HLA-DR, -A,-B and -C, in order to verify our results in another population with a possibly different genetic background. Italian controls included medical students, university and hospital staff, blood donors (female:male ratio 1:1.1) matched for age and regional origin with the MS patients.

MATERIALS AND METHODS

Subjects

Patients (83%) and controls (91%) mostly overlap with those included in our previous paper [START_REF] Bergamaschi | HLA-class I markers and multiple sclerosis susceptibility in the Italian population[END_REF] and were already typed for HLA-A*02, Cw*05 and DRB1*15. Selection for inclusion was based on DNA availability.

We also analysed a UK data set composed of 721 MS patients, 408 healthy matched controls and 480 family trios. These samples were described in detail elsewhere. [START_REF]A second major histocompatibility complex susceptibility locus for multiple sclerosis[END_REF] Patients were diagnosed according to McDonald et al. criteria. [START_REF] Mcdonald | Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis[END_REF] All the samples, both Italian and British, were of European ancestry.

Features of the analyzed samples are shown in Table 1. All the samples were collected after informed consent and appropriate ethical approvals.

HLA typing of Italian samples

HLA typing was performed using specific reactions in order to obtain information about the four The methods used to analyze HLA-A*02, HLA-Cw*05 and DRB*15 were described in detail elsewhere. [START_REF] Bergamaschi | HLA-class I markers and multiple sclerosis susceptibility in the Italian population[END_REF] conditions of the 12 th International Histocompatibility Workshop. [START_REF] Tonks | Genetic Diversity of HLA: Functional and Medical Implications[END_REF] All these protocols, with the exception of A*02, did not allow to distinguish between homozygotes and heterozygotes but only whether the samples were positive or negative for the tested markers.

These methods were validated testing 45 samples previously typed with standard protocols. The results were consistent for all tested samples.

HLA class I and class II typing of UK samples

All the UK samples had been previously completely typed for the HLA-A, -C, -B and -DRB1 loci as described in detail elsewhere. [START_REF]A second major histocompatibility complex susceptibility locus for multiple sclerosis[END_REF] The trio families were genotyped with a medium resolution method for all HLA class I (two-digit)

and class II (four-digit) alleles. In particular for the HLA-B locus, all the family members were

Statistical analysis

The association of each marker, haplotype or phenotype combination with the disease was measured by χ 2 test, the Odds Ratio (OR) and its 95% Confidence Intervals (95% CI). Allele and haplotype analyses of UK samples were performed with the UNPHASED v 3.0.13 program using the "certain haplotype only" option for family trios and the "uncertain haplotypes" option for the case/control cohort. [START_REF] Dudbridge | Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data[END_REF] Linkage disequilibria (LD) were calculated from phenotypes according to Mattiuz et al. [START_REF] Mattiuz | New approaches to the population genetics and segregation analysis of the HLA system[END_REF].

Logistic regression was carried out to determine the effect of the considered markers on MS susceptibility. The association of each polymorphism with the disease was measured by the Odds Ratio (OR) and its 95% Confidence Intervals (95% CI). We used likelihood ratio tests (LRT) to assess the statistical significance of the models and to compare the models. The contribution of each variable was evaluated by comparing the LRTs derived from a model with and without the variable.

Reported p values were not corrected for the number of comparisons. 2). The complete results including all the other possible combinations of HLA-A, -B, -C tested alleles are shown in the Supplementary Table 1.

RESULTS

Case/Control association analysis in the Italian

A*02-Cw*05 conferred a strong protection particularly in the presence of B*44 (OR = 0.28; p = 4.1x10 -6 ) but also of B*18 (OR = 0.41; p = 0.004). Conversely, in their absence, the protection conferred by A*02-Cw*05 was weaker and not statistically significant, although, owing to the low frequency of this combination, this analysis had only limited power (α = 0.05; β = 0.21) and cannot be considered informative. The OR values for the different phenotypic combinations in the whole population and in DR15 negative individuals were substantially the same (Table 2).

Association analysis in UK family trios

To 2. A comparison of the results of the various tested populations and data sets is summarized in Table 5. HLA-B*18 was not included because in the British population some of its combinations with A*02 and Cw*05 were very rare and none was significantly associated to MS (Table 3 and Supplementary Tables 2,3, 4) and in both populations the logistic regression analysis ruled out a protective role of this marker (Table 4).

Dissection of the protective effect of the

The main conclusions that can be drawn from Table 5 are the following:

-The A*02-Cw*05-B*44/12 combination conferred a significant protection in both populations.

Actually, this was the only allele combination consistently associated in both populations and in all the tested panels.

-A significant protection was also observed for the Cw*05-B*12 combination in the absence of A*02, but only in the UK case/control (OR = 0.31, 95% CI: 0.10-0.81, p = 0.0324, Supplementary

Table 3) and combined haplotype (OR = 0.37, p = 0.0087) panels.

- studies. [START_REF] International | Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases[END_REF][START_REF] Jager | Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci[END_REF][START_REF] Healy | HLA B*44: protective effects in MS susceptibility and MRI outcome measures[END_REF] Several interpretations of these results were possible: i) the stronger protection we detected for A*02 -Cw*05 was secondary to linkage disequilibrium with B*4402; ii) a primarily involved protective factor is carried by the 44.1 haplotype; iii) the presence of an epistatic interaction (i.e. an interaction between 2 alleles (in cis or trans) in which the risk associated with a particular allele depends on the presence or absence of a second allele, [START_REF] Lincoln | Epistasis among HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci determines multiple sclerosis susceptibility[END_REF]) between A*02 and Cw*05 and/or B*4402.

To answer to these questions we evaluated the association of the above involved HLA markers in The typing strategy utilized for the Italian sample did not distinguish homozygotes from heterozygotes, making it impossible to deduce the haplotypic combinations of the tested alleles.

Therefore the detected associations were with phenotypic rather than haplotypic allele combinations and it was not possible to determine whether the involved alleles were carried in cis or in trans. The second population compensated for this limitation, since in the UK samples it was possible to determine the allele phase, either by family segregation (trios) or by computer algorithms (casecontrol). The protective effect of Cw*05 evident in the UK population was not replicated in the Italian sample and was not distinguishable from that of B*44 because the LD between these two markers is particularly high in the UK population (Figure 1). While our results clearly show that the A*02-Cw*05 combination maintains a protective effect independently of the tested B alleles, we were not able to elucidate whether this protective effect was due to the presence of a non analyzed factor characterizing the HLA extended haplotype/s carrying A*02 and Cw*05 (haplotypic effect) or to a direct interaction between the two alleles. There is a preliminary indication in favour of the "haplotypic effect" hypothesis derived from the observation that the Cw*05-B*44 haplotype not carrying A*02 still maintains a significant protective effect. This result would be against the hypothesis of an A*02/Cw*05 interaction and indicates the presence of a protective factor in the region of the haplotype closer to HLA-B and HLA-C loci. However, this information mainly comes from the UK case/control data set where the haplotypic combinations are not certain (ascertained through family segregation) but are deduced by an algorithm.

Association analysis in the

Further studies are needed to definitively demonstrate which of the two hypotheses is correct. A direct cooperation between A*02 and Cw*05 molecules in MS protection remains an attractive hypothesis since they present antigenic peptides and interact with NK receptors. [START_REF] Moretta | Killer immunoglobulin-like receptors[END_REF][START_REF] Thananchai | Cutting Edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B[END_REF] ). However, it is likely that its highly protective effect is genuine, and not a spurious signal due to its low frequency or to stratification, since it was replicated in two independent samples belonging to two populations with a different genetic background.

The identification of the primarily associated factor/s requires an extended analysis of the involved haplotype/s in populations with different genetic background. This will help to elucidate the mechanism mediating the protective effect and hence to shed new light on MS pathogenesis.

FIGURE LEGEND

Map of the HLA alleles analysed in this study and their linkage disequilibrium relationship. Pairwise D', r 2 and p values were calculated according to Mattiuz et al. [START_REF] Mattiuz | New approaches to the population genetics and segregation analysis of the HLA system[END_REF]

A

  total of 1445 Italian MS patients and 973 controls were analyzed for HLA-A*02, -Cw*05, -B*18 and -B*44 class I markers and for DRB1*15. Of these, 1433 patients and 950 controls were also typed for B*44 subtypes (4402 and 4403). The majority of the samples were recruited from North-West Italy and the remaining from Central Italy. Patients with Sardinian ancestors were excluded to avoid the introduction of a confounding source of heterogeneity.

  HLA class I markers (HLA-A*02, -Cw*05, -B*44 and -B*18) and DRB1*15.

  typed for both HLA-B*12 subtypes, namely B*44 and B*45. The case/control samples were analyzed with a low resolution method for all class I and class II alleles. This method did not allow discrimination between the two HLA-B*12 subtypes. However, since B*44 is by far the most frequent B*12 subtype in the British population (0.185 versus 0.006 in the non transmitted haplotypes of the 480 family trios) and is in strong linkage disequilibrium with Cw*05 (D'=0.85, r 2 =0.66, Fig.1), it is likely that most B*12 positive and practically all B*12-Cw*05 positive individuals were B*44 positive.

  population. A total of 1445 MS patients and 973 controls, previously typed for HLA-DRB1*15, A*02 and Cw*05, were further typed for B*18 and B*44. A schematic map of the HLA genes analysed in this study is shown in Figure 1 together with the pairwise linkage disequilibria (LD) between the tested alleles. The association with MS of the different phenotypic combinations carrying both HLA-A*02 and HLA-Cw*05 (A*02-Cw*05) with or without B*18 and B*44 was then analysed (Table

  Figure 1. The large majority of haplotypes carrying both A*02 and Cw*05 were B*44 positive and conferred a strong protection (OR = 0.36; p = 5.5x10 -4 ), comparable to that seen in the Italian population. The association with the A*02-Cw*05-B*44 haplotype remained significant (p=0.0057) after conditioning on the DRB1 locus by the UNPHASED program, main effect option. The A*02-Cw*05-B*18 and A*02-Cw*05-B*44/B*18neg haplotypes were very rare in the UK population and would require testing of a much larger panel of families to yield informative results. The complete results including all the other possible combinations of HLA-A*02, -B*44, -B*18 and -Cw*05 alleles are shown in the Supplementary Table2.

  A*02-Cw*05 haplotypes To dissect the effect of the different components of the A*02-Cw*05 protective combinations both in the Italian and UK samples we a) performed a logistic regression analysis and b) tested the association of all the possible combinations of the A*02, Cw*05 and B* alleles. The same procedure was applied to the analysis both of Italian phenotypes and UK haplotypes. a) For the logistic regression analysis (

  To elucidate whether the effect of A*02-Cw*05 was distinct from that of A*02 alone, we analysed the odds ratio of the A*02+Cw*05+ vs the A*02+Cw*05-combination. The combined presence of A*02 and Cw*05 was significantly more protective than A*02 without Cw*05 in both populations (Italy: OR = 0.49, 95% CI = 0.34-0.73, p=0.0004; UK: OR = 0.69, 95% CI = 0.50-0.95, p = 0.023), after adjustment for DRB*1501. When adjusting also for B*44/12, the protection contributed by the A*02+Cw*05+ combination remained significantly higher than that of A*02 without Cw*05 in the Italian population (OR = 0.55, 95% CI = 0.37-0.82, p = 0.003) while it was no longer significant in UK (p = 0.3). The above results indicate the presence of three distinct protective effects, namely A*02-Cw*05, A*02 and B*44 with a decreasing protective strength While this is evident in Italians, in the UK population only two effects are clearly detectable since it is not possible to distinguish the effect of B*12 from that of A*02-Cw*05. This is likely explained by the fact that most (>90%) A*02-Cw*05 haplotypes carry B*44 in the British as against about 50% in the Italian population.b) In order to better understand the effect of the different allele combinations we tested their association in DR15 negative Italian (1005 MS cases and 848 controls) and UK (261 MS patients, 293 controls and 131 DR15 negative families) samples. The complete results including all the possible combinations of HLA-A*02, -B*44, -B*18 and -Cw*05 alleles of the Italian and British data sets are shown in the Supplementary Tables1, 2, 3 and 4.

  The A*02-Cw*05 combination in the absence of B*44 was significantly protective only in the Italian population (OR = 0.36, p = 0.007). This haplotypic combination was practically absent (<1%) in the British population (supplementary Tables 2, 3, 4). -In both populations A*02 also conferred a significant protection in the absence of Cw*05 and B*44. The OR conferred by A*02 was significantly different from that of the A*02-Cw*05-B*44 combination in the DR15 negative Italian dataset (p= 0.002) and in the UK family trios (p= 0.007) suggesting the presence of two distinct protective effects, while this difference was not statistically different in the UK case-control cohort. -B*44 in the absence of A*02-Cw*05 consistently showed OR values <1 although, at difference from the logistic regression results, the lower number of included samples (only those DR15 negative) did not allow to reach statistical significance. In the Italian samples it was possible to test the association with MS also of the two B*44 subtypes (B*4402 and B*4403), (Supplementary

  two genetically different populations. Specifically, in the UK population the A*02-Cw*05 positive combination is carried mostly by the A*02-Cw*05-B*44 haplotype (91% of A*02-Cw*05 control haplotypes) while, the Italian A*02-Cw*05 positive individuals frequently carry this combination also with B*18 (41%) or in the absence of both B markers (8%).

  Italian population showed a very strong protective effect of the A*02-Cw*05-B*44 phenotypic combination, independently of DRB1*15. The protective effect of the A*02-Cw*05-B*44 haplotype was replicated in British family trios and shown to be independent of DRB1 locus. In this dataset the A*02-Cw*05-B*44 haplotype conferred a protection similar to the effect observed for the phenotypic allele combination in Italy. In both populations the protection was significantly higher than that of A*02 alone, i.e. in the absence of Cw*05 and B44 (p = 0.002 and 0.007 respectively in Italy and UK trios), indicating two distinct protective effects. The logistic regression analysis showed that the A*02-Cw*05 combination maintained a significant protective effect in both populations when adjusting for the B locus indicating that this effect was not secondary to linkage disequilibrium with B*44. Moreover, in the Italian population A*02-Cw*05 maintained a significant protective effect also in the absence of B*44, suggesting that this effect was not restricted to the A*02-Cw*05-B*44 combination. This result cannot be replicated in the UK sample though, since the A*02-Cw*05 combination without B*44 is virtually absent in this population. Finally, A*02-Cw*05 was significantly more protective than A*02 without Cw*05 in both populations.In both populations B*44 showed significant protection independent of the A*02 effect, paralleling the result recently published in a US sample. However, B*44 maintained only a borderline effect relative to A*02-Cw*05, indicating that most of its protection is mediated by the haplotype. The recent studies[START_REF] International | Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases[END_REF][START_REF] Jager | Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci[END_REF][START_REF] Healy | HLA B*44: protective effects in MS susceptibility and MRI outcome measures[END_REF] reporting the protection of B*44 did not evaluate the effect of the A*02-Cw*05 haplotype. Since two of the above studies[START_REF] International | Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases[END_REF][START_REF] Jager | Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci[END_REF] included a large overlap of individuals from UK where the majority of A*02-Cw*05 haplotypes also carry the B*44 allele, it is possible that their data indicate the same effect observed in the present paper. These results highlight the importance of testing the association of allele combinations in addition to single alleles within the HLA region as well as highlight the usefulness of testing populations with different haplotype frequencies to dissect the associated alleles.

e 5x10 - 7 a

 7 Presence of the indicated alleles b Frequency of individuals carrying the indicated alleles c Calculated from 2x2 tables versus all the other possible allele combinations d HLA-B was considered as a triallelic locus including HLA-B*44, HLA-B*18 or none of them. The A*02 -Cw*05 individuals positive for both B*44 and B*18 were counted twice (cases N=2, controls N=6 in the total samples; cases N=1, controls N=6 in DR15 negative samples). e Reference for OR calculation.

Table 4 )

 4 all Italian individuals and UK haplotypes were categorized according to the presence or absence of each of the considered alleles (A*02, Cw*05, B*18, B*44) and also of the A*02-Cw*05 combination. DRB1*15 was always included in the model. To increase the power of the UK study, haplotype frequencies deduced from an additional case-control dataset of the UK population were combined with those of family trios, with a total of 2106 case and 1676 control haplotypes (see note to Table 4) fully typed for HLA-DR, -A, -B and -In the Italian sample, the significant protection conferred by A*02 (OR=0.63; CI95% 0.53-0.74) and by the A*02-Cw*05 combination (OR=0.39; CI95% 0.27-0.57) remained similar after accounting for B*44 and B*18. In addition we observed a significant protective effect for B*44 (OR=0.70; CI95% 0.56-0.88) that was not substantially modified by adjustment for the other markers included in the model. No significant effect was detected for B*18 and for Cw*05. The contribution to the model of A*02 and of A*02-Cw*05 relative to B*44, as calculated by the Likelihood Ratio Test

	(LRT), was highly significant (p=1.3x10 -7 and p=7.3x10 -6 respectively). As for B*44, its
	contribution was still significant but weaker relative to the effect of A*02 (p=0.005) and borderline
	relative to A*02-Cw*05 (p=0.050).
	In the UK sample we replicated the protective effect of A*02 (OR=0.72; CI95%0.62-0.83) and of
	A*02-Cw*05 (OR=0.56; CI95% 0.42-0.76) which remained similar after accounting for the other

C. This second UK database was typed for B*12 instead of B*12 subtypes, B*44 and B*45 (see Materials and Methods).

markers. A*02 showed a significant contribution to the model relative to Cw*05 (p=0.0003) and B*12 (p=0.00013), while, at difference from Italians, the A*02-Cw*05 combination yielded only a modest (p=0.0272) contribution relative to B*12. B*12, in its turn, showed a significant protection (OR=0.70; CI95% 0.58-0.85) which was not modified by adjustment for A*02 but whose contribution was no longer significant after accounting for Cw*05 (p=0.06). The contribution of B*12 to MS protection maintained a borderline effect relative to A*02-Cw*05 (p=0.047), similarly to what seen in the Italian sample. The protection of Cw*05 was statistically significant, at difference from the Italians, but it was no longer significant after adjustment for B*12 (p=0.06) or both B*12 and A*02 (p=0.21). No protective effect was observed for B*18.

Table 1 )

 1 . The two subtypes were differently distributed according to the presence or absence of Cw*05: about 86% of Cw*05 positive as against about 35% of Cw*05 negative individuals were . On the contrary, in the Italian population it behaved as a strong risk factor, confirming our

	previous results.[8]
	DISCUSSION
	This work stems from our previous study which found that the combination of two HLA class I
	alleles, HLA-A*02 and -Cw*05, conferred a significantly stronger protective effect towards MS than
	A*02 alone.[8] The A*02-Cw*05 combination is carried by two HLA extended haplotypes, namely
	44.1 ([HLA A*0201-Cw*0501-B*4402-DRB1*0401] and 18.3 [HLA A*0201-Cw*0501-B*1801-
	DRB1*1102], respectively. Our result was particularly intriguing since a strong protective
	association with the B*4402 allele characterizing the 44.1 haplotype was recently reported in three

B*4402, similarly in patients and in controls. In the absence of A*02-Cw*05 neither of the two B*44 subtypes was significantly associated with MS (Supplementary Table

1

). sets

  In conclusion, this study contributes to the demonstration that the HLA class I region contains additional factors modulating MS susceptibility, independently of the well established DRB1 locus. At least two independent protective effects are proposed which are tagged by the A*02-Cw*05 combination and the A*02 allele, respectively. The protective effect of the A*02-Cw*05-B*44 haplotype has a strength comparable to that of DRB1*15, which is the strongest MS risk factor, thus behaving as an important modulator of MS susceptibility. The A*02-Cw*05-B44 combination is relatively rare in both tested populations (4.6% and 15.0% of DR15 negative Italian and British controls respectively

	. Evidence
	in other autoimmune or infective diseases indicate that molecules encoded by different HLA loci
	might cooperate in the disease mechanism.[19, 22, 23] A direct functional effect of A*02 is
	suggested by recent results showing that A*02 prevents an MS-like disease in transgenic humanized
	mice.[24]

Table 1 :

 1 in the Italian (IT) and UK controls. Significant values are in boldface. Regarding B*44/B12 , results are referred to B*44 for the Italians and B*12 for the UK . Characteristics of the patients

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a of whom 480 with both parents RR: Relapsing Remitting; SP: Secondary Progressive; PP: Primary Progressive; PR: Progressive Relapsing.

Table 2 :

 2 Combinations of the "HLA-A*02-HLA-Cw*05" phenotype with HLA-B in Italian MS patients and controls

	HLA phenotype	a	Sample	Frequency b	OR (95%CI) P value c
	A*02 -Cw*05	B*	d	MS patients Controls
			total	N = 1445 N = 973
	+	-		0.008	0.013 0.59 (0.24-1.41) 0.317
	+	44	0.013	0.044 0.28 (0.15-0.50) 4.1x10 -6
	+	18	0.014	0.032 0.41 (0.22-0.75) 0.0040
	all other combinations		0.966	0.917	1	e	2x10 -7
			DR15 negative N = 1005 N = 848
	+	-		0.007	0.012 0.56 (0.19-1.59) 0.400
	+	44	0.013	0.046 0.27 (0.13-0.52) 3.3x10 -5
	+	18	0.013	0.037 0.33 (0.16-0.67) 0.0015
	all other combinations		0.968	0.913	1

Table 3 :

 3 Combinations of the "HLA-A*02-HLA-Cw*05" haplotype with HLA-B in UK family trios Haplotypes were calculated with the UNPHASED v 3.0.13 program using the "certain haplotype only" option.[START_REF] Dudbridge | Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data[END_REF] All typing information, including DR, were utilised to deduce haplotypes.

	HLA Haplotype a	Frequency
			Transmitted	Non transmitted	OR (95% CI)	p b
	A*02 -Cw*05	B* c	N = 531 d	N = 720 d
	+	-	0.0018	0.0041	0.43 (0.01-5.40)	0.641
	+	44	0.028	0.075	0.36 (0.19-0.67)	5.5x10 -4
	+	18	0.0094	0.0028	3.24 (0.53-34.12)	0.142
	all other combinations	0.960	0.918	1 e	0.0036

a b Calculated from 2x2 tables versus all the other possible allele combinations. c HLA-B was considered as a triallelic locus including HLA-B*44, HLA-B*18 or none of them. d HLA-DRB1*15 negative haplotypes from 480 families. e Reference for OR calculation.

ACKNOWLEDGEMENTS

This work was supported by: the Italian Foundation for Multiple Sclerosis (FISM grants 2001/R/44, 2002/R/40 and 2005/R/10, 2008/R/11); CARIPLO Foundation, Regione Piemonte Ricerca Sanitaria Finalizzata (grants 2003 2004, 2007, 2008, 2009), Regione Piemonte CIPE 2004, Italian MIUR Ministry (PRIN 2008), Italian Ministry of Health (ricerca finalizzata grant 2004.80, "Giovani Ricercatori 2008", Ricerca Corrente 2008-2009), Eastern Piedmont University, Compagnia di San Paolo (Turin), Fondazione CRT (Turin), Cambridge NIHR Biomedical Research Centre. NB and LC were supported by a fellowship from FISM (2003/B/2, 2009/B/1). L.B. was supported by a PhD Lagrange Fellowship.

We are grateful to the patients and their parents.

Italian individuals and UK haplotypes were categorized according to the presence or absence of the indicated alleles Each marker in row is adjusted for markers in columns p-values for adjusted models are obtained from Likelihood Ratio Test (LRT) comparing the likelihood of the two-gene additive vs the single marker model considering as single marker the marker used for adjustment. NA = not applicable a Results are referred to B*44 for the Italian individuals and B*12 for the UK haplotypes b values adjusted for DRB1*15 plus the indicated additional two class I markers c subjects positive for both A*02 and Cw*05 (A2+ and Cw5+). d 1445 MS patients and 973 controls carrying the indicated alleles. e since the two markers are allelic, they were considered as a single variable. Italian individuals and UK haplotypes were categorized according to the presence or absence of any of them. f 2106 cases (860 transmitted haplotypes in family trios +1246 haplotypes from MS patients) and 1676 controls (860 non-transmitted haplotypes in family trios + 816 haplotypes from controls). Haplotypes were calculated from individuals, fully typed for HLA-A, -Cw, -B and -DRB1 loci, with the UNPHASED v 3.0.13 program using the "certain haplotype only" option in family trios and the "uncertain haplotypes" option in the case/control cohort. [START_REF] Dudbridge | Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data[END_REF] All typing informations, including DR, were utilised to deduce haplotypes.

NA = not applicable


MD Iacopo Sardi
email: i.sardi@meyer.it

Giancarlo Marca

Maria Grazia Giovannini

Sabrina Malvagia

Renzo Guerrini

Lorenzo Genitori

Maura Massimino

Maurizio Aricò

Giancarlo La Marca

Sardi

Detection of doxorubicin hydrochloride accumulation in the rat brain after morphine treatment by mass spectrometry

Keywords: blood-brain barrier, doxorubicin, morphine, rodent model, mass spectrometry

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.

Introduction

Despite recent advances in the cure of childhood cancer, brain tumor still lags behind other cancer types [START_REF] Bondy | Brain tumor epidemiology: consensus from the Brain Tumor Epidemiology Consortium[END_REF][START_REF] Buckner | Central nervous system tumors[END_REF]. Although the reason for treatment failure may depend on several issues, inadequate efficacy of chemotherapy probably plays a major role, compared to what has been achieved in other cancer types.

Based on in vivo and in vitro studies, brain tumors might be expected to be sensitive to chemotherapy agents, as anthracyclines [START_REF] Neuwelt | Pharmacology and toxicity of intracarotid adriamycin administration following osmotic blood-brain barrier modification[END_REF][START_REF] Wolff | Chemosensitivity of glioma cells in vitro: a meta analysis[END_REF]. Yet, most patients fail to achieve adequate disease control due to insufficient cytoreduction of unresectable tumors, or develop tumor relapse despite apparent gross-total resection and/or irradiation [START_REF] Buckner | Central nervous system tumors[END_REF].

One major obstacle to efficient tumor cytoreduction may be insufficient drug delivery to the tumor site. The Blood-Brain Barrier (BBB) is a physical and metabolic barrier between systemic circulation and the brain. It maintains homeostasis and protects the brain from toxic insults. Yet, BBB may represent a real limiting factor to delivering chemotherapy agents within the brain tumor mass.

Morphine is the most used narcotic analgesic. In a recent report Sharma and coworkers using a rodent model documented that morphine induced a transient alteration of BBB permeability to large molecules [START_REF] Sharma | Alterations in blood-brain barrier function by morphine and methamphetamine[END_REF]. We hypothesized that a reversible modulation of the BBB by morphine might act as permeability enhancer for chemotherapy agents and designed an experimental rat model with the aim to achieve permeability of the BBB to doxorubicin by morphine pretreatment. Here we demonstrate that doxorubicin concentration in the brain is greatly enhanced when doxorubicin is administered in the presence of therapeutic plasma levels of morphine.

Methods

Drugs and Reagents

Doxorubicin (Adriblastina, Farmitalia Carlo Erba, Milan, Italy) 12 mg/kg in sterile H 2 O was administered i.p. in a volume of 6 ml/kg [START_REF] Johansen | Doxorubicin pharmacokinetics after intravenous andintraperitoneal administration in the nude mouse[END_REF]. Morphine (10 mg/kg) was dissolved in sterile saline and administered i.p. in a volume of 4 ml/kg. Chemical standard of Doxorubicin hydrochloride (purity 98%) was purchased from Sigma-Aldrich (Steinheim, Germany); the internal standard, Doxorubicin-13 C 4 (99.8% D purity) was from Medical Isotopes Inc. (Pelham, USA). Stock solutions of both were made in HPLC grade methanol (2000 mg/L). Further dilutions were made using HPLC grade water. All chemicals and solvents were of the highest purity available from commercial sources and used without further purification.

Animals

Male adult Wistar rats (260-280 g) were used (Harlan Italia, Milano, Italy). All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive 86/609/EEC). All efforts were made to minimize animal sufferings and to use only the number of animals necessary to produce reliable scientific data. Rats were randomly allocated to one of the following experimental groups (5 animals per group): Group 1 (D+M): on day 1 rats were treated twice with morphine (10 mg/kg, i.p., at 10.00 a.m. and again at 5.00 p.m.); on day 2 at 10.00 a.m., they received a third dose of morphine; one hour later they received i.p. doxorubicin. Group 2 (D): rats were treated with doxorubicin alone. Group 3 (Ctr): naïve rats received no drugs.

Neurological test

Neurological evaluation of motor sensory functions was carried out 10 days after doxorubicin administration on two groups of rats: a) rats treated as group 1 animals (n=4) and saline-treated rats (n=4) as controls. The examiners were blind as to the procedure that the rat had undergone.

Evaluations were performed in the afternoon. The neurological examination consisted of six tests: 1) spontaneous activity; 2) symmetry in the movement of four limbs; 3) forepaw outstretching; 4) climbing; 5) body proprioception; 6) response to vibrissae touch. The score assigned to each rat at completion of the evaluation equals the sum of all six test scores. The final minimum score was 3 and the maximum was 18 [START_REF] Garcia | Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats[END_REF][START_REF] Melani | The selective A2A receptor antagonist SCH 58261 reduces striatal transmitter outflow, turning behavior and ischemic brain damage induced by permanent focal ischemia in the rat[END_REF]. Rats were weighted every day.

Collection of tissues

One hour after administration of doxorubicin, rats were deeply anesthetized with chloral hydrate (400 mg/kg, i.p.). The heart was exposed and blood was withdrawn by intracardiac puncture, collected into a pre-heparinized plastic tube; plasma was separated from blood cells. Animals were then perfused transcardially with ice-cold saline (500 ml, 50 ml/min). At the end of perfusion, the cerebral hemispheres, the cerebellum, and brainstem were dissected; heart and kidney were collected; all tissues were rinsed in ice-cold saline and transferred in screw-capped plastic tubes.

All tissues were immediately frozen on dry ice and kept at -80°C until assay.

Quantitative analysis of doxorubicin by mass spectrometry

Doxorubicin determination on plasma. To quantify plasma concentration of doxorubicin, samples (25 µL) were spotted on filter paper (903 , Whatman GmbH, Dassel Germany) and air-dried. Dried plasma spots were punched and two 3.2 mm circles (containing about 1.75 µL of plasma each) were extracted together with 200 µL of 20/80 water/methanol solution containing 10 µmol/L of 13 C 4 -doxorubicin. Samples were put in an orbital shaker and kept at 37° C for 20 min.

For the setting-up of this study, a pooled mixture of plasma samples from untreated rats was spiked with increasing concentrations of doxorubicin and 25 µL were spotted on 903 filter paper. Tissue sample preparation. Thawed brain sections (cerebral hemispheres, brainstem, and whole brain) were weighed and spiked with 2 µL of 13 C 4 -doxorubicin (10 µmol/L) directly injected into the tissue. After 10 min, the sections were diluted with 2.5 mL H 2 O. Sample homogenization was performed using a mechanical homogenizer (UltraTurrex, IKA-Werke, Staufen, Germany) followed by 10 min of intermittent ultrasonication in the ice bath. Samples were defatted using 2 mL of chloroform with vigorous shaking for 5 min, followed by centrifugation at 3,000 rpm for 10 min. Optimal CE and CXP were found at 16 V and 10 V, respectively (Fig. 1).

Quantitative analysis was undertaken using an HPLC Series 1100 Agilent Technologies (Waldbronn, Germany). Liquid chromatography was performed using a Phenomenex Synergi 4µm Polar-RP 80A 4µm, 2 x 150 mm HPLC column (Phenomenex, Anzola Emilia, Italy) injecting 5 µL of the extracted sample. Column flow was 0.2 mL/min using an isocratic aqueous solution of 80% methanol containing 0.1% formic acid. The eluent from the column was directed to the TurboIonSpray probe without split ratio.

LDH activity and tissue lipid peroxidation

Lactate dehydrogenase (LDH) activity was measured in rat plasma using the Cytotoxicity Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) and absorbance read at 490 nm. Tissue lipid peroxidation in terms of malondialdehyde (MDA) production (thiobarbituric acidreactive species) was determined as previously described [START_REF] Uchiyama | Determination of malondialdehyde precursor in tissues by thiobarbituric acid test[END_REF].

Statistical analysis

Statistical significance was evaluated using one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test as a post-hoc analysis or by Student's t test, as appropriate. A P value of 0.05 or less was taken as the criterion for statistically significant difference.

Results

The concentration of doxorubicin within the brain tissue was significantly higher in rats pre-treated with morphine than in those treated with doxorubicin alone. Doxorubicin was undetectable in control samples from untreated rats (Fig. 2). Doxorubicin concentration was significantly higher in all the brain areas under study of animals pretreated with morphine (Fig. 3).

Cerebral hemispheres: the mean concentration of doxorubicin in the cerebral hemispheres of rats receiving doxorubicin alone was 5.88±0.34 ng/g fresh tissue (n=5), versus 18.8±1.01 ng/g fresh tissue in those treated with doxorubicin plus morphine (n=5), while control animals had no doxorubicin (0.002±0.001 ng/g fresh tissue, n=3). The estimated limit of detection of doxorubicin (signal to noise ratio >3) in the brain was 1 ng/g fresh tissue, and the limit of quantitation (signal to noise ratio >5) was 2 ng/g fresh tissue. This difference was statistically significant (one-way ANOVA F [2,10] = 168.3, P < 0.0001; *** P < 0.001 vs all other groups, Newman-Keuls post hoc test).

Cerebellum: the mean concentration of doxorubicin in the cerebellum of rats receiving doxorubicin alone was 5.18±0.38 ng/g fresh tissue (n=5), versus 15.94±0.371 ng/g fresh tissue in those treated with doxorubicin plus morphine (n=5), while control animals had no doxorubicin (n=3). This difference was statistically significant (one-way ANOVA F[2,10]= 482.0, P < 0.0001; *** P < 0.001 vs all other groups, Newman-Keuls post hoc test).

Brainstem: the mean concentration of doxorubicin in the brainstem of rats treated with doxorubicin alone was 5.71±0.38 ng/g fresh tissue (n=5), versus 14.6±0.25 ng/g fresh tissue in those treated with doxorubicin plus morphine (n=5). Control animals had no detectable doxorubicin (0.001±0.001 ng/g fresh tissue, n=5). This difference was statistically significant (one-way ANOVA F[2,10]= 521.2, P < 0.0001; *** P <0.001 vs all other groups , Newman-Keuls post hoc test).

Pretreatment of rats with lower doses of morphine (5 or 2.5 mg/kg, three times in 24 h, i.p.) failed to facilitate doxorubicin entry into the brain (n=3; data not shown).

Mean plasma levels of doxorubicin in animals pretreated with morphine (12.89±0.71 µmol/L plasma, n=5) did not differ from that found in rats receiving doxorubicin alone (13.26±2.12 µmol/L plasma, n=5) (P= 0.8711, Student's t test) (Fig. 3A).

Cardiac and renal toxicity after treatment with an anthracycline is a matter of great concern. We therefore investigated whether pretreatment with morphine might increase the doxorubicin concentrations in the heart and kidney, thus predicting increased toxicity in these two target organs.

Pretreatment with morphine did not increase the levels of doxorubicin in either tissue at 1 h after administration (P> 0.05, Student's t test, Fig. 3B andC).

We addressed the issue of cardiac and renal toxicity by analysis of plasma LDH activity and MDA levels. Doxorubicin induced a 60% increase of plasma LDH activity 1 h after treatment in treated versus control rats (Table 1, P=n.s.). No difference in LDH activity was found between rats treated with doxorubicin alone and those receiving morphine plus doxorubicin.

Mean MDA plasma levels were not statistically different among the three groups (Table 1).

In order to clarify whether the concomitant administration of doxorubicin and morphine might cause any late neurological toxicities/mortalities, we followed a group of rats for 10 days after the administration of the two drugs. We did not observe any clear sign of clinical toxicity nor any of the animals died after treatment. Rats treated with doxorubicin plus morphine did not show any neurological deficit in comparison to the control group 10 days after treatment (final maximum score ± SEM: 17,50±0,29 vs 17,75±0,25). Rats treated with morphine plus doxorubicin had significant body weight loss for the first 6 days after treatment, after which they started to gain weight.

Discussion

The main result of our study is that pretreatment with morphine at therapeutic dose allows delivery of doxorubicin within the brain tissue, which is otherwise precluded by the blood brain barrier.

Penetration of doxorubicin appears to be unrestricted to the different areas of the brain.

A prerequisite for the efficacy of a cytotoxic drug for the treatment of cancer is its capacity to reach the tumor cell at therapeutic concentrations. For brain tumors, this goal is usually hampered by BBB, a structure with the delicate duty to preserve, as far as possible, the integrity of brain tissue from chemical or infectious hazards.

Recently, Sharma et al. reported that rats treated with morphine exhibited leakage of albumin in several brain areas. This phenomenon was much more pronounced on the second and third day after morphine withdrawal, and was associated with ultrastructural changes in the nerve and glial cells [START_REF] Sharma | Alterations in blood-brain barrier function by morphine and methamphetamine[END_REF]. The authors hypothesized that neurochemical mediators and vasoactive agents act on the vessels through signal transduction pathways leading to the opening of the BBB. Based on these data, we decided to investigate the possibility of morphine mediated active BBB permeabilization.

Morphine is a substrate of the MDR1 isoform of P-glycoprotein (P-gp) efflux transporter, which very efficiently removes several molecules and drugs from the CNS, thus limiting their entry into the brain. Morphine mediated "accumulation" of doxorubicin into the brain might result from its reduced efflux mediated by P-gp at the level of the BBB cells [START_REF] Löscher | Drug transporters in the epileptic brain[END_REF]. P-gp is localized mostly at the apical side of brain endothelial cell membrane. P-gp substrates flowing into the endothelial cells are rapidly pumped back into the blood, thus preventing them from entering the brain. This hypothesis is supported by evidence that P-gp blockade by specific inhibitors results into a significantly increased brain concentration of some antiepileptic drugs [START_REF] Löscher | Drug transporters in the epileptic brain[END_REF]. Whether alternative or additional mechanisms may mediate the impact of morphine and other opioid drugs on the BBB remains to be elucidated further [START_REF] Dagenais | Variable modulation of opioid brain uptake by Pglycoprotein in mice[END_REF][START_REF] Schinkel | P-glycoprotein in the bloodbrain barrier of mice influences the brain penetration and pharmacological activity of many drugs[END_REF][START_REF] King | Transport of opioids from the brain to the periphery by P-glycoprotein: peripheral actions of central drugs[END_REF].

Data on the effects of chronic exposure to morphine are conflicting [START_REF] Aquilante | Increased brain P-glycoprotein in morphine tolerant rats[END_REF][START_REF] Yousif | Effect of chronic exposure to morphine on the rat blood-brain barrier: focus on the Pglycoprotein[END_REF]. In rats chronically treated with morphine, in situ perfusion of sucrose did not allow its penetration into the BBB [START_REF] Yousif | Effect of chronic exposure to morphine on the rat blood-brain barrier: focus on the Pglycoprotein[END_REF]. These data might be considered at odds with our results. Yet, we documented that an antiblastic drugdoxorubicinaccumulated into the brain and was detected by mass spectrometry, a highly precise, reliable and sensitive method. Furthermore, several studies demonstrate that morphine and doxorubicin use the same efflux channels [START_REF] Löscher | Drug transporters in the epileptic brain[END_REF][START_REF] Fricker | Modulation of drug transporters at the blood-brain barrier[END_REF], whereas sucrose crosses BBB by diffusion. We consider these data not to be an argument against our results.

On the other side, our group has recently reported unexpected respiratory distress as enhanced toxicity of dimethylsulfoxide (DMSO) on the central nervous system of children receiving morphine during hematopoietic stem cell transplantation. While this finding was apparently unexplained, we now consider that DMSO may have had undesired access to the brain tissue due to concurrent morphine-mediated temporary alteration of the BBB [START_REF] Caselli | Respiratory depression and somnolence in children receiving dimethylsulfoxide and morphine during hematopoietic stem cells transplantation[END_REF].

Delivering specific drugs beyond the BBB may open novel therapeutic approaches for several human diseases [START_REF] Doolittle | New frontiers in translational research in neurooncology and the blood-brain barrier: report of the tenth annual Blood-Brain Barrier Disruption Consortium Meeting[END_REF]. Pioneer experience in the treatment of childhood leukemia brought to intrathecal injection [START_REF] Pinkel | Nine years' experience with "total therapy[END_REF]. Yet, intrathecal chemotherapy can be adopted with great caution, and only for a very limited number of drugs. Inadvertent intrathecal administration of anthracycline turned into catastrophic toxicity [20,21]. Intracarotid infusion of hyperosmolar mannitol has been used to disrupt the BBB with the aim to increase the delivery of water-soluble agents into the brain and peritumoral areas [22]. Although this method gave some promising result, it requires an invasive surgical approach, with high risk of adverse effects which limit its potential clinical application on a large scale. More recently, nanoparticles have been adopted to enhance the transport of therapeutic agents across the BBB, providing refined drug delivery [23]. Doxorubicin accumulation was achieved after pretreatment with therapeutic dose of morphine.

When we tried to document this phenomenon with lower doses of morphine pretreatment, (25 and 50% dose) we did not document any accumulation compared to control rats, not pretreated with morphine. Thus, permissive effect of morphine appears to be active only beyond a threshold dose. Moreover, our data demonstrate that the concentration of doxorubicin after morphine pretreatment significantly higher in cerebral hemispheres than in cerebellum and brainstem (Fig. 3). This result may depend upon a different structure of BBB or on a different distribution of P-gp isoforms in these brain areas, making the brainstem less accessible to many molecules.

Although doxorubicin is a very effective anti-cancer drug, its cardiac toxicity has to be considered as a potential limitation to unrestricted use. To evaluate whether doxorubicin toxicity might be enhanced by co-administration of morphine, thus leading to dangerous acute and/or chronic side effects, we quantified doxorubicin levels in heart and kidney of morphine pretreated rats. Coadministration of morphine did not increase the levels of doxorubicin in either tissue 1 hour after administration. We also evaluated plasma LDH activity and MDA levels, as markers of acute cardiotoxicity, and found no difference between rats treated with doxorubicin alone and those also receiving morphine.

In conclusion, our preliminary results indicate that morphine pre-treatment mediates doxorubicin delivery beyond the BBB to the brain, in the absence of signs of increased acute toxicity. This finding might provide the rationale for clinical applications in the treatment of refractory brain tumors an pave the way to novel applications of active but currently inapplicable drugs. It remains to be assessed whether this observation will also facilitate studies exploring the brain delivery of other active agents, such as monoclonal antibodies or other macromolecules for the treatment of brain tumors or other human brain disease. Values are the mean ± SEM. Statistical significance between means was analyzed using one-way ANOVA followed by the Newman-Keuls post hoc multiple comparison test. Differences among groups were not statistically significant.

LEGEND TO FIGURES

  The aqueous upper phase was transferred to a new tube, and the chloroform extraction step was repeated 6 times. After the last extraction acetonitrile (400 µL) was added and precipitates removed by centrifugation at 13,200 rpm for 3 min. The samples were dried out and rehydrated with acidified methanol (0.1% formic acid, v/v) 300 µL. To determine the extraction yield of doxorubicin, brain tissue samples from an untreated rat were spiked with doxorubicin (5 µmol/L) before and after extraction process.Mass Spectrometry. An Applied Biosystems-Sciex (Toronto, Canada) API 4000 bench-top TurboIonSpray-Triple-Quad Mass Spectrometer (MS/MS) was used for this study. The ion source operated under positive ion mode (5,500 V).Declustering Potential (DP), Collision Exit Potential (CXP) and Collision Energy (CE) were automatically optimized for doxorubicin and 13 C 4 -doxorubicin. The resulting DP was 35 V.

  childhood acute lymphocytic leukemia. Pediatrics 50:246-251 20. Aricó M, Nespoli L, Porta F, Caselli D, Raiteri E, Burgio GR (1990) Severe acute encephalopathy following inadvertent intrathecal doxorubicin administration. Med Pediatr Oncol 18:261-263 21. Patel MM, Goyal BR, Bhadada SV, Bhatt JS, Amin AF (2009) Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs 23:35-58 22. Gumerlock MK, Belshe BD, Madsen R, Watts C (1992) Osmotic blood-brain barrier disruption and chemotherapy in the treatment of high grade malignant glioma: patient series and literature review. J Neurooncol 12:33-46 23. Sarin H (2009) Recent progress towards development of effective systemic chemotherapy for the treatment of malignant brain tumors. J Transl Med 7:77.

Figure 1 .

 1 Figure 1. MS/MS spectrum obtained by fragmenting the precursor ion (544.4 Th) of doxorubicin and the precursor ion (548.4 Th) of internal standard ( 13 C 4 -doxorubicin) under the above described conditions. From these experiments, the resulting ion-pair transition for the quantitative experiment (MRM) is 544.4>397.2 for doxorubicin and 548.4>401.2 for 13 C 4 -doxorubicin. The peaks corresponding to doxorubicin and internal standard eluted from the chromatographic column at 4 min. No interferences were revealed at the same retention time. The choice of a binary mobile phase was made considering the chromatographic column conditions.

Figure 2 .

 2 Figure 2. Extracted ion chromatograms of doxorubicin and internal standard (IS) in cerebral hemisphere (2a), cerebellum (2b) and brainstem (2c). A doxorubicin signal was very close to baseline in all control samples and therefore not detectable. Red signal: internal standard; blu signal: doxorubicin.

Figure 3 .

 3 Figure 3. Levels of doxorubicin in cerebral hemispjheres (A), cerebellum (B) and brainstem (C) of the rat 1 h after a single administration of doxorubicin (12 mg/kg, i.p.). Doxorubicin was administered to different groups of rats either alone (D, grey bars, n=5) or after pre-treatment with 3 doses of morphine (10 mg/kg each, three times in 24 h, D+M, open bars, n=5). Control animals (Ctr) received neither drug (black bars, n=3). Tissue levels of doxorubicin are expressed as µg/g fresh tissue (mean ± SEM). Statistical analysis was performed separately within each brain area investigated. Significance: *** P < 0.001 vs all other groups (one-way ANOVA and Newman-Keuls post hoc test). Levels of doxorubicin in the cerebral hemispheres of rats treated with morphine plus doxorubicin were significantly higher than in cerebellum or brainstem (P<0.01, oneway ANOVA and Newman-Keuls post hoc test).

Figure 4 .

 4 Figure 4. Levels of doxorubicin in plasma (A), heart (B) and kidney (C) of the rat 1 h after a single administration of doxorubicin (12 mg/kg, i.p.). Doxorubicin was administered to different groups of rats either alone (D, grey bars) or after pre-treatment with 3 doses of morphine (10 mg/kg each, three times in 24 h, D+M, open bars). Panel A: plasma levels of doxorubicin are expressed as µmol/L plasma; D+M: n=5; D: N=5. Panels B and C: Tissue levels of doxorubicin are expressed as µg/g fresh tissue. Panel B: D+M: n=3; D: n=3.Panel C: D+M: n=3; D: n=3. Data presented are the

Table 1 .

 1 Levels of LDH activity and MDA production in plasma of control rats, rats treated with doxorubicin alone or after pre-treatment with morphine.

	GROUP	LDH	MDA
		(U/ml plasma)	(nmol/ml plasma)
	Control	0.72 ± 0.08	323.2 ± 64.7
	Doxorubicin	1.15 ± 0.26	294.0 ± 60.1
	Doxorubicin + morphine	1.16 ± 0.15	290.0 ± 73.2
	Doxorubicin was administered to different groups of rats either alone (12 mg/kg, i.p., doxorubicin,
	n=7) or after pre-treatment with 3 doses of morphine (10 mg/kg each, i.p., three times in 24 h,
	Doxorubicin + Morphine, n=6). Control animals (n=6) received neither drug.

Acknowledgements

Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC), grant RG-6232; "NOI PER VOI" Onlus; Fondazione Anna Meyer. We thank Dr. Richard E. Kast for his valuable support and Dr. Lisa Giovannelli for her help in setting up the methods for LDH and TBARs assay.

mean ± SEM. Statistical analysis was performed separately on data from each tissue investigated.

Statistical analysis showed no significant differences between each pair of data (P > 0.05, Student's t test).


H Boschker

M Huijben

A Vailionis

J Verbeeck

S Van Aert

M Luysberg

S Bals

G Van Tendeloo

E P Houwman

G Koster

D H A Blank

G Rijnders

Optimized fabrication of high quality La 0.67 Sr 0.33 MnO 3 thin films considering all essential characteristics

Keywords: numbers: 75.47.Gk, 75.47.Lx, 75.70.Ak, 68.37.Ma, 81.15.Fg

Optimized fabrication of high-quality La0.67Sr0.33MnO3 thin films considering all essential characteristics

H Boschker, M Huijben,

Introduction

The discovery of colossal magnetoresistance in the perovskite manganites in 1993 resulted in an extensive amount of research [START_REF] R Von Helmolt | Giant negative magnetoresistance in perovskitelike La 2/3 Ba 1/3 MnO x ferromagnetic-films[END_REF][START_REF] Jin | Thousandfold change in resistivity in magnetoresistive La-Ca-Mn-O films[END_REF], because of the potential for data storage applications. Among the perovskite manganites, La 0.67 Sr 0.33 MnO 3 (LSMO) has the largest single electron bandwidth and the highest Curie temperature. These properties, together with the 100% spin polarization, make LSMO an interesting material for application in spintronic devices. Examples are magnetic tunnel junctions [START_REF] Jz Sun | Observation of large low-field magnetoresistance in trilayer perpendicular transport devices made using doped manganate perovskites[END_REF][START_REF] Donnell | Colossal magnetoresistance magnetic tunnel junctions grown by molecular-beam epitaxy[END_REF][START_REF] Bowen | Nearly total spin polarization in La 2/3 Sr 1/3 MnO 3 from tunneling experiments[END_REF][START_REF] Ogimoto | Tunneling magnetoresistance above room temperature in La 0.7 Sr 0.3 MnO 3 /SrTiO 3 /La 0.7 Sr 0.3 MnO 3 junctions[END_REF], Schottky devices [START_REF] Fm Postma | Epitaxial diodes of a half-metallic ferromagnet on an oxide semiconductor[END_REF][START_REF] Nakagawa | Magnetocapacitance and exponential magnetoresistance in manganite-titanate heterojunctions[END_REF][START_REF] Hikita | Termination control of the interface dipole in La 0.7 Sr 0.3 MnO 3 /Nb:SrTiO 3 (001) Schottky junctions[END_REF] and magnetoelectric devices [START_REF] Eerenstein | Giant sharp and persistent converse magnetoelectric effects in multiferroic epitaxial heterostructures[END_REF][START_REF] Hajo | Magnetoelectric Effects in Complex Oxides with Competing Ground States[END_REF][START_REF] Garcia | Giant tunnel electroresistance for non-destructive readout of ferroelectric states[END_REF][START_REF] Yu | Interface Ferromagnetism and Orbital Reconstruction in BiFeO 3 -La 0.7 Sr 0.3 MnO 3 Heterostructures[END_REF][START_REF] Wu | Reversible electric control of exchange bias in a multiferroic field-effect device[END_REF][START_REF] Borisevich | Suppression of Octahedral Tilts and Associated Changes in Electronic Properties at Epitaxial Oxide Heterostructure Interfaces[END_REF]. Furthermore, LSMO is also used to investigate other materials, e.g. by spin injection into cuprate superconductors [START_REF] Sawa | Spinpolarized tunneling of La 0.67 Sr 0.33 MnO 3 /YBa 2 Cu 3 O 7-δ junctions[END_REF][START_REF] Zy Chen | Spin-polarized transport across a La 0.7 Sr 0.3 MnO 3 /YBa 2 Cu 3 O 7-x interface: Role of Andreev bound states[END_REF][START_REF] Pena | Giant magnetoresistance in ferromagnet/superconductor superlattices[END_REF][START_REF] Van Zalk | Magnetization-induced resistance-switching effects in La 0.67 Sr 0.33 MnO 3 /YBa 2 Cu 3 O 7-δ bi-and trilayers[END_REF], to probe spin polarization at the BaTiO 3 /Fe interface [START_REF] Garcia | Ferroelectric Control of Spin Polarization[END_REF] and to study spin dependent transport in organic materials [START_REF] Barraud | Unravelling the role of the interface for spin injection into organic semiconductors[END_REF].

All these applications require LSMO thin films and the device performance will mainly depend on the thin film quality. The optimization of the LSMO thin films is not straightforward, as different parameters exist which can be optimized, e.g. the magnetization and the electrical conductivity. In addition to these parameters, a smooth surface morphology is required for most applications. Focussing on ideal layer-by-layer growth can compromise the functional properties of the film [START_REF] Huijben | Critical thickness and orbital ordering in ultrathin La 0.7 Sr 0.3 MnO 3 films[END_REF]. Other detrimental effects are Mn 3 O 4 precipitates on LSMO surfaces, which are the result of off-stoichiometric deposition [START_REF] Higuchi | Mn 3 O 4 precipitates in laser-ablated manganite films[END_REF], and Sr segregation towards the surface [START_REF] Herger | Structure determination of monolayer-by-monolayer grown La 1-x Sr x MnO 3 thin films and the onset of magnetoresistance[END_REF].

Summarizing, a high quality LSMO film combines an atomically smooth surface morphology with a large magnetization and a low residual resistivity, while avoiding precipitates and surface segregation. In literature, typically only a few of these issues are adressed, e.g., by focussing only on the electrical conductivity. Here, we show that high quality films, combining good ferromagnetic properties with a high conductivity can be realized. As discussed in a previous paper [START_REF] Huijben | Critical thickness and orbital ordering in ultrathin La 0.7 Sr 0.3 MnO 3 films[END_REF], we optimized the LSMO films on a combination of the magnetic properties and two dimensional (2D) layerby-layer growth. We found that the properties of the films are very sensitive to the amount of oxygen present during the deposition. Too little oxygen resulted in inferior ferromagnetic properties, while too much oxygen resulted in three dimensional growth. In this paper, we present a thorough characterization of the resulting films and compare them to examples found in literature. The films have the highest reported saturation magnetization. The optimization on ferromagnetism and 2D layer-by-layer growth resulted in a high conductivity and a lack of surface segregation as well.

This article is organized as follows. First, the growth of the films is described, together with an analysis of the surface morphology with reflection high energy electron diffraction (RHEED) and scanning probe microscopy (SPM). Then the crystal structure is discussed, followed by the functional properties, magnetization and electrical conductivity. In the next section, in situ x-ray photoemission spectroscopy (XPS) measurements are presented, which are used to study the possibility of surface segregation. The interface atomic structure was measured with scanning transmission electron microscopy (STEM). In the final section, the properties of the LSMO thin films are compared with films described in literature.

Thin film growth

In this section, the growth of the LSMO thin films is described. First, the treatment of the substrates used as template for the growth is discussed. Then, the growth of the thin films by pulsed laser deposition is described. In situ RHEED measurements are presented from which it is concluded that the initial growth occurs in the two dimensional layer-by-layer growth mode, and that a transition to a steady state growth mode occurs for thicker films. Finally, the surface morphology was characterized with scanning probe microscopy, which showed smooth surfaces without the presence of precipitates.

Substrates

Several substrates are commercially available for the growth of oxide thin films. A number of substrates which have comparable lattice parameters to LSMO can be used as a template for the growth of the LSMO thin films. They include NdGaO 3 (NGO), (LaAlO 3 ) 0,3 -(Sr 2 AlTaO 6 ) 0,7 (LSAT), SrTiO 3 (STO) and DyScO 3 (DSO). The pseudocubic lattice parameters are respectively 3.85, 3.87, 3.905 and 3.95 Å. STO and LSAT are cubic crystals while NGO and DSO have an orthorhombic crystal structure. For the studies in this article, we used STO (001) c substrates, as STO is the standard substrate used for LSMO growth in literature and this allows for better comparison between the samples. (The subscripts o, c and pc denote the orthorhombic, cubic and pseudocubic crystal structure, respectively.)

The substrates were first ultrasonically cleaned with acetone and ethanol. Singly TiO 2 terminated (B-site) STO (001) c substrates were obtained with the procedure developed by Koster et al. [START_REF] Koster | Quasi-ideal strontium titanate crystal surfaces through formation of strontium hydroxide[END_REF]. In some cases, the annealing step of 2 hours at 950 • C resulted in some Sr segregation from the bulk towards the surface, due to reduced STO quality [START_REF] Bachelet | Atomically flat SrOterminated SrTiO 3 (001) substrate[END_REF][START_REF] Je Kleibeuker | Atomically Defined Rare-Earth Scandate Crystal Surfaces[END_REF]. This could be observed with atomic force microscopy (AFM), either in the phase contrast or with the presence of straight stepedges aligned with the principal crystal directions [START_REF] Koster | Surface morphology determined by (001) single-crystal SrTiO 3 termination[END_REF]. These substrates were treated with H 2 O and the HF solution again, followed by a 30 minute anneal at 920 • C, after which no SrO termination could be detected.

Pulsed laser deposition

The LSMO thin films were grown with pulsed laser deposition (PLD) (TSST system). The substrate temperature during growth was 750-800 • C in an oxygen environment of 0.27 mbar. The laser beam was produced by a 248-nm-wavelength KrF excimer laser (LPXPro TM from Coherent, Inc.) with a typical pulse duration of 20-30 ns. With a 4 by 15 mm rectangular mask the most homogeneous part of the laser beam was selected. An image of the mask was created on the stoichiometric target (Praxair electronics) with a lens, resulting in a spotsize of 2.3 mm 2 (0.9 by 2.5 mm). The beam energy was controlled with a variable attenuator or with the laser voltage, yielding a fluence at the target of 2 J/cm 2 . The repetition rate was 1 Hz and the substrate was placed at 5 cm distance directly opposite to the target. Before deposition, the target was pre-ablated for 2 minutes at 5 Hz to remove any possible surface contamination. After deposition, the PLD chamber was flooded with pure oxygen (typically 100 mbar) and the samples were cooled down by switching of the heater power. Typically, the cooldown required 2 hours.

Growth mode

The growth of the films was studied in situ with RHEED during the growth. The substrate RHEED pattern is shown in figure 1 on the top left. The main specular spot is very intense compared to the two side spots. This is the typical signature of TiO 2 terminated STO [START_REF] Koster | Surface morphology determined by (001) single-crystal SrTiO 3 termination[END_REF]. Kikuchi lines are visible as well, indicating the smoothness of the substrate. The side spots are doubled, which is due to the additional periodicity at the surface from the regularly spaced terrace steps. Figure 1, top right, shows the RHEED image of the LSMO film after the deposition of 5 unit cell layers. Clear two dimensional spots are visible, but also 2D streaks are present. The latter are attributed to the scattering of the RHEED beam off the unit cell high steps at the surface. Similar RHEED images were observed after the completion of films with thicknesses up to 40 nm.

The main graph in figure 1 shows the intensity of the specular reflection as measured during the initial growth of LSMO. The intensity shows oscillations which correspond to the growth of the individual unit cell layers. Within the oscillations, recovery of the intensity after the sudden decrease during the laser burst is observed, as shown more clearly in the inset of the graph. The oscillation amplitude decreases with the amount of material deposited during the first part of the growth. This decrease of the intensity is due to the difference in reflectivity of the STO surface and the LSMO surface, due to the increased scattering from the heavy La ions in the lattice. Finally, the full width at the half maximum (FWHM) of the intensity of the specular spot, measured along the (10) direction, is presented as well. During the growth, the RHEED spots are periodically more streaky, because more stepedge scattering is present at half unit cell layer coverage compared to full unit cell layer coverage. Therefore, the FWHM oscillates as well. The FWHM depends only on the shape of the intensity distribution and not on the total intensity and it is therefore a better indicator of the surface morphology than the main intensity of the reflection. As can be seen in the figure, the FWHM during the growth is almost equal to the FWHM of the substrate reflection indicating a smooth surface morphology. From these measurements it is concluded that the initial stage of the LSMO growth proceeds in the ideal 2D layer-by-layer growth mode.

During the growth of the film, an increase of the RHEED oscillation maximum intensity was observed, as presented in figure 2. The oscillation intensity maximum typically peaked around 20 to 25 unit cell layers. The origin of the maximum in intensity is not understood. The maximum in intensity was not observed during growth on an Asite terminated substrate surface (SrRuO 3 film grown on STO [START_REF] Rijnders | Enhanced surface diffusion through termination conversion during epitaxial SrRuO3 growth[END_REF]), so it might indicate a termination conversion. After this peak the oscillation intensity maximum decreased rapidly and the oscillation amplitude became comparable to the intensity variations in the laser pulse recovery cycles. It is concluded that the LSMO growth mode during the latter part of the growth is close to the steady state growth mode which is characterized by a relatively constant step density, which is large compared to the step density of the initial substrate surface.

Surface morphology

The surface morphology of the films was studied with atomic force microscopy (AFM).

The images showed smooth surfaces as shown in the examples presented in figure 3.

The step and terrace structure of the substrate is still observed on the surface of the films. Figure 3a shows the surface morphology of a 20 unit cell layer film which was completely grown in the 2D layer-by-layer growth mode, while figure 3b shows the surface morphology of a 30 unit cell layer film where the growth changed to the steady state growth mode. For the latter film islands with unit cell height are observed on the terraces. The root mean square roughness of the samples is 0.15 nm (20 uc sample) and 0.21 nm (30 uc sample) respectively. For LSMO, it is known that off-stoichiometric deposition results in precipitate formation on the surface [START_REF] Higuchi | Mn 3 O 4 precipitates in laser-ablated manganite films[END_REF]. Such precipitates have not been observed.

Crystal structure

The crystal structure of the LSMO thin films was analyzed with the use of xray diffraction (XRD) measurements. The measurements were performed using a PANalytical X'Pert materials diffractometer or at the beamline 7-2 of the Stanford Synchrotron Radiation Laboratory.

The LSMO bulk rhombohedral crystal structure [START_REF] Pg Radaelli | Structural effects on the magnetic and transport properties of perovskite A (1-x) A (x) MnO 3 (x=0.25, 0.30)[END_REF] is incommensurate with the square/rectangular surface unit cells of the substrates. The LSMO is therefore under both normal strain as well as shear strain [START_REF] Farag | Modeling of twinning in epitaxial (001)-oriented La 0.67 Sr 0.33 MnO 3 thin films[END_REF]. To accommodate this strain LSMO adopts a different crystal symmetry, namely a distorted (monoclinic) orthorhombic unit cell with space group P2 1 /m [START_REF] Vailionis | Misfit strain accommodation in epitaxial ABO 3 perovskites: lattice rotations and lattice modulations[END_REF]. The orthorhombic unit cell is (110) o oriented out-of-plane with [110] o and [001] o in-plane orientations. To confirm that the LSMO layers were grown in a fully coherent fashion with respect to the underlying substrate, Next to the peak, finite thickness fringes are observed whose period corresponds well to the 40 nm film thickness as estimated from counting the RHEED oscillations. The clear oscillations indicate a high quality sample with a smooth substrate film interface and a smooth film surface, as was also concluded from the AFM measurements.

Functional properties

The functional properties of LSMO are its magnetization and electrical transport. Here, measurements are presented of an 11.5 nm LSMO film grown on STO (001) c . The sample thickness was determined by counting RHEED oscillations and was confirmed with an XRD reflectivity measurement. The sample has a Curie temperature of 350 K, a low temperature saturation magnetization of 4.0±0.05 µ B /Mn and a low temperature resistivity of 70 µΩcm.

Magnetization

The magnetization of the sample was measured with a vibrating sample magnetometer (VSM) (Physical Properties Measurement System (PPMS) by Quantum Design). Figure 6a shows the low temperature magnetization loop, obtained at 10 K with the field applied along the [100] pc axis and after subtraction of a diamagnetic background from the substrate. The saturation magnetization is reached for an applied field strength larger than 50 kA/m (≈630 Oe) and is equal to 4.0±0.05 µ B /Mn. This implies that next to the expected 3.7 µ B /Mn spin angular momentum also 0.3 µ B /Mn orbital angular momentum is present in the LSMO. Saturation values of 4 or more µ B /Mn have been observed earlier in single crystals with doping x=0.1 and x=0.2 [START_REF] Urushibara | Insulator-metal transition transition and giant magnetoresistance in La 1-x Sr x MnO 3[END_REF] and in LSMO polycrystals [START_REF] Koide | Close correlation between the magnetic moments, lattice distortions, and hybridization in LaMnO3 and La1-xSrxMnO3+delta: Doping-dependent magnetic circular X-ray dichroism study[END_REF]. The magnetization at remanence, 2.7 µ B /Mn, is significantly smaller than the saturation magnetization. This is partially because the measurement is along the magnetic hard axis [START_REF] Mathews | Step-induced uniaxial magnetic anisotropy of La 0.67 Sr 0.33 MnO 3 thin films[END_REF], and partially because the weak magnetic anisotropy of LSMO on STO results in nanoscale domain formation [START_REF] Houwman | Out-of-plane magnetic domain structure in a thin film of La 0.67 Sr 0.33 MnO 3 on SrTiO 3 (001) observed by magnetic force microscopy[END_REF]. The magnetization is very soft with a coercivity of 1.6 kA/m (≈20 Oe).

The temperature dependence of the magnetization is presented in figure 6b. For each datapoint in the graph, a full hysteresis loop between 240 and -240 kA/m (≈3000 Oe) was measured and the saturation magnetization was calculated after the background subtraction. The Curie temperature of the sample is 350 K. The saturation magnetization follows the Brillouin functional dependence for a Weiss ferromagnet [START_REF] Chikazumi | Physics of ferromagnetism[END_REF]. The curve calculated using J=4, J is the total angular momentum, results in the best fit of the data. This indicates either that the spins in the LSMO behave not completely classically (the case of J=∞) and not completely quantum mechanically (J= 1 2 ), but show intermediate behaviour or that spin wave excitations play an important role in the temperature dependence of the magnetization.

Electrical transport

The resistivity of the samples was measured in the van der Pauw configuration [START_REF] Van Der Pauw | A method of measuring the resistivity and Hall coefficient on lamellae of arbitrary shape[END_REF] (PPMS by Quantum Design). In order to obtain ohmic contacts between the aluminium bonding wires and the LSMO layer, gold contacts were deposited on the corners of the sample with the use of a shadow mask. Measurements were performed as a function of temperature (10-350 K) and magnetic field (0-2.5 T). The temperature dependent resistivity of the sample is shown in figure 7. The sample shows metallic behaviour with a residual resistivity at 10 K of 70 µΩcm. For thicker films (≥20 nm), a residual resistivity of 60 µΩcm was measured. The low temperature part of the resistivity curve can be described with the relation:

ρ(T ) = ρ 0 + aT 2 + bT 5 , (1)
in which ρ 0 is the temperature independent impurity scattering and the T 2 (T 5 ) term describes electron electron (electron phonon) scattering. A fit using equation 1 is shown in the figure and it decribes the curve up to a temperature of 300 K well. At 300 K a discontinuity in the slope of the resistivity curve is observed and above 300 K the resistivity increases faster compared to the model, indicating the approach of the metal insulator transition at T C . Figure 7 also presents the magnetoresistivity as a function of temperature. At each temperature the magnetic field is swept between -2.5 and 2.5 T and the magnetoresistivity at 2.5 T (ρ(2.5T ) -ρ(0))/ρ(0) is shown. The magnetoresistance is negative and largest, -16 %, at the Curie temperature. Hall effect measurements determined the charge carriers to be holes, as expected for LSMO. The observed Hall coefficient R H =4.6•10 -4 cm 3 /C implies a carrier density of approximately 0.8 hole/unit cell, which is in reasonable agreement with earlier measurements on single crystals [START_REF] Asamitsu | Hall effect in La 1-x Sr x MnO 3[END_REF].

Photoelectron spectroscopy

Several LSMO thin films were characterized with in situ x-ray and ultra violet photoelectron spectroscopy (XPS and UPS). The measurements were performed with an XPS/UPS system designed by Omicron Nanotechnology GmbH, equipped with an EA 125 electron energy analyzer. For XPS an Al Kα source (XM 1000) was used and the UV light source is a He plasma lamp (HIS 13) operated at the HeI (21.22 eV) excitation edge. The base pressure of the system was below 10 -10 mbar. The analyzer was calibrated with the use of an in situ sputter cleaned Au sample.

Figure 8 shows a survey scan of a 10 unit cell layer LSMO sample grown on (001) c STO. The main features include the La 3d peaks at 850 eV, the oxygen 1s peak at 531 eV, the Mn 2p peaks at 640 eV and the Sr and La peaks at low binding energy. Next to this a small Ti peak originating from the substrate can be seen at 460 eV. No indications for the presence of impurity atoms, including carbon contamination, was found. This is attributed to the in situ measurement. No attempts have been made to quantify the composition of the film, as large uncertainty margins exist for the element sensitivity factors required to normalize measured peak intensities. From XRD and functional properties measurements it is clear that the composition of the films is equal to the desired LSMO composition within the rather large error margin of the XPS measurements.

The inset of figure 8 shows the valence band spectrum as measured with UPS at room temperature. No Fermi edge is observed, the photoelectron count increases linearly with binding energy up to a value of 1.5 eV, after which a steep increase is observed. The absence of the Fermi edge is not surprising. In literature, several studies have observed a Fermi edge, but only at low (<50K) temperature [START_REF] Park | Direct evidence for a half-metallic ferromagnet[END_REF][START_REF] Dessau | Direct electronic structure measurements of the colossal magnetoresistive oxides[END_REF] or using resonant photoemission spectroscopy [START_REF] Horiba | In vacuo photoemission study of atomically controlled La 1-x Sr x MnO 3 thin films: Composition dependence of the electronic structure[END_REF]. LSMO's electron density at the Fermi energy is much reduced compared to band theory due to the presence of the pseudogap [START_REF] Dessau | Direct electronic structure measurements of the colossal magnetoresistive oxides[END_REF][START_REF] Yd Chuang | Fermi surface nesting and nanoscale fluctuating charge/orbital ordering in colossal magnetoresistive oxides[END_REF] and disappears above the Curie temperature. It is therefore natural that the Fermi edge cannot easily be observed in a room temperature measurement. The steep increase of the electron density below 1.5 eV is in good agreement with the literature [START_REF] Park | Direct evidence for a half-metallic ferromagnet[END_REF].

Several authors have suggested that Sr or Ca segregation towards the surface of the film is an intrinsic effect in LSMO (or La 0.67 Ca 0.33 MnO 3 (LCMO) respectively) thin films [START_REF] Simon | Diffusion and segregation effects in doped manganite/titanate heterostructures[END_REF][START_REF] Herger | Structure determination of monolayer-by-monolayer grown La 1-x Sr x MnO 3 thin films and the onset of magnetoresistance[END_REF]. Angle dependent XPS measurements can be used to study changes in the composition of a thin film in the surface layer. The results obtained for a 10 unit cell layer film grown on (001) c STO are presented in table 1. A film thickness of ten unit cell layers was chosen for this experiment in order to directly compare with the 1 to 9 unit cell layer experiments by Herger et al. [START_REF] Herger | Structure determination of monolayer-by-monolayer grown La 1-x Sr x MnO 3 thin films and the onset of magnetoresistance[END_REF]. If thinner LSMO layers would be used the XPS Sr signal would gain relative intensity due to a substrate contribution, affecting the measurement. As the relative intensity of peaks measured at different take-off angles does not depend on the element sensitivity factors, quantitative analysis is possible. The Sr 3d peak at 135 eV was compared with the La 4d peak at 105 eV and for both elements, an intensity ratio between bulk and surface sensitive measurements of 1.5±0.05 was found. This limits the off-stoichiometry of the surface to within 6%. In the example in literature [START_REF] Herger | Structure determination of monolayer-by-monolayer grown La 1-x Sr x MnO 3 thin films and the onset of magnetoresistance[END_REF] it is mentioned that the topmost layer has a La/Sr ratio of 0.5 instead of 2, while the subsequent layers are stoichiometric. Based on an electron escape depth of 2 nm for electrons with kinetic energy of 1350 eV [45], Herger's 9 unit cell layer sample would show an off-stoichiometry of 33% in the XPS measurement. Therefore, it is concluded that Sr segregation is much reduced in these samples as compared to the samples of Herger et al.. It is known that the Sr segregation depends on the oxygen partial pressure [START_REF] Fister | In situ characterization of strontium surface segregation in epitaxial la0.7sr0.3mno3 thin films as a function of oxygen partial pressure[END_REF]. Therefore, the lack of Sr segregation can be attributed to the high Table 1. Integrated XPS peak intensity for a 10 unit cell layer LSMO sample grown on (001) c STO. The XPS intensity is in arbitrary units and for peaks split by e.g. spin orbit coupling the intensities of all the components have been added. The error margins are estimated on the quality of the background subtraction routines. oxygen pressure used during the deposition.

Peak

Scanning transmission electron microscopy

A multilayer LSMO/STO sample was characterized with scanning transmission electron microscopy (STEM), to study the interface atomic structure. The STEM data presented was measured with a FEI Titan microscope. The sample is comprised of several layers, from substrate to surface: 4 nm LSMO, 2 nm STO, 16 nm LSMO, 2 nm STO and 8 nm LSMO.

Figure 9a presents a low magnification HAADF image of the different layers in the sample. In HAADF microscopy, the observed intensity of a column of atoms scales with the atomic weight of the elements within the columns. Therefore, the LSMO is brighter [START_REF] Huijben | Critical thickness and orbital ordering in ultrathin La 0.7 Sr 0.3 MnO 3 films[END_REF][START_REF] Gray | Interface properties of magnetic tunnel junction La 0.7 Sr 0.3 MnO 3 /SrTiO 3 superlattices studied by standing-wave excited photoemission spectroscopy[END_REF] in the image than the STO. Figure 9b presents a higher magnification image obtained from the layers close to the substrate. Both the Aand B-site columns are observed. The multilayer structure is grown coherently and following a quantitative analysis by statistical parameter estimation [START_REF] Van Aert | Quantitative atomic resolution mapping using high-angle annular dark field scanning transmission electron microscopy[END_REF], it can be concluded that the interfaces are well defined with a chemical roughness of maximum 1 unit cell.

Comparison to other groups

Table 2 presents an overview of the most important properties of LSMO thin films. Data taken from different research groups is included in order to compare the thin film quality. For reference also the properties of a single crystal are mentioned. All films were grown on STO (001) c substrates by PLD, except for the samples grown in Illinois, for which MBE was used. Most groups published AFM measurements of the surface morphology, which showed smooth surfaces with the step and terrace structure of the substrate clearly visible. Two groups, the Illinois group and the Rome group, did not publish images of the film surface morphology, but they did present a RHEED image from which it was concluded that the films were smooth [START_REF] Kavich | Nanoscale suppression of magnetization at atomically assembled manganite interfaces: XMCD and XRMS measurements[END_REF][START_REF] Tebano | High-quality in situ manganite thin films by pulsed laser deposition at low background pressures[END_REF]. The Rome group mentions that their films have a metal-insulator transition temperature of 370 K. This temperature is defined in their papers as the temperature at which the resistivity of the films has a maximum and it is not equal to the Curie temperature. The value mentioned in the table is deduced from the temperature dependence of the magnetization plot in [START_REF] Tebano | Evidence of orbital reconstruction at interfaces in ultrathin La 0.67 Sr 0.33 MnO 3 films[END_REF]. The data from the 20 unit cell layer sample was used, which has a similar metalinsulator transition temperature as their thick samples.

In general, a similar crystal structure with an out-of-plane lattice parameter of 3.83 to 3.85 Å and a similar surface morphology is obtained. Nevertheless, differences in the functional properties are present. We therefore suspect that these differences are due to small variations in the stoichiometry of the films, e.g. the La/Sr ratio and the degree of oxygenation of the film.

A high quality sample should combine a high T C with a high saturation magnetization and a low residual resistivity. The samples described here have the highest saturation magnetization, a high T C and a low residual resistivity. Only the samples from the Illinois group have a lower residual resistivity while both the Illinois and the Tokyo group obtained a slightly higher T C . We obtained a higher saturation magnetization, 4.0 µ B /Mn in contrast to the 3.2 µ B /Mn reported by the Illinois group. The Tokyo group did not report the saturation magnetization, but the magnetization value at 200 K can be compared; 2.7 µ B /Mn (Tokyo [START_REF] Jong | Enhanced thermodynamic stability of epitaxial oxide thin films[END_REF]) versus 3.2 µ B /Mn (this work). Combining all three properties it is concluded that the samples of this work combine both a high magnetization with a low residual resistivity and have the highest quality reported so far in literature.

Conclusion

In this article, an overview of the fabrication and the properties of high quality LSMO thin films was presented. The films have a saturation magnetization of 4.0 µ B /Mn, a Curie temperature of 350 K and a residual resistivity of 60 µΩcm. XPS measurements show the absence of chemical impurities and no evidence for surface segregation. STEM measurements show that multi-layer structures with sharp interfaces were realized. These results indicate that high quality films, combining both large magnetization and small residual resistivity, were realized. For LSMO devices, the thin film quality is crucial for the device performance. Therefore, this research is important for the application of LSMO in devices.

Figure 1 .

 1 Figure 1. RHEED specular spot intensity and FWHM as monitored during the initial growth of LSMO on STO (001) c . The inset shows an expanded view of the growth of the first unit cell layer. Two RHEED images are presented, the left image was taken before deposition (at low pressure) and the right image was taken after deposition of 5 unit cell layers.

Figure 2 .

 2 Figure2. RHEED specular spot intensity and FWHM as monitored during the growth of a relatively thick film of LSMO on STO (001) c . The graph shows the 7 th to 32 nd oscillation. A recovery of the RHEED oscillation intensity maximum, followed by a rapid decrease, is observed around the 20 th unit cell layer.

Figure 3 .

 3 Figure 3. Surface morphology as measured with ex situ AFM. a) 20 unit cell layers of LSMO grown on STO. b) 30 unit cell layers of LSMO grown on STO. Both images are 2 by 2 µm.

Figure 4 .

 4 Figure 4. Room temperature XRD RLMs around the (260) o , (444) o , (620) o and (444) o reflections of a 40 nm LSMO film grown on STO.

Figure 5 .

 5 Figure 5. Room temperature XRD HK scan (along the out-of-plane direction) of a 40 nm LSMO/STO sample. The substrate and film Bragg peaks have been indicated and multiple thickness fringes are observed.

Figure 6 .

 6 Figure 6. Magnetization measurements of 11.5 nm LSMO grown on STO (001) c . a) Hysteresis loop obtained at 10 K with the field applied in the plane of the film parallel to the [100] pc LSMO lattice direction. b) Temperature dependence of the saturation magnetization. Datapoints are indicated with black triangles and the curves are calculated from the Brillouin functional dependence of a Weiss ferromagnet. The shape of the curve depends on the value of J, J= 1 2 ,1,2,4 and ∞ respectively, following the direction of the arrow.

Figure 5

 5 Figure5shows an HK scan of the (220) o LSMO and (001) c STO Bragg peaks. Next to the peak, finite thickness fringes are observed whose period corresponds well to the 40 nm film thickness as estimated from counting the RHEED oscillations. The clear oscillations indicate a high quality sample with a smooth substrate film interface and a smooth film surface, as was also concluded from the AFM measurements.

Figure 7 .

 7 Figure 7. Electrical transport measurements of 11.5 nm LSMO grown on STO (001) c . Temperature dependent resistivity and magnetoresistance. The solid black line is a fit to the low temperature resistivity data, as discussed in the main text.

Figure 8 .

 8 Figure 8. An XPS survey scan of a 10 unit cell layer LSMO sample grown on (001) c STO. The La 3d, Mn 2p, O 1s, Sr 3d and La 4d peaks are indicated. The inset shows the photoelectron intensity in the region close to the Fermi energy measured with UPS. The measurements were performed at room temperature.

Figure 9 .

 9 Figure 9. HAADF-STEM images of an LSMO/STO heterostructure. a) Low magnification overview. b) High magnification image of the area close to the substrate.

Table 2 .

 2 Overview of the thin film properties. T C is the Curie temperature, ρ 0 is the residual resistivity at 10 K and M sat is the saturation magnetization at 10 K.

	Group	[001] pc axis ( Å)	T C (K)
	Deposition	ρ 0 (µΩcm)	M sat (µ B /Mn)	References
	Tokyo (Tokura et al.)	-	3 7 0
	Single crystal	90	3.5	[33]
	Tsukuba (Kawasaki et al.)	3 . 8 3	3 4 0
	PLD	200	3.6	[48, 49, 42, 50]
	Illinois (Eckstein et al.)	3 . 8 5	3 5 5
	MBE	40	3.2	[4, 51, 52]
	Orsay Cedex (Contour et al.)	-	3 5 0
	PLD	-	3.7	[53, 54, 5]
	Rome (Balestrino et al.)	3 . 8 5	3 0 0
	PLD	1000	3.5	[55, 56, 57]
	Tokyo (Hwang et al.)	3 . 8 4	3 6 0
	PLD	60	-	[58, 59, 60]
	Twente (This work)	3.845	350
	PLD	60	4.0

Acknowledgments

This research was financially supported by the Dutch Science Foundation, by NanoNed, a nanotechnology program of the Dutch Ministry of Economic Affairs and by the NanOxide program of the European Science Foundation. This work is supported in part by the Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, under contract DE-AC02-76SF00515.