Carlos Afonso
email: afonso@ccr.jussieu.fr

Yetrib Hathout

Catherine Fenselau

Evidence for Zinc Ion Sharing in Metallothionein Dimers Provided by Collision Induced Dissociation

Nanospray and collisionally induced dissociation are used to evaluate the presence and absence of interstrand co-chelation of zinc ions in dimers of two kinds of metallothionein. As was reported in a previous publication from this laboratory, co-chelation stabilizes the dimer to collisional activation, and facilitates asymmetrical zinc ion transfers during fragmentation. In the case of metallothionein, dimers of the holoprotein are found to share zinc ions, while dimers of metallothionein, in which one domain has been denatured, do not. Zinc ions are silent to most physicochemical probes, e.g., NMR and Mossbauer spectroscopies, and the capability of mass spectrometry to provide information on zinc complexes has widespread potential application in biochemistry.

In recent years electrospray ionization has been very useful for analysis of metal/protein complexes [1][2][3][4]. Applications to metalloproteins have been successful, largely because metal ions are usually complexed to proteins by strong electrostatic forces, and electrostatic forces are preserved and even reinforced in the water-free environment of the gas phase [5,6]. Electrospray and nanospray can provide information on identity and stoichiometry, e.g., the nature and number of metal ions present, with high mass accuracy and high sensitivity [7]. When relevant, information can also be obtained on the oxidation state of the metal ion [8,9]. More recently, collisionally induced dissociation (CID) has been used to provide information on cooperative binding within protein complexes, and on overall conformation [10,11]. We have previously proposed that CID can be used to distinguish zinc ion sharing or mixed ligand co-chelation in complexes of metallothionein and glutathione [4]. Metallothionein (MT) comprises two distinct domains, the N-terminal domain ( -domain) in which 9 cysteine residues coordinate three bivalent metal ions and the C-terminal domain (domain) in which 11 cysteines coordinate four metal ions. Metallothionein functions mainly as a zinc ion reservoir, providing zinc ions to other zinc depleted metalloenzymes. Jiang et al [START_REF] Jiang | Proc. Natl. Acad. Sci. USA[END_REF] showed that each domain reacts differently. The -domain is a better zinc donor than thedomain. It has also been demonstrated experimentally that metallothionein transfers zinc ions to other proteins, including proteins with lower zinc ion affinities, via intermolecular complexes [START_REF] Otvos | [END_REF][START_REF] Hathout | [END_REF][15]. It seems likely that zinc ions are transferred via rapid formation of intermolecular ligand fields [START_REF] Otvos | [END_REF]16].

Mass spectrometry is one of the few physical techniques that can provide information on biological zinc complexes, which are silent to most physicochemical probes [17]. Recently nanospray has been used to demonstrate that metal ions are rapidly redistributed between two molecules of MT, monitoring the scrambling that occurs after rapid mixing of Zn 7 MT with Cd 7 MT [17] Otvos et al. used NMR spectroscopy to provide evidence that this occurs via a transient dimeric intermediate [START_REF] Otvos | [END_REF]. No transient intermediate dimers could be detected by mass spectrometry. However, a stable dimeric complex of methallothionein as been characterized after isolation by size-exclusion chromatography, which appears to be stabilized by disulfide bridging [17].

The stoichiometry of this stable MT dimer in the gas phase is investigated here, and CID is used to address the question of interchain co-chelation of zinc ions. Control experiments are performed with a partially denatured MT dimer, to verify the specificity of the observed complexes and the validity of the CID method for characterizing "shared" zinc ions.

Experimental

Materials

Rabbit liver metallothionein 2a, zinc atomic absorption standard solution and ammonium bicarbonate were obtained from Sigma (St Louis, MO). Deionized water (18.2 M was obtained with a MilliQ (Millipore, Bedford, MA) apparatus. HPLC grade methanol and acetonitrile were obtained from Fisher Scientific (Pittsburgh, PA).

Sample preparation

Rabbit liver metallothionein 2a was purified by reverse phase HPLC and reconstituted with zinc ions, following a published procedure [START_REF] Hathout | [END_REF]. Briefly, metallothionein is denatured using hydrochloric acid and dipicolinic acid, and then the 2a isoform is purified by reverse-phase high pressure liquid chromatography (discarding the contaminating MT-IIb isoform). After lyophilization, the holoprotein Zn 7 MT is reconstituted by the addition of ZnCl 2, raising the pH slowly under argon to avoid oxidative dimerization. Excess zinc ions are eliminated by centrifugation using 5 kDa cutoff centrifugal ultrafiltration cartridge (Ultrafree, Millipore, Bedford, MA). Acetic acid (0.1 %) was added to this solution when partial denaturation was required.

An Applied Biosystems (Foster City, CA) Q-Star hybrid quadrupole/time of flight mass spectrometer equipped with a Protana (Odense, Denmark) nanoelectropray source was used for all the experiments. About 2 l of sample was loaded into a Protana metallized tip using Eppendorf GELoaders 1-10 l (Westbury, NY). The instrument was set to work in the positive ion mode with an ionization voltage set at 900 V. The declustering potential was varied between 30 and 100 V. For tandem mass spectrometry, argon was used as collision gas and the collision energy was varied between 20 and 50 V. External calibration was performed using a polypropylene glycol 3000 solution (Applied Biosystems, Foster City, CA). The quadrupole mass filter was tuned to transmit ions in the 200-2500 m/z range. Simulation of isotopic distributions, and calculation of apoprotein monoisotopic and average masses were performed with an ICR-2LS v2.66.79 program (EMSL). Determination of protein formulae was performed using GPMAW 4.12 (Lighthouse data, Odense, Denmark).

Results

Under physiologic conditions (pH 7.4) in ammonium bicarbonate buffer, the ionized protein carrying 7 zinc ions was observed as expected (6569 Da in Figure 1). This mass is in good agreement with the theoretical average mass calculated from the amino acid sequence, plus seven zinc ions [7,18]. Excess zinc ions were eliminated before the analysis by ultrafiltration, and no nonspecific adducts are detected in the spectra. The narrow charge distribution observed here (centered around the 5+ ions) is typical for this stable folded protein [2]. Ions detected at m/z 1878 are assigned to the stable dimeric form of Zn 7 -metallothionein carrying 7 protons. This dimeric species was found to be stable at relatively high declustering potentials (i.e. 100 V in Figure 1).

When the dimeric species carrying fourteen zinc ions was selected for collisional activation 50 V collision energy was required (Figure 2), and two complementary cluster ion distributions appeared, one with a +3 charge state and the other with a +4 charge state. The masses of each series correspond to the monomeric form of the protein with a continuum number of zinc ions. Although seven charges cannot be divided equally between the two CID product ions, the distribution observed indicates that the two polypeptides in the dimer have similar conformations and flexibility. The monomeric product ions with three charges carry from 7 to 12 zinc ions, with the 10 zinc specie as the most abundant. The monomeric protein product ions with four charges carry from 1 to 10 zinc ions. The 4 zinc specie is the most abundant. This experiment was repeated with a solution of metallothionein that had been partially denatured at pH 3.5 by addition of diluted acetic acid (see Experimental). It has been shown previously that the two domains of MT have different stabilities [START_REF] Jiang | Proc. Natl. Acad. Sci. USA[END_REF]. Below pH 3 both domains are unfolded and all zinc ions are released. At pH 3.5 the -domain unfolds and releases three zinc ions, whereas the -domain remains folded, binding four zinc ions. Under this condition the mass spectrum displays mainly metallothionein carrying 4 zinc ions (presumably those cooperatively bound in the alpha-domain). When a low declustering potential (30 V in Figure 3) is used, a dimeric form is also observed, at m/z 1823, (Figure 3), carrying eight zinc ions and seven excess protons.

Under collisional dissociation (Figure 4), the dimer of partially denatured metallothionein is fragmented into MT monomers at two charge states (+4 and +3), each carrying four zinc ions.

No evidence for any inter-cluster ion transfer was detected in the decompositon of this dimer.

Electrostatic interactions involving interstrand co-chelation of multiple zinc ions are expected to be relatively strong, and to stabilize the complex. It should be noted that higher CID energies were required to fragment the dimer of the fully metalated holoprotein, than were required to fragment the dimer of the partially denatured protein. A collision energy of 50 V was required to obtain the CID spectrum shown in Figure 2, while 30 V provided the CID spectrum in Figure 4.

Discussion

In an earlier study, CID was used to distinguish between Zn 7 MT complexes with glutathione derivatives that are stabilized by co-chelation of zinc ions, and those that are not [4],

This distinction is based on differences in the collision energy required for dissociation, and more importantly, on differences in the distribution of zinc ions between the dissociation products. In the present study, differences are observed on both counts in the dissociation of the dimer of fully metalated MT and the dimer of the partially demetalated protein. The disulfide linkage postulated in the stable dimer of Zn 7 MT may contribute to the requirement for higher collision energies for dissociation. However, the asymmetric distribution of zinc ions in the products argues strongly for co-chelation of some or all between the two polypeptide strands. Reflecting on the distribution of 10 and 4 zinc ions in the most abundant dissociation products, one can speculate that the three metal ions in each of the labile beta-domains are involved in interstrand ligands to a larger extent than those in the more stable alpha domains, and thus are more readily transferred between polypeptides. This is also consistent with the absence of interstrand transfer between dimers of Zn 4 MT. Co-chelation and transfer of metal ions from each beta domain would be symmetrically achieved in the head-to-tail conformation predicted for Zn 7 MT dimers based on the x-ray crystallographic structure [19] and supported by recent theoretical studies [20].

Figure 1 :

 1 Figure 1: Mass spectrum of Zn 7 MT in a pH 7.4 ammonium bicarbonate solution.

Figure 2 :

 2 Figure 2: Product ion spectrum of [2Zn 7 MT+7H] 7+ ions from the spectrum in Figure 1, obtained

Figure 3 :

 3 Figure 3: Mass spectra of Zn 7 MT partially denatured at pH 3.5.

Figure 4 :

 4 Figure 4: Product ion spectrum of [2Zn 4 MT+7H] 7+ from the spectrum in Figure 3, recorded with

Figure 4

 4 Figure 4

Acknowledgment

The authors thank Dr X. Yao and K. Reynolds for helpful discussions. This work was supported financially by the National Institutes for Health (GM 21248).


Daniel Li

Compact composition operators on Hardy-Orlicz and Bergman-Orlicz spaces *

Keywords: Bergman spaces, Bergman-Orlicz spaces, Blaschke product, Carleson function, Carleson measure, compactness, composition operator, Hardy spaces, Hardy-Orlicz spaces, Nevanlinna counting function MSC. Primary: 47B33, Secondary: 30H10, 30H20, 30J10, 46E15

, that every composition operator which is compact on the Hardy space H p , 1 ≤ p < ∞, is also compact on the Bergman space B p = L p a (D). In this survey, after having described the above known results, we consider Hardy-Orlicz H Ψ and Bergman-Orlicz B Ψ spaces, characterize the compactness of their composition operators, and show that there exist Orlicz functions for which there are composition operators which are compact on H Ψ but not on B Ψ .

f B p = D |f (z)| p dA(z) 1/p
, * These results come from joint works with P. Lefèvre, H. Queffélec and L. Rodríguez-Piazza ( [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], [START_REF] Lefèvre | Nevanlinna counting function and Carleson function of analytic maps[END_REF], [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF]). It is an expanded version of the conference I gave at the ICM satellite conference Functional Analysis and Operator Theory, held in Bangalore, India, 8-11 august 2010.

C ϕ : H p → H p resp. C ϕ : B p → B p ,
thanks to Littlewood's subordination principle (see [START_REF] Duren | Theory of H p Spaces[END_REF], Theorem 1.7). The function ϕ is called the symbol of C ϕ .

Compactness

The compactness of composition operators on Bergman spaces had been characterized in 1975 by D. M. Boyd ( [START_REF] Boyd | Composition operators on the Bergman space[END_REF]) for p = 2 and by B. MacCluer and J. Shapiro in 1986 for the other p's ( [START_REF] Maccluer | Angular derivatives and compact composition operators on the Hardy and Bergman spaces[END_REF], Theorem 3.5 and Theorem 5.3): Theorem 1.1 (Boyd (1976), MacCluer-Shapiro (1986)) Let ϕ be an analytic self-map of D. Then, for 1 ≤ p < ∞, one has:

C ϕ : B p → B p compact ⇐⇒ lim |z|→1 1 -|ϕ(z)| 1 -|z| = +∞ . (1)
That means that ϕ(z) approaches the boundary of D less quickly that z. That means also (but we do not need this remark in the sequel) that ϕ has no finite angular derivative on ∂D (ϕ has a finite angular derivative at ω ∈ ∂D if the angular limits ϕ * (ω) = lim z→ω ϕ(z) and ϕ ′ (ω) = ∠ lim z→ω ϕ(z)-ϕ * (ω) z-ω exist, with |ϕ * (ω)| = 1; by the Julia-Carathéodory Theorem, the non-existence of such a derivative is equivalent to the right-hand side of (1): see [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], § 4.2). We actually prefer to write [START_REF] Boyd | Composition operators on the Bergman space[END_REF] in the following way:

C ϕ : B p → B p compact ⇐⇒ lim |z|→1 1 -|z| 1 -|ϕ(z)| = 0 . (2)
On the other hand, it is not difficult to see ([14], Theorem 2.1, or [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], § 3.5):

Proposition 1.2 [START_REF] Shapiro | Compact, nuclear, and Hilbert-Schmidt composition operators On H 2[END_REF]) Let ϕ be an analytic self-map of D. Then, for 1 ≤ p < ∞, one has:

C ϕ : H p → H p compact =⇒ lim |z|→1 1 -|z| 1 -|ϕ(z)| = 0 . ( 3
)
(this is actually an equivalence when ϕ is univalent ( [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], § 3.2), or more generally boundedly-valent, but there are Blaschke products for which the converse of (3) is not true: see [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], § 10.2, or [START_REF] Lefèvre | Some revisited results about composition operators on Hardy spaces[END_REF], Theorem 3.1, for a more general result, with a simpler proof).

Hence:

Corollary 1.3 For 1 ≤ p < ∞, one has:

C ϕ : H p → H p compact =⇒ C ϕ : B p → B p compact.
The converse is not true.

Goal

Our goal is to replace the classical Hardy spaces H p and Bergman spaces B p by Hardy-Orlicz spaces H Ψ and Bergman-Orlicz spaces B Ψ and compare the compactness of composition operators C ϕ :

H Ψ → H Ψ and C ϕ : B Ψ → B Ψ .
We shall detail that in Section 3.

Some comments

To prove Proposition 1.2, J. H. Shapiro and P. D. Taylor used the following result ([14], Theorem 6.1):

Theorem 1.4 (Shapiro-Taylor (1973)) If the composition operator C ϕ is compact on H p0 for some 1 ≤ p 0 < ∞, then it is compact on H p for all 1 ≤ p < ∞.
Proof. First, by Montel's Theorem, C ϕ is compact on H p if and only if C ϕ (f n ) p converges to 0 for every sequence (f n ) in the unit ball of H p which converges uniformly to 0 on compact subsets of D. One then uses Riesz's factorization Theorem: if f n is in H p , we can write f n = B n g n , where B n is a Blaschke product and g n has no zero in D. By Montel's Theorem, we may assume that (g n ) n converge uniformly on compact sets of D. Setting h n = g p/p0 n , we get a function which is in the unit ball of H p0 . Since (g n ) n converges uniformly on compact sets of D, so does (h n ) n . Its limit h belongs also to the unit ball of H p0 and, by the compactness of

C ϕ , C ϕ (h n ) n converges to C ϕ (h) in H p0 .
Now the compactness of C ϕ on H p0 implies that |ϕ * | < 1 almost everywhere (ϕ * is the boundary values function of ϕ). In fact, let P n (z) = z n , since (P n ) is in the unit ball of H p0 and converges uniformly to 0 on compact subsets of D, one has

C ϕ (P n ) p0 → 0. But, if E ϕ = {ξ ∈ ∂D ; |ϕ * (ξ)| = 1}, one has C ϕ (P n ) p0 p0 ≥ Eϕ |ϕ * (ξ)| np0 dm(ξ) ≥ m(E ϕ )
, where m is the normalized Lebesgue measure on ∂D. Hence m(E ϕ ) = 0.

It follows that the sequence (

h n • ϕ) * n = (h n • ϕ * ) n converges almost everywhere to h • ϕ * on ∂D. Since C ϕ (h n ) n converges in the norm of L p0 (T), Vitali's convergence Theorem gives: lim m(E)→0 sup n E |h n • ϕ * | p0 dm = 0 . But E |f n • ϕ * | p dm ≤ E |g n • ϕ * | p dm = E |h n • ϕ * | p0 dm , so Vitali's convergence Theorem again gives f n • ϕ p → 0, since f n • ϕ * tends to 0 a. e. on ∂D.
Actually, the proof of Proposition 1.2 can be made without using Theorem 1.4, but we gave it to see that Riesz's factorization Theorem is the main tool.

Proof of Proposition 1.2. For every z ∈ D, the evaluation map e z : f ∈ H p → f (z) is a continuous linear form and e z ≤ 2 1/p

(1-|z|) 1/p (see [START_REF] Duren | Theory of H p Spaces[END_REF], lemma in § 3.2, page 36). But actually

e z = 1 (1-|z| 2 ) 1/p . Indeed, let u z (ζ) = 1-|z| 1-zζ 2/p , |ζ| < 1.
Then, using the Parseval formula, u z p p = 1-|z| 1+|z| . Therefore

e z ≥ |u z (z)| u z p ≥ 1 (1+|z|) 2/p 1-|z| 1+|z| 1/p = 1 (1 -|z| 2 ) 1/p •
On the other hand, it is clear, by the Cauchy-Schwarz inequality, that |h(z)| ≤ 1/(1 -|z| 2 ) 1/2 for every h in the unit ball of H 2 ; hence, if f is in the unit ball of H p , and we write f = Bg, where B is the Blaschke product associated to the zeroes of f , we get, since h = g p/2 is in the unit ball of

H 2 : |f (z)| ≤ |g(z)| = |h(z)| 2/p ≤ 1/(1 -|z| 2 ) 1/p . Now, ez ez w * -→ |z|→1 0 in (H p ) * , because e z (P )(1 -|z|) 1/p = P (z)(1 -|z|)
= 1-|z| 2 1-|ϕ(z)| 2 1/p ≥ 1 2 1/p 1-|z| 1-|ϕ(z)| 1/p .
For Bergman spaces, the necessary condition of compactness in Theorem 1.1 follows the same lines as in the Hardy case. B. D. MacCluer and J. H. Shapiro ([9], Theorem 5.3) proved the sufficient condition, in showing that the compactness of C ϕ on one of the Bergman spaces B p0 , 1 ≤ p 0 < ∞, implies its compactness for all the Bergman spaces B p , 1 ≤ p < ∞ (see Theorem 1.5 below), and then used Boyd's result for p = 2 (actually, they gave a new proof of Boyd's result). To do that, since there is no Bergman version of Riesz's factorization Theorem, they had to use another tool and they used the notion of Carleson measure, that we shall develop in the next section. Before that, let us give a proof of Boyd's result. We follow [START_REF] Zhu | Operator Theory in Function Spaces[END_REF], Theorem 10.3.5.

Proof of Theorem 1.1 (for p = 2). We only have to show that lim

|z|→1 1-|z| 1-|ϕ(z)| = 0 implies the compactness of C ϕ on B 2 .
We may assume that ϕ(0

) = 0. Indeed, if ϕ(0) = a, let ψ = ϕ a • ϕ, with ϕ a (z) = a-z 1-āz . One has ψ(0) = 0 and C ϕ = C ψ • C ϕa , since ϕ = ϕ a • ψ. Hence the compactness of C ψ implies the one of C ϕ . Moreover, one has |ψ(z)| ≤ |ϕ(z)|, so that the condition lim |z|→1 1-|z| 1-|ϕ(z)| = 0 implies that lim |z|→1 1-|z| 1-|ψ(z)| = 0.
Let (f n ) be a sequence in the unit ball of B 2 which converges to 0 uniformly on compact subsets of D. Then so does (f ′ n ). Using Taylor expansion, one has a constant C > 0 such that:

D |f (z)| 2 dA(z) ≤ C |f (0)| 2 + D (1 -|z| 2 ) 2 |f ′ (z)| 2 dA(z) for every analytic function f : D → C. It follows that C ϕ (f n ) 2 B 2 ≤ C |(f n • ϕ)(0)| 2 + D (1 -|z| 2 ) 2 |(f n • ϕ) ′ (z)| 2 dA(z) for every n ≥ 1. Since f n [ϕ(0)] -→ n→∞ 0
, it remains to show that the integral tends to 0.

For every ε > 0, we may take, by hypothesis, some δ > 0, with δ < 1, such

that 1-|z| 2 1-|ϕ(z)| 2 ≤ ε for δ ≤ |z| < 1.
This implies that:

D (1 -|z| 2 ) 2 |(f n • ϕ) ′ (z)| 2 dA(z) ≤ |z|<δ (1 -|z| 2 ) 2 |(f ′ n • ϕ)(z)| 2 |ϕ ′ (z)| 2 dA(z) + ε δ≤|z|<1 (1 -|ϕ(z)| 2 )(1 -|z| 2 )|ϕ ′ (z)| 2 |(f ′ n • ϕ)(z)| 2 dA(z) .
Denote by I n the first integral and by J n the second one. Since f ′ n [ϕ(z)] tends to 0 uniformly for |z| ≤ δ, I n tends to 0. It remains to show that the sequence (J n ) is bounded. Since 1 -|z| 2 ≤ 2 log 1/|z|, the change of variable formula (see [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], p. 179, or [START_REF] Zhu | Operator Theory in Function Spaces[END_REF], Proposition 10.2.5) gives:

J n ≤ 2 D (1 -|ϕ(z)| 2 )|ϕ ′ (z)| 2 |(f ′ n • ϕ)(z)| 2 log 1 |z| dA(z) = 2 D (1 -|w| 2 )|f ′ n (w)| 2 N ϕ (w) dA(w).
Since ϕ(0) = 0, Littlewood's inequality (see [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], § 10.4) reads as N ϕ (w) ≤ log 1/|w| for w = 0. We get, since 2 log 1/|w| ≈ 1 -|w| 2 as |w| → 1, a constant C > 0 such that:

J n ≤ C D (1 -|w| 2 ) 2 |f ′ n (w)| 2 dA(w) ,
and that ends the proof, since one easily sees (using Taylor expansion) that this last integral is less or equal than D |f n (w)| 2 dA(w) ≤ 1.

Carleson measures

If D is the open unit disk of the complex plane and T = ∂D is the unit circle, we denote by A the normalized area measure on D and by m the normalized Lebesgue measure on T.

The Carleson window W (ξ, h) centered at ξ ∈ T and of size h, 0 < h ≤ 1, is the set

W (ξ, h) = {z ∈ D ; |z| > 1 -h and | arg(z ξ)| < πh} .
The point is that the two dimensions of the window W (ξ, h) are proportional.

An α-Carleson measure (Carleson measure if α = 1) is a measure µ on D such that:

ρ µ (h) := sup |ξ|=1 µ[W (ξ, h)] = O (h α ) .
We say that ρ µ is the Carleson function of µ. We denote by A ϕ the pull-back measure of A by ϕ and by m ϕ the pull-back measure of m by ϕ * , ϕ * being the boundary values function of ϕ. Recall that these pull-back measures are defined by m ϕ (E) = m[ϕ * -1 (E)] and A ϕ (E) = A[ϕ -1 (E)] for every Borel set E of ∂D or of D, respectively. We write ρ ϕ and ρ ϕ,2 the Carleson functions of m ϕ and A ϕ respectively. We call them the Carleson function of ϕ and the Carleson function of order 2 of ϕ.

Carleson's Theorem (see [START_REF] Duren | Theory of H p Spaces[END_REF], Theorem 9.3) says that, for p < ∞, the inclusion map

I µ : f ∈ H p → f ∈ L p (µ)
: H p → L p (D, m ϕ ), resp. J ϕ : B p → L p (D, A ϕ ), since: C ϕ (f ) p H p = D |f | p dm ϕ and C ϕ (f ) p B p = D |f | p dA ϕ .
Hence the continuity of the composition operator C ϕ , both on H p and B p , implies that m ϕ is a Carleson measure and A ϕ is a 2-Carleson measure. It should be stressed that in the Hardy case, m ϕ is a measure on D, and not on D in general, so we have to adapt the previous notations in this case.

For compactness, one has the following result:

Theorem 1. 5 (MacCluer (1985), MacCluer-Shapiro (1986)) For 1 ≤ p < ∞, and every analytic self-map ϕ of D, one has:

C ϕ : H p → H p compact ⇐⇒ ρ ϕ (h) = o (h), as h → 0, and
C ϕ : B p → B p compact ⇐⇒ ρ ϕ,2 (h) = o (h 2 ), as h → 0.
Recall that when the composition operator C ϕ : H p → H p is compact, one has |ϕ * | < 1 a.e., and hence m ϕ is supported by D. Since these characterizations do not depend on p, one recovers Theorem 1.4 and get its Bergman counterpart.

We shall see in the next section how this theorem changes when we replace the classical Hardy and Bergman spaces by their Orlicz generalizations.

Proof of Theorem 1.5. We only prove the Bergman case; the Hardy case being analogous.

1) Assume that C ϕ is compact on B p . Consider, for every a ∈ D, the Berezin kernel

H a = (1 -|a| 2 ) 2 |1 -az| 4 •
One has H a B 1 = 1 and

H a ∞ = (1 -|a| 2 ) 2 (1 -|a|) 4 = (1 + |a|) 2 (1 -|a|) 2 ≤ 4 (1 -|a|) 2 ; hence, writing a = (1 -h)ξ, 0 < h ≤ 1, |ξ| = 1, we get H a B p ≤ (4/h 2 ) 1-
C ϕ (f a ) p B p = D |f a • ϕ| p dA ≥ W (ξ,h) |f a | p dA ϕ ≥ C p p h 2 A ϕ [W (ξ, h)] . Hence ρ ϕ,2 (h) = o (h 2 ).
2) Conversely, assume that ρ ϕ,2 (h) = o (h 2 ), and let (f n ) be a sequence in the unit ball of B p converging uniformly to 0 on compact subsets of D, and ε > 0.

For every measure µ on

D, let K µ,2 (h) = sup 0<t≤h ρµ(t) t 2 and K ϕ,2 (h) = K Aϕ,2 (h).
By hypothesis, there is a δ > 0 such that K ϕ,2 (δ) ≤ ε. Let µ be the measure 1I D\D(0,1-δ) .A ϕ . One has K µ,2 (1) ≤ 2K ϕ,2 (δ), because, for δ < h ≤ 1, the intersection of a window of size h with the annulus {z ∈ D ; 1 -δ < |z| < 1} can be covered by less than 2(h/δ) windows of size δ. Now, the Bergman version of Carleson's Theorem (see [START_REF] Stegenga | Multipliers of the Dirichlet space[END_REF], proof of Theorem 1.2, bottom of the page 117, with α = 1/2) says that:

D |f (z)| p dµ ≤ C p K µ,2 (1) f p B p
for every f ∈ B p . Hence:

D |f n • ϕ| p dA = D(0,1-δ) |f n (z)| p dA ϕ (z) + D\D(0,1-δ) |f n (z)| p dA ϕ (z) ≤ ε + 2C p ε ,
for n large enough, since (f n ) converges uniformly to 0 on D(0, 1 -δ). It follows that C ϕ (f n ) B p converges to 0.

2 Hardy-Orlicz and Bergman-Orlicz spaces

Orlicz spaces

An Orlicz function is a function Ψ : [0, ∞) → [0, ∞) which is positive, nondecreasing, convex and such that Ψ(0) = 0, Ψ(x) > 0 for x > 0 and Ψ

(x) -→ x→∞ ∞. Examples. Ψ(x) = x p ; Ψ(x) = x p log(x + 1), 1 ≤ p < ∞; Ψ(x) = e x q -1; Ψ(x) = exp[ log(x + 1) q ] -1, q ≥ 1.
If (S, T , µ) is a finite measure space, the Orlicz space L Ψ (µ) is the space of classes of measurable functions f : S → C such that, for some C > 0:

S Ψ(|f |/C) dµ < ∞ .
The norm is defined by:

f Ψ = inf{C ; S Ψ(|f |/C) dµ ≤ 1} .
For Ψ(x) = x p , we get the classical Lebesgue space: L Ψ (µ) = L p (µ).

Hardy-Orlicz and Bergman-Orlicz spaces

We define the Bergman-Orlicz space B Ψ by:

B Ψ = {f ∈ L Ψ (D, A) ; f analytic} , with the norm f B Ψ = f L Ψ (D,A) .
The Hardy-Orlicz space H Ψ can be defined as in the classical case (see [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], Definition 3.2), but is is easier to define it by:

H Ψ = {f ∈ H 1 ; f * ∈ L Ψ (T, m)} , with the norm f H Ψ = f * L Ψ (m) .
As in the classical case (because Ψ(|f |) is subharmonic), Littlewood's subordination principle implies that every analytic ϕ : D → D induces bounded composition operators C ϕ : B Ψ → B Ψ and C ϕ : H Ψ → H Ψ . Theorem 2.1 Let µ be a finite positive measure on D, and assume that the identity maps I µ : H Ψ → L Ψ (µ) and J µ : B Ψ → L Ψ (µ) are defined. Then:

Compactness

1) lim h→0 Ψ -1 (1/h) Ψ -1 [1/hK µ (h)] = 0 ⇒ I µ compact ⇒ lim h→0 Ψ -1 (1/h) Ψ -1 [1/ρ µ (h)] = 0; 2) lim h→0 Ψ -1 (1/h 2 ) Ψ -1 [1/h 2 K µ,2 (h)] = 0 ⇒ J µ compact ⇒ lim h→0 Ψ -1 (1/h 2 ) Ψ -1 [1/ρ µ (h)] = 0, where K µ (h) = sup 0<t≤h ρ µ (t) t and K µ,2 (h) = sup 0<t≤h ρ µ (t) t 2 •
Actually, the sufficient conditions imply the existence of the identity maps I µ and J µ .

The conditions in 1), resp. 2), are equivalent if Ψ is "regular" (namely, if Ψ satisfies the condition ∇ 0 , whose definition is given after Proposition 4.3 below), as Ψ(x) = x p , for which case both conditions in 1) read as

ρ µ (h) h -→ h→0 0 and in 2) as ρ µ,2 (h) h 2 -→ h→0
0, but examples show that there is no equivalence in general (see [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], pp. 50-54 and [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF], § 2).

Compactness for composition operators

Nevertheless, for composition operators C ϕ , the following theorem (see [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], Theorem 4.19 and [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF], Theorem 3.1), which is one of the main result of this survey, says that:

K ϕ (h) ≈ ρ ϕ (h)/h and K ϕ,2 (h) ≈ ρ ϕ,2 (h)/h 2 .
Theorem 2.2 There is a universal constant C > 0 such that, for every analytic self-map ϕ : D → D, one has, for every 0 < ε < 1 and h > 0 small enough, for every ξ ∈ T:

1) m[ϕ * ∈ W (ξ, εh)] ≤ C ε m[ϕ * ∈ W (ξ, h)]; 2) A[ϕ ∈ W (ξ, εh)] ≤ C ε 2 A[ϕ ∈ W (ξ, h)].
For fixed h, this expresses that the measure m ϕ is a Carleson measure and A ϕ is a 2-Carleson measure. The theorem says that this is true at "all scales".

It follows that:

Theorem 2.3 For every analytic self-map ϕ : D → D, one has:

1) C ϕ : H Ψ → H Ψ compact ⇐⇒ lim h→0 Ψ -1 (1/h) Ψ -1 [1/ρ ϕ (h)] = 0; 2) C ϕ : B Ψ → B Ψ compact ⇐⇒ lim h→0 Ψ -1 (1/h 2 ) Ψ -1 [1/ρ ϕ,2 (h)] = 0.
Let us give a very vague idea of the proof of Theorem 2.2, in the Bergman case (the proof in the Hardy case, though following the same ideas, is different, and actually more difficult). By setting f = h/(1 -ϕ), it suffices to show that

A({|f | > λ}) ≤ K λ 2 A({|f | > 1}) for λ ≥ λ 0 > 0 , (4)
for every analytic function f :

D → Π + = {z ∈ C ; Re z > 0} such that |f (0)| ≤ α 0
, for some α 0 > 0. But the fact that A ϕ is a 2-Carleson measure writes:

A({|f | > λ}) ≤ C λ 2 |f (0)| , λ > 0 , (5)
where f = h/(1 -ϕ). One has hence to replace |f (0)| in the majorization by A({|f | > 1}). To that effect, one splits the disk D into pieces which are "uniformly conform" to D and on which we can use [START_REF] Lefèvre | Nevanlinna counting function and Carleson function of analytic maps[END_REF]. However, it is far from being so easy, and we refer to [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF] (and [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF] for the Hardy case) for the details.

3 Compactness on H Ψ versus compactness on B Ψ Thanks to Theorem 2.3, in order to compare the compactness of the composition operator C ϕ on H Ψ and on B Ψ , we have to compare ρ ϕ (h) and ρ ϕ,2 (h). But if one reads their definitions:

ρ ϕ (h) = sup |ξ|=1 m[ϕ * ∈ W (ξ, h)] and ρ ϕ,2 (h) = sup |ξ|=1 A[ϕ ∈ W (ξ, h)] ,
that does not seem straightforward. We shall compare them in an indirect way, by using the Nevanlinna counting function.

Nevanlinna counting function

The Nevanlinna counting function counts how many pre-images each element has, with a weight which decreases when this pre-image approaches ∂D. Namely:

N ϕ (w) = ϕ(z)=w log 1 |z|
for w ∈ ϕ(D) and w = ϕ(0). One sets N ϕ (w) = 0 for the other w ∈ D.

Our second main theorem asserts that the Nevanlinna counting function of ϕ is equivalent to its Carleson function (see [START_REF] Lefèvre | Nevanlinna counting function and Carleson function of analytic maps[END_REF]). Theorem 3.1 There exists a universal constant C > 1 such that, for every analytic self-map ϕ : D → D and for h > 0 small enough, we have:

1 C sup w∈W (ξ,h/C) N ϕ (w) ≤ m ϕ [W (ξ, h)] ≤ C A[W (ξ, Ch)] W (ξ,Ch) N ϕ (z) dA(z) .
Now, if one defines the Nevanlinna function of order 2 by:

N ϕ,2 (w) = ϕ(z)=w log 1 |z| 2
for w ∈ ϕ(D) and w = ϕ(0) and N ϕ,2 (w) = 0 for the other w ∈ D, one has easily (see [START_REF] Shapiro | The essential norm of a composition operator[END_REF], Proposition 6.6):

N ϕ,2 (w) = 2 1 0 N ϕ (r, w) dr r ,
where

N ϕ (r, w) = ϕ(z)=w,|z|<r log r |z| is the restricted Nevanlinna function. But, since N ϕ (r, w) = N ϕr (w) with ϕ r (z) = ϕ(rz)
, one gets, by integrating in polar coordinates:

Corollary 3.2 There is some universal constant C > 1 such that, for every analytic self-map ϕ : D → D and for h > 0 small enough:

1 C ρ ϕ,2 (h/C) ≤ sup |w|≥1-h N ϕ,2 (w) ≤ C ρ ϕ,2 (Ch)
But now, it is easy to compare N ϕ and N ϕ,2 : one has:

N ϕ,2 (w) ≤ [N ϕ (w)] 2
(simply because the ℓ 2 -norm is smaller than the ℓ 1 -norm). Therefore:

Theorem 3.
3 There is a universal constant C > 1 such that, for every analytic self-map ϕ : D → D, one has for h > 0 small enough and every ξ ∈ T:

A ϕ [W (ξ, h)] ≤ C m ϕ [W (ξ, Ch)] 2
We can now compare the compactness on H Ψ and on B Ψ .

Theorem 3.4 Under some condition on Ψ, one has:

C ϕ : H Ψ → H Ψ compact =⇒ C ϕ : B Ψ → B Ψ compact. -1 0 G
This condition has not a very nice statement:

∀A > 0, ∃x A > 0, ∃B ≥ A : Ψ[AΨ -1 (x 2 )] ≤ Ψ[BΨ -1 (x)] 2 , x ≥ x A (though, setting χ A (x) = Ψ[AΨ -1 (x)], it writes better χ A (x 2 ) ≤ [χ B (x)] 2
), but it is satisfied in many cases:

-if Ψ grows moderately; namely, satisfies the condition ∆ 2 , i.e. Ψ(2x) ≤ C Ψ(x) for x large enough; this is the case for Ψ(x) = x p , and we recover the classical case of Corollary 1.3;

-if Ψ grows quickly; namely, satisfies the condition ∆ 2 , i.e. for some α > 1, [Ψ(x)] 2 ≤ Ψ(αx) for x large enough; for instance if Ψ(x) = e x q -1, q ≥ 1;

-but also for Ψ(x) = exp[ log(x + 1) 2 ] -1.

Nevertheless ( [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF], Theorem 4.2):

Theorem 3.5 There exists a symbol ϕ and an Orlicz function Ψ such that the composition operator C ϕ : H Ψ → H Ψ is compact, and moreover in all Schatten classes S p (H 2 ), p > 0, whereas C ϕ : B Ψ → B Ψ is not compact.

The symbol ϕ is a conformal map from D onto the domain G, represented on the picture, delimited by three circular arcs of radii 1/2. The Carleson function of ϕ is "small" whereas its Carleson function of order 2 is "small":

ρ ϕ (h) ≤ C e -π/4h ρ ϕ,2 (h) ≥ (1/C) e -π/h
Now, it remains to construct a concave and piecewise linear function F (and Ψ will be F -1 ) in such way that:

lim x→∞ F (x) F (e πx/4 ) = 0 and lim sup x→∞ F (x 2 ) F (e πx ) > 0 , in order that lim h→0 Ψ -1 (1/h) Ψ -1 [1/ρ ϕ (h)] = 0, but lim sup h→0 Ψ -1 (1/h 2 ) Ψ -1 [1/ρ ϕ,2 (h)] > 0.
Remark. Let us stress that the compactness of C ϕ : H Ψ → H Ψ always implies the compactness of C ϕ :

B Ψ 2 → B Ψ 2 . Indeed, if Ψ(x) = [Ψ(x)] 2 , then Ψ-1 (t 2 ) = Ψ -1 (t), so we get Ψ-1 (1/h 2 )/ Ψ-1 1/ν ϕ,2 (h) ≤ Ψ -1 (1/h)/Ψ -1 1/ν ϕ (h) , since one has ν ϕ,2 (h) ≤ [ν ϕ (h)] 2
, where ν ϕ (h) = sup |w|≥1-h N ϕ (w).

4 Final remarks

Modulus of the symbol and compactness on Bergman-Orlicz spaces

What about the compactness of C ϕ on the Bergman-Orlicz spaces and the behaviour of the modulus of its symbol ϕ?

We prove in [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], Theorem 5.7, that the compactness of C ϕ : B Ψ → B Ψ implies that:

lim |a|→1 Ψ -1 1 (1 -|ϕ(a)|) 2 Ψ -1 1 (1 -|a|) 2 = 0 . ( 6
)
The proof is essentially the same as the proof of Proposition 1.2 given in Section 1.3. But it follows also from Theorem 2.3 and Corollary 3.2. Indeed, these results have the following consequence:

lim h→0 Ψ -1 (1/h 2 ) Ψ -1 1/ν ϕ,2 (h) = 0 , (7)
where ν ϕ,2 (h) = sup |w|≥1-h N ϕ,2 (w). Now, since N ϕ,2 ϕ(z) ≥ log(1/|z|) Let us recall that ϕ bounded-valent means that there is an integer L ≥ 1 such that the equation ϕ(z) = w has at most L solution(s) in D, for every w ∈ D; we then have N ϕ,2 (w) ≤ L(1 -|z|), where ϕ(z) = w, with |z| > 0 minimal, and Theorem 4.2 follows.

Compactness of C ϕ also is equivalent to [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF], for every symbol ϕ, if one adds a condition on the Orlicz function Ψ. Condition ∇ 0 is the "regularity" condition mentioned after Theorem 2.1, which gives the equivalence between the necessary and sufficient conditions. Ψ satisfies ∇ 0 if (see [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], Definition 4.5 and Proposition 4.6): there is x 0 > 0 such that, for every B > 1, there exists c B > 1 such that:

Ψ(Bx) Ψ(x) ≤ Ψ(c B By) Ψ(y) for x 0 ≤ x ≤ y. (8)
Let us point out that it is satisfied, in particular, when log Ψ(e x ) is convex, or if Ψ satisfies the condition ∆ 2 ([4], Proposition 4.7).

Proof. We may assume that ϕ(0) = 0. Then Littlewood's inequality writes N ϕ (w) ≤ log 1/|w|, w = 0, and gives, for some constant C > 1:

N ϕ,2 (w) ≤ C sup ϕ(z)=w (1 -|z|) ϕ(z)=w log 1 |z| ≤ C 2 (1 -|w|) sup ϕ(z)=w (1 -|z|) .
By hypothesis, for every A > 0, we have, with w = ϕ(z):

Ψ -1 1 (1 -|z|) 2 ≥ A Ψ -1 1 (1 -|ϕ(z)|) 2
for |z| close enough to 1. With h = 1 -|w|, this writes:

1 -|z| ≤ 1/ Ψ AΨ -1 (1/h 2 ) .
We get hence:

ν ϕ,2 (h) ≤ C 2 h Ψ AΨ -1 (1/h 2 )
if h > 0 is small enough. It follows that we shall have:

lim h→0 Ψ -1 (1/h 2 ) Ψ -1 1/ν ϕ,2 (h) = 0
if for every B > 1, we can find A > 0 such that:

Ψ -1 Ψ[AΨ -1 (1/h 2 )] C 2 h ≥ BΨ -1 (1/h 2 ) .
Setting x = Ψ -1 (1/h 2 ), it suffices to have:

Ψ(Bx) 2 ≤ Ψ(x) Ψ (A/C 4 )x , (9)
for x > 0 big enough, since Ψ (A/C 4 )x ≤ Ψ(Ax)/C 4 by convexity.

When Ψ ∈ ∇ 0 , (8) gives ( 9) with y = Bx and A = C 4 c B B 2 .

Blaschke products

We may ask about the converse implication: does the compactness of C ϕ on B Ψ imply its compactness on H Ψ ? Even in the Hilbertian case B 2 -H 2 , this is not the case (see [START_REF] Shapiro | Composition Operators and Classical Function Theory[END_REF], pp. 183-185): there is a Blaschke product B (whose associated composition operator is an isometry from H 2 into itself) with no angular derivative, so C B is compact on B 2 . Another example (a Blaschke product also) is given in [START_REF] Lefèvre | Composition operators on Hardy-Orlicz spaces[END_REF], when Ψ(x) = e x 2 -1. More generally: Theorem 4.4 For every Orlicz function Ψ satisfying the condition ∇ 0 , there is a Blaschke product B, whose associated composition operator C B is an isometry from H Ψ into itself, but such that C B is compact on B Ψ .

Proof. Indeed ( [START_REF] Lefèvre | Some revisited results about composition operators on Hardy spaces[END_REF], Theorem 3.1), for every function δ : (0, 1) → (0, 1/2] such that δ(t) -→ t→0 0, there is a Blaschke product B such that:

1 -|B(z)| ≥ δ(1 -|z|),
for all z ∈ D .

By replacing B(z) by zB(z), we may assume that B(0) = 0 (note that 1 -|zB(z)| ≥ 1 -|B(z)|). Then C B is an isometry from H Ψ into itself. Indeed, one can see ( [START_REF] Nordgren | Composition operators[END_REF], Theorem 1) that if ϕ is an inner function, then the pull-back measure m ϕ is equal to P a .m, when P a is the Poisson kernel at a = ϕ(0). When ϕ(0) = 0, one has m ϕ = m and C ϕ is an isometry. Hence, taking, for t > 0 small enough:

δ(t) = 1 Ψ Ψ -1 (1/t 2 )
, we get

Ψ -1 1 (1 -|B(z)|) 2 Ψ -1 1 (1 -|z|) 2 ≤ 1 Ψ -1 1 (1 -|z|) 2
, and we get [START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF]. Hence, assuming that Ψ satisfies the condition ∇ 0 , C B is compact on B Ψ , by Proposition 4.3.

Remark. Another, but different, survey on this topic can be found in [START_REF] Queffélec | On Hardy type and Bergman type spaces[END_REF]

1 Introduction

 1 Let D = {z ∈ C ; |z| < 1} be the open unit disk of the complex plane. For 1 ≤ p < ∞, consider the Hardy space H p = {f : D → C ; f analytic and f H p < +∞} , where f H p = sup r<1 1 2π 2π 0 |f (re it )| p dt 1/p , and the Bergman space B p (otherwise denoted by A p or L p a ) B p = {f : D → C ; f analytic and f ∈ L p (D, A)} , whose norm is defined by

1 where

 1 dA = dxdy π is the normalized area measure on D. Every analytic self-map ϕ : D → D (such a function is also known as a Schur function, or function of the Schur-Agler class) defines a composition operator f → C ϕ (f ) = f • ϕ which is a bounded linear operator

1 p and the function f a = h 2 4 1 - 1 p

 1211 H a is in the unit ball of B p . Moreover, f a tends to 0 as |a| → 1 uniformly on compact subsets of D. Since C ϕ is compact on B p , one has C ϕ (f a ) B p -→ |a|→1 0. But it is easy to see that |1 -āz| ≤ 5h when z ∈ W (ξ, h). Hence |f a (z)| ≥ C p /h 2/p when z ∈ W (ξ, h) and

Theorem 1 .

 1 5 has the following generalization ([4], Theorem 4.11) and [6], Theorem 2.5):

Theorem 4 . 1

 41 For every analytic self-map ϕ : D → D and for every Orlicz function Ψ, the composition operator C ϕ : B Ψ → B Ψ is compact if and only if:

Proposition 4 . 3

 43 Assume that the Orlicz function Ψ satisfies the condition ∇ 0 . Then the composition operator C ϕ : B Ψ → B Ψ is compact if and only if[START_REF] Lefèvre | Compact composition operators on Bergman-Orlicz spaces[END_REF] holds.

  is defined and bounded if and only if µ is a Carleson measure. A Bergman version has been proved by W. W. Hastings in 1975 ([3]): J µ : f ∈ B p → f ∈ L p (µ) is defined and bounded if and only if µ is a 2-Carleson measure. Now, composition operators C ϕ : H p → H p , resp. C ϕ : B p → B p , may be seen as inclusion maps I ϕ


MSc Marian E Michielsen
email: m.michielsen@erasmusmc.nl

MD Marion Smits

MD, PhD Gerard M Ribbers

MD, PhD Henk J Stam

PhD Jos N Van Der Geest

Johannes Bj Bussmann

PhD Ruud W Selles

PhD Johannes B J Bussmann

The neuronal correlates of mirror therapy: an fMRI study on mirror induced visual illusions in stroke patients

Keywords: Stroke, Upper-Limbs, Rehabilitation, fMRI, Mirror Therapy

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

INTRODUCTION

Mirror therapy was first introduced by Ramachandran and co-workers to alleviate phantom limb pain in amputees. [START_REF] Ramachandran | Touching the phantom limb[END_REF] By showing patients the reflection of their unimpaired arm in a mirror, they retrieved the sensation of their amputated arm without pain. Since then, the paradigm of mirror therapy has also been studied in other pain related syndromes (especially chronic regional pain syndrome) [START_REF] Mccabe | A controlled pilot study of the utility of mirror visual feedback in the treatment of complex regional pain syndrome (type 1)[END_REF] and motor related syndromes (stroke, hand surgery). [START_REF] Yavuzer | Mirror therapy improves hand function in subacute stroke: a randomized controlled trial[END_REF][START_REF] Rosen | Training with a mirror in rehabilitation of the hand. Scandinavian journal of plastic and reconstructive surgery and hand surgery / Nordisk plastikkirurgisk forening [and[END_REF] The focus of these studies has been on potential clinical effects; reduction of pain and improvement of motor function. Relatively little research has focussed on the mechanisms that underlie the effects of mirror therapy.

Presumably, different working mechanisms are behind the effects of mirror therapy on pain and motor symptoms. For the latter category, the focus of the current study, a number of mechanisms have been proposed. Ramachandran originally hypothesised that paralysis following stroke might have a 'learned' component, which could possibly be 'unlearned' by means of the mirror illusion. [START_REF] Ramachandran | Phantom limbs, neglect syndromes, repressed memories, and Freudian psychology[END_REF] Others suggested that mirror therapy might be a form of visually guided motor imagery. [START_REF] Stevens | Using motor imagery in the rehabilitation of hemiparesis[END_REF] Motor imagery itself has proven to be effective in the rehabilitation of patients with hemiparesis [START_REF] Sharma | Motor imagery: a backdoor to the motor system after stroke?[END_REF] and the mirror induced visual feedback of the imagined movement might further facilitate this. In addition, it has been hypothesized that the observation of the mirror illusion might trigger the mirror neuron system (MNS). [START_REF] Yavuzer | Mirror therapy improves hand function in subacute stroke: a randomized controlled trial[END_REF] Mirror neurons were initially discovered by Di Pelligrino and co-workers in monkeys [START_REF] Di Pellegrino | Understanding motor events: a neurophysiological study[END_REF] and are a particular type of neurons that discharge both with performance of a motor action and with observation of another individual performing similar motor actions. [START_REF] Rizzolatti | The mirror-neuron system[END_REF] As single cell studies are not normally performed in the human brain, there is not yet direct evidence for the existence of a mirror neuron system in humans. However, brain imaging data do suggest the existence of a similar system. [START_REF] Iacoboni | Cortical mechanisms of human imitation[END_REF][START_REF] Rizzolatti | Neurophysiological mechanisms underlying the understanding and imitation of action[END_REF] Previous research has indicated the potential of the MNS in motor recovery by showing that the observation of movements performed by others improves motor performance in stroke patients. [START_REF] Ertelt | Action observation has a positive impact on rehabilitation of motor deficits after stroke[END_REF] It is conceivable that observing one's own mirrored movement promotes recovery in a similar way.

A number of studies have evaluated the neuronal correlates of mirror therapy by examining the observation of the mirror reflection of a moving hand in healthy subjects. These studies were based on the hypothesis that the mirror illusion would increase excitability or activity in primary motor areas in the hemisphere ipsilateral to the moving hand. Using transcranial magnetic stimulation (TMS) [START_REF] Garry | Mirror, mirror on the wall: viewing a mirror reflection of unilateral hand movements facilitates ipsilateral M1 excitability[END_REF][START_REF] Funase | Increased corticospinal excitability during direct observation of self-movement and indirect observation with a mirror box[END_REF][START_REF] Fukumura | Influence of mirror therapy on human motor cortex[END_REF], magnetoencephalography (MEG) [START_REF] Tominaga | A mirror reflection of a hand modulates stimulus-induced 20-Hz activity[END_REF], electroencephalography (EEG) [START_REF] Touzalin-Chretien | Motor cortex activation induced by a mirror: evidence from lateralized readiness potentials[END_REF] and functional magnetic resonance imaging (fMRI) [START_REF] Matthys | Mirrorinduced visual illusion of hand movements: a functional magnetic resonance imaging study[END_REF] the authors compared neuronal activity or excitability ipsilateral to the moving hand with or without observing its mirror reflection. The MEG study reported the mirror illusion to suppress 20-Hz activity, indicating increased activation of the primary motor cortex [START_REF] Tominaga | A mirror reflection of a hand modulates stimulus-induced 20-Hz activity[END_REF], while the EEG study reported that the mirror illusion of movement induced lateralized readiness potentials, indicating cortical motor preparation for the non-moving hand. [START_REF] Touzalin-Chretien | Motor cortex activation induced by a mirror: evidence from lateralized readiness potentials[END_REF] On the other hand, the TMS studies either found no effect of the mirror illusion on motor cortex excitability [START_REF] Garry | Mirror, mirror on the wall: viewing a mirror reflection of unilateral hand movements facilitates ipsilateral M1 excitability[END_REF][START_REF] Funase | Increased corticospinal excitability during direct observation of self-movement and indirect observation with a mirror box[END_REF] or indicated that the mirror illusion needs to be combined with motor imagery in order to increase motor cortex excitability. [START_REF] Fukumura | Influence of mirror therapy on human motor cortex[END_REF] Finally, an fMRI study from our research group [START_REF] Matthys | Mirrorinduced visual illusion of hand movements: a functional magnetic resonance imaging study[END_REF] found no increased activity in sensorimotor areas as a result of the mirror illusion, but did find an increase in activity in the superior temporal sulcus (STS), presumed to be due to involvement of the MNS. [START_REF] Iacoboni | The mirror neuron system and the consequences of its dysfunction[END_REF] So far, no studies on the neuronal correlates of the mirror illusion have been performed in patient groups. It seems obvious that caution has to be taken when generalizing results from mirror studies in healthy participants to stroke patients. Patients have a damaged hemisphere, and alteration of activity within that hemisphere might not be as easily achieved as in healthy participants. Furthermore, stroke patients performing mirror therapy are generally instructed to practice bimanually, moving affected and un-affected limb together. [START_REF] Yavuzer | Mirror therapy improves hand function in subacute stroke: a randomized controlled trial[END_REF][START_REF] Altschuler | Rehabilitation of hemiparesis after stroke with a mirror[END_REF][START_REF] Dohle | Mirror Therapy Promotes Recovery From Severe Hemiparesis: A Randomized Controlled Trial[END_REF] As stroke patients move asymmetrically, placing a mirror between their hands will give them a sudden illusion of normal movement of the involved hand, creating an incongruence between task performance and visual feedback. This situation can not be created similarly in healthy controls, and is conceptually different from the experiments in which healthy controls only move one hand.

In summary, while clinical trials have presented promising results of mirror therapy in several patients groups, the working mechanisms have not yet been investigated in patients. Additionally, results of the studies on the working mechanisms in healthy participants have not been conclusive and effects that have been found in these studies cannot be generalized to stroke patients. In the present study we therefore investigated the neuronal correlates of the mirror illusion in stroke patients. We used fMRI to compare two different sets of conditions: 1) moving the unaffected hand while observing it directly versus moving the unaffected hand while observing its mirror reflection and 2) moving both hands while observing the affected hand directly versus moving both hands while observing the mirror reflection of the unaffected hand in place of the affected hand. We hypothesised that observing the mirror reflection would increase neuronal activity in the affected hemisphere.

MATERIALS & METHODS

Participants

Patients that took part in this experiment were selected participants of a randomized controlled trial (RCT) investigating the effects of a rehabilitation program of mirror therapy (www.trialregister.nl NTR1052). In this trial, 40 stroke patients were included and randomly assigned to either an experimental (mirror) group or a control group. Patients from the trial that were eligible to be scanned in an MRI scanner were asked to take part in the present study. This study took place ahead of the start of the clinical trial, before patients had been allocated to a treatment group. Inclusion criteria for the RCT were knowledge of the Dutch language, a Brunnstrom Score for the upper-extremity between III and V, home dwelling status and at least one year post stroke. Patients with neglect, co-morbidities that influenced upper-extremity usage or who had suffered multiple strokes were excluded from participation. For patients to be able to take part in the present study, the following additional inclusion criteria applied: a Brunnstrom score of IV or V and standard MRI exclusion criteria. Application of these criteria resulted in a total of 22 eligible patients. The study was approved by the Medical Ethics Committee of the Erasmus MC Rotterdam and all patients gave written informed consent before participating in the study. Before the fMRI experiment started the Fugl Meyer [START_REF] Fugl-Meyer | The post-stroke hemiplegic patient. 1. a method for evaluation of physical performance[END_REF] assessment of upper-extremity function was administered to all participants for descriptive purposes.

fMRI experiment

The fMRI paradigm we used was based on one previously designed in our laboratory. [START_REF] Matthys | Mirrorinduced visual illusion of hand movements: a functional magnetic resonance imaging study[END_REF] We performed two separate experiments each involving two conditions within a single scanning session.

In the first experiment patients were instructed to only move their unaffected hand (unimanual), either while looking directly at it (no mirror condition) or while observing its reflection in a mirror (mirror condition). In the second experiment patients were instructed to move both their hands (bimanual) either while looking directly at their affected hand (no mirror condition) or while observing the mirror reflection of their unaffected hand in place of their affected hand (mirror condition) (see Figure 1). In all four conditions, patients could see two hands. In the no mirror conditions of both experiments, patients had a direct view of both their affected and their unaffected hand. In the mirror conditions, patients had a direct view of their unaffected hand and saw the reflection of their unaffected hand in place of their affected hand, also leading to visual feedback of two hands.

During scanning, patients lay on their back in the scanner with their upper arms comfortably resting on the scanner table alongside their torso and their elbows flexed in such a way that their hands were 20 centimetres apart above their waists. By means of two mirrors attached to the head coil above the head, patients were able to look in the direction of their feet and could thus view both their hands.

All four conditions were performed using a block design, consisting of 10 alternating 30s periods of 5 rest blocks and 5 active blocks. In the active blocks patients had to open and close either their unaffected hand or both hands, in the rest blocks patients had to hold their hands still. Patients were instructed to pace the opening of their hands to a metronome with a rhythm of 0.5 Hz. The onsets of the rest and active conditions were indicated verbally using simple words (start, rest) generated by a computer program (Matlab 7.1; Mathworks, Sherborn, Mass). Auditory stimuli were presented to the patients through MRI compatible headphones. The hand movement was practiced before the scan session started.

The four conditions were presented to the patients in random order. During the mirror conditions, a large mirror was placed between the subjects' hands in such a way that the mirror image of the unaffected hand was superimposed on the position of the affected hand. The large mirror was made of MRI-compatible material (plexiglass) and was shaped in such a way that it fitted inside the scanner bore and fully obstructed the view of the hand behind the mirror (see Figure 2). In this way, a visual illusion of two normal hands was created. Before the scanning session, patients practiced outside the scanner with a regular mirror as used during mirror therapy in order to make sure they experienced the visual illusion. While it is hard to objectively quantify the presence or strength of the illusion, all subjects reported that the illusion of seeing the affected hand moving in an unimpaired fashion was similar to their experience during the mirror exercises outside the MRI scanner.

Imaging was performed on a 3T MR system (HD platform, GE Healthcare, Milwaukee, Wis).

For anatomical reference, a high-resolution, 3-dimensional, inversion recovery, fast spoiled gradient echo, T1-weighted image was acquired (TR/TE/TI 10.7/2.2/300 ms, 18° flip angle, matrix 416×256, and field of view 250×175 mm 2 ). For functional imaging, a single-shot, T2*-weighted, gradient echo echo-planar imaging (EPI) sequence was used (TR/TE 3000/30 ms, 75° flip angle, matrix 64×96, field of view 220×220 mm 2 ). An fMRI acquisition lasted for 5m15s, including 15 seconds of dummy scans that were discarded. For each of the four conditions 100 volumes were collected. The imaging volume covered the entire brain including the cerebellum.

Statistical analyses

The imaging data were analyzed using statistical parametric mapping software (SPM5, Wellcome All functional images for each participant were realigned to the first scan of each condition and then coregistered to the T1-weighted anatomical scan. Subsequently, images were transformed to standard Montreal Neurologic Institute space. To prevent warping around the lesions, we used a segmentation-based normalization approach. [START_REF] Crinion | Spatial normalization of lesioned brains: performance evaluation and impact on fMRI analyses[END_REF] Finally, normalized images were spatially smoothed by using a Gaussian filter of 8-mm FWHM.

Preliminary analyses showed that the realignment parameters estimated during spatial preprocessing were sometimes correlated with the task design. Therefore, we decided not to model the realignment parameters in the design matrix as regressors of no interest, as this would have resulted in cancelling out task-related activation. Instead, we used the ArtRepair Toolbox [START_REF] Mazaika | Artifact Repair for fMRI Data from High Motion Clinical Subjects[END_REF], which evaluates all volumes, detects the ones most affected by movement, and deweights these in the general linear model estimation. The experimental block design was convolved with the canonical hemodynamic response function, and the resulting model was estimated using a high-pass filter at 128 s in order to remove low-frequency artifacts.

In the first-level analysis, statistical maps were calculated for each of the four task blocks (i.e.

movement with mirror, movement without mirror, rest with mirror, rest without mirror) for each patient and each experiment (bimanual and unimanual) separately. Statistical maps of patients with left-sided lesions were flipped about the midsagittal plane, so that the affected hemisphere corresponded to the right side of the brain for all patients. The statistical maps were used for second level analyses.

Second level analysis

We performed a two factorial analysis with movement (activity versus rest) and mirror (mirror versus no mirror) as main factors for both the unimanual and the bimanual experiments separately. Main effects of movement and mirror as well as the interaction between movement and mirror were investigated. Significance was set at p<.05 (FDR corrected) with a minimum cluster size of 20.

RESULTS

Four patients were discarded from further analysis, one due to scanner failure, and three due to scanner artefacts in their data sets. The remaining analyses were thus conducted using data sets of 18 patients. The characteristics of these patients are presented in Table 1.

DISCUSSION

In an attempt to unravel the working mechanism of mirror therapy, this study investigated the neuronal correlates of the mirror-induced visual illusion in stroke patients. In this first study, to our knowledge, in which stroke patients participated instead of healthy volunteers, we showed increased activity as a result of the mirror illusion during bimanual movement in two areas: the precuneus and the posterior cingulate cortex. We found no differential effect on neuronal activity of the mirror illusion during unimanual movement, nor did we find evidence for the mirror illusion to increase activity in motor areas or the mirror neuron system.

A network including both the precuneus and posterior cingulate cortex is reported to be associated with mental representation of the self. [START_REF] Fransson | The precuneus/posterior cingulate cortex plays a pivotal role in the default mode network: Evidence from a partial correlation network analysis[END_REF] More specifically, research showed that the precuneus is activated when actions are being interpreted as being controlled by the self as well as during self centred mental imagery strategies [START_REF] Cavanna | The precuneus: a review of its functional anatomy and behavioural correlates[END_REF] whereas the cingulate cortex becomes activated during spatial navigation [START_REF] Moffat | Aging and spatial navigation: what do we know and where do we go?[END_REF] and has been found to process information about the spatial positions of the limbs in monkeys. [START_REF] Bakola | Cortical Connections of Parietal Field PEc in the Macaque: Linking Vision and Somatic Sensation for the Control of Limb Action[END_REF] The mirror illusion of a normal moving affected hand thus seems to increase alertness and spatial attention towards this hand. The fact that we did not find this activation during the unimanual condition, suggests that it is not so much the illusion of a virtual moving hand that causes this activation, but the mismatch between the movement one performs and the movement that is observed. Research showing that the cingulate cortex becomes activated during conflict monitoring, specifically during action conflicts, supports this. [START_REF] Botvinick | Conflict monitoring and cognitive control[END_REF] Two previous studies in healthy participants have reported increased neuronal activity in similar areas as a result of an incongruence between movement observation and action.[30-32] Dohle et al. [START_REF] Dohle | Body scheme gates visual processing[END_REF] found that such incongruence increased activation in occipital and posterior parietal areas, amongst them the precuneus, which they suggest to play a decisive role during mirror therapy. [START_REF] Dohle | Mirror Therapy Promotes Recovery From Severe Hemiparesis: A Randomized Controlled Trial[END_REF] In an earlier experiment Fink et al. [START_REF] Fink | The neural consequences of conflict between intention and the senses[END_REF] also reported on the neural correlates of a conflict between visual and proprioceptive information. Using positron emission tomography (PET) they investigated the effect of the mirror illusion during either in-phase movements or out-of-phase movements that are perceived as in-phase movements due to the mirror. Their results showed an effect of the mirror in the dorsolateral prefrontal cortex (DLPCF) and in the superior posterior parietal cortex (Brodmann's area (BA) 7). BA 7 is a point of convergence between vision and proprioception and plays a role in visuomotor coordination. The precuneus, which in our study became activated during the bimanual mirror condition, is part of BA 7 [START_REF] Cavanna | The precuneus: a review of its functional anatomy and behavioural correlates[END_REF] but is located more medial than the area of activation reported by Fink et al. The main difference between our study and Fink et al.'s is that in the latter participants actively had to create the motor-sensory conflict (by moving out-of-phase), while in our patient group this resulted from the involved arm not being able to perform similar movements as the non-involved arm. The situation created by Fink et al. likely induces a larger cognitive burden for the participants, which may explain increased DLPCF activation, while the main effect of the mirror they observed in BA 7 is in line with our findings of precuneus activation in the presence of a motor-sensory conflict, albeit under different experimental settings.

The question is how the involvement of the areas we found activated by the mirror illusion relates to the reported improvements in motor function following mirror therapy. [START_REF] Yavuzer | Mirror therapy improves hand function in subacute stroke: a randomized controlled trial[END_REF][START_REF] Dohle | Mirror Therapy Promotes Recovery From Severe Hemiparesis: A Randomized Controlled Trial[END_REF] Possibly, by increasing the spatial attention towards the affected limb the mirror illusion might help in overcoming the learned non-use phenomenon [START_REF] Dohle | Mirror Therapy Promotes Recovery From Severe Hemiparesis: A Randomized Controlled Trial[END_REF][START_REF] Taub | Somatosensory deafferentation research with monkeys: implications for rehabilitation medicine[END_REF], and as a result of the ensuing increased use of the limb improve motor performance. An alternative hypothesis, supported by the fact that we only observed increased cortical activation during the bimanual experiment, might be that the effects of the mirror lie in an enhancement of spatial coupling between limbs. It is well known that when two arms move simultaneously, movements become more temporally and spatially stable. [START_REF] Kelso | Patterns of human interlimb coordination emerge from the properties of non-linear, limit cycle oscillatory processes: theory and data[END_REF] In stroke rehabilitation, this phenomenon has been exploited in the form of bimanual training programs. Several studies have shown that bimanual training strategies have a favourable effect over unimanual training [START_REF] Stoykov | A review of bilateral training for upper extremity hemiparesis[END_REF],

demonstrating that spatial coupling can cause the affected limb to take on the properties of the noneffected limb, thereby improving motor performance. The hypothesis that the mirror illusion enhances this spatial coupling is supported by studies on healthy volunteers, showing that the mirror illusion increased the tendency of one limb to take on the spatial properties of the other limb. [START_REF] Altschuler | Interaction of vision and movement via a mirror[END_REF][START_REF] Franz | Fooling the brain into thinking it sees both hands moving enhances bimanual spatial coupling[END_REF] So far, several reviews and clinical studies have attributed the effects of mirror therapy to activation of motor areas or the mirror neuron system (MNS). [START_REF] Yavuzer | Mirror therapy improves hand function in subacute stroke: a randomized controlled trial[END_REF][START_REF] Ramachandran | The use of visual feedback, in particular mirror visual feedback, in restoring brain function[END_REF][START_REF] Ezendam | Systematic review of the effectiveness of mirror therapy in upper extremity function[END_REF][START_REF] Rizzolatti | Mirror neurons and their clinical relevance[END_REF] As mentioned, in the present study we did not observe activation resulting from the mirror illusion in these areas (e.g. M1, PMC, SMA, Broca's area), neither in the unimanual experiment nor in the bimanual experiment. Research performed in healthy subjects has so far also been unable to provide convincing evidence for the activation of these areas by the mirror illusion, only the fMRI study of Matthys et al. showed some evidence for MNS activation by reporting increased activation within STS. [START_REF] Matthys | Mirrorinduced visual illusion of hand movements: a functional magnetic resonance imaging study[END_REF] However, although STS is reported to be related to the MNS [START_REF] Iacoboni | The mirror neuron system and the consequences of its dysfunction[END_REF], this area has been associated with many different behaviours and its exact function remains poorly understood. [START_REF] Hein | Superior temporal sulcus--It's my area: or is it?[END_REF] Matthys et al. also reported activation in the superior occipital gyrus, an area connected with the PPC trough the dorsal stream.

Activation of this area may reflect increased attentional demands for the integration of vision and proprioception induced by the mirror, which is in line with our present results. It has to be noted that the analysis strategy of Matthys et al. differed from the strategy employed in the present paper.

Contrary to our approach, Matthys et al. did not apply an ANOVA design and thus did not examine the effect of the mirror, the hand motor performance and its interaction separately. As a final note on the proposed activation of the MNS or motor system by the mirror illusion, a previous study showed that whereas observation of actions attributed to another individual activated the motor system, observation of identical actions linked to the self did not. [START_REF] Schutz-Bosbach | Self and other in the human motor system[END_REF] The MNS thus seems to distinguish between observing actions linked to the self and actions linked to others. This finding further undermines the notion that the mirror illusion of self-performed movements might trigger the motor or mirror neuron system.

We acknowledge that our study has some limitations. In general, detecting study-related effects in fMRI experiments in stroke patients is difficult as the neuronal circuitry may be distorted and heterogeneous between subjects. Some authors try to get around this issue by including only patients with minor lesions, which is a major source of selection bias. In the current study, in order to enlarge the contrast between mirror and no mirror conditions in the bimanual experiment, we explicitly enrolled patients with larger motor deficits. Consequently, the within-group variability was considerable, which may have decreased the power to detect differences between conditions. Another issue, related to the severity of their motor deficit, is that some patients had problems keeping their head still during the experimental task. As these movements were task-related, we could not simply regress out head motion-related activation as this would have cancelled out task-related activation as well. However, we used an alternative, sensitive method to deal with this issue (see Methods section), and activation patterns we observed are in accordance with the expected activation for uni-and bimanual hand motor tasks, implicating the validity of our analysis. A general limitation of this and similar studies is that it is difficult to objectify the strength of the illusion patients experience when inside the scanner. To maximize the mirror illusion during scanning, all patients practiced with a standard mirror used for mirror therapy outside the scanner, and they all reported similar illusion strength during the measurements inside the scanner as outside.

In conclusion, the present study showed that during bimanual movement, the mirror illusion alters neuronal activation in the precuneus and posterior cingulate cortex, areas related to alertness and spatial-awareness. We found no differential effect on neuronal activity of the mirror illusion during unimanual movements, nor did we find evidence for the mirror illusion to increase activity in motor areas. By increasing awareness of the affected limb, possibly due the mismatch between action and observation, the mirror illusion might reduce learned non-use. The fact that we did not observe any activation in areas belonging to the motor or mirror neuron system questions popular theories that contribute the clinical effects of mirror therapy to these systems. As research into the working mechanism of mirror therapy has so far mainly focussed on these systems, we suggest that future research should adopt a broader perspective, amongst other things taking the ideas as proposed in this paper into account. Since a better understanding of why and how mirror therapy works may lead to a more effective application and might help in selecting patients for which mirror therapy will be most effective, it is important that efforts to unravel the neuronal correlates of mirror therapy continue.

Figure 1

Schematic representation of the four conditions. FE indicates which hands perform the flexionextension movements, and the arrow is used to indicate the direction of gaze in each condition.

Figure 3

Activation map of the interaction effect of movement x mirror for the bimanual experiment (p<.05, FDR corrected, minimum cluster size 20). Label A: posterior cingulate cortex; Label B: precuneus.

  Department of Cognitive Neurology, University College London, UK) implemented in MATLAB version 7.1 (Mathworks, Sherborn, MAS).

Table 1 Patient characteristics of the study participants

 1

	223
	224
	225
	226
	227
	228
	229
	230
	231
	232
	233
	234
	235
	236

* Values are mean ± standard deviation † Brainstem lesions are located in the pons, above the crossing of the cortico-spinal tract mirror for both experiments. Observed activation patterns for the main effect of movement were in accordance with the expected activation for uni-and bimanual hand motor tasks. Activity was observed in the pre-and postcentral gyrus (primary motor and sensory cortex), the medial superior Characteristic Total *

Table 2 Areas of activation (unimanual experiment: main effect of movement)

 2

	Anatomical location	Side	Cluster size	Z-score		MNI
					x	y	z
	Pre-and Post central Gyrus	Unaffected	2103	7.57	-34	-26	54
	Cerebellum	Unaffected	1287	7.28	16	-56	-20
	Middle Occipital/Temporal gyrus Affected	612	6.18	42	-64	4
	Medial Frontal Gyrus, Superior	Affected,
			448	6.17	-6	-6	56
	Frontal Gyrus, Cingulate Gyrus	Unaffected
	Middle Occipital/Temporal gyrus Unaffected	491	5.98	-48	-70	-2
	Precentral Gyrus	Unaffected	36	5.72	-52	0	8

Table 3 Areas of activation (bimanual experiment: interaction movement x mirror)

 3

	Anatomical location	Side	Cluster size	Z-score		MNI
					x	y	z
		Affected,
	Precuneus	Unaffected,	78	5.28	2	-58	58
		Central
	Posterior Cingulate Cortex	Unaffected	117	4.68	-6	-36	8
	Areas are tresholded at p < .05 (FDR corrected) with a minimum cluster size of 20 voxels. MNI =
	Montreal Neurological Institute.

ACKNOWLEDGMENTS

We want to thank Martine Eckhardt for her help with the measurements and organization of the study and Maarten Bobbert, Daphne Heemskerk and David Warmerdam for their help with the analysis of the data and interpretation of results. Also, we would like to acknowledge the two anonymous reviewers for their valuable suggestions on the data analysis and the manuscript. This study was supported by Hersenstichting Nederland, Fonds NutsOhra, Innovatiefonds Zorgverzekeraars, Prinses Beatrix Fonds and Wetenschappelijk College Fysiotherapie.

Areas of activation (bimanual experiment: main effect of movement)

Areas are tresholded at p < .05 (FDR corrected) with a minimum cluster size of 20 voxels. MNI = Montreal Neurological Institute.

Analysis of the main effect of mirror (mirror versus no mirror) showed no significant areas of activation in either of the two experiments. The interaction of mirror x movement showed no significant activation for the unimanual experiment but it did show significant activation in the bimanual experiment; in the precuneus and the posterior cingulate cortex. Post-hoc analysis revealed that this was caused by increased activity in the movement with mirror condition versus the movement without mirror condition. Table 3 and Figure 3 show the activated clusters for the interaction effect of movement x mirror in the bimanual experiment.

Competing Interest: None declared

COPYRIGHT LICENCE STATEMENT

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive license (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in Journal of Neurology, Neurosurgery & Psychiatry and any other BMJPGL products and to exploit all subsidiary rights, as set out in our license.

Figure 2

Participant lying in the scanner during the mirror condition. The unaffected hand is not visible, as it is positioned in front of the mirror.


Ukena Sn

Mischak-Weissinger E, Buchholz S, Stadler M, Ganser A, J Grosse

Anke Franzke
email: franzke.anke@mh-hannover.de

Acute but not chronic graft-versus-host disease is associated with a reduction of circulating CD4 + CD25 high CD127 low/-regulatory T cells

Keywords: Regulatory T cells, GvHD, stem cell transplantation, reconstitution

Defects in central and peripheral tolerance are thought to contribute to life threatening graftversus-host disease (GvHD), a severe complication following allogeneic stem cell transplantation (SCT). Recent investigations have demonstrated regulatory T cells (Tregs) to suppress allogeneic immune reactions. Therefore, SCT patients with no or critically low numbers of Tregs may have an increased risk of GvHD. To address this hypothesis, we analyzed the recovery of CD4 + CD25 high CD127 low/-Tregs in the peripheral blood of patients who have never developed GvHD (n=6), patients who developed acute/ chronic GvHD (n=18) and patients who developed chronic GvHD without an earlier acute manifestation (n=5) every 30 days for the first six months after peripheral blood SCT (PBSCT). The number of Tregs continuously improved in acute/ chronic GvHD patients, but always remained lower than Tregs quantified in patients who never developed a GvHD. In contrast, chronic GvHD patients who did not develop acute GvHD earlier displayed significantly increased Treg cell numbers at the timepoint of chronic inflammation. These results indicate that numerically deficient Tregs following PBSCT are associated with the development of acute but not chronic GvHD.

Introduction

Allogeneic stem cell transplantation (SCT) is an effective strategy for treating haematological malignancies. However, graft-versus-host disease (GvHD) following SCT occurs in about 1/3 of HLA-matched related donor transplantations and in about 2/3 of patients who are transplanted from an HLA-mismatched and/ or -matched unrelated donor [START_REF] Przepiorka | Chronic graft-versus-host disease after allogeneic blood stem cell transplantation[END_REF] with a mortality rate up to 50-60% [START_REF] Przepiorka | Risk factors for acute graft-versus-host disease after allogeneic blood stem cell transplantation[END_REF]. T cell depletion from the graft represents an effective approach but favors graft-rejection and aborts the graft-versus-leukemia effect. Evidence demonstrating the pivotal role that regulatory T cells (Tregs) play in GvHD development include the observation that adoptive transfer of Tregs in rodent models ameliorates this allogeneic immune response and depletion of Tregs from the stem cell graft accelerates GvHD [START_REF] Cohen | CD4(+)CD25(+) immunoregulatory T cells: New therapeutics for graft-versus-host disease[END_REF][START_REF] Edinger | CD4(+)CD25(+) regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation[END_REF][START_REF] Hoffmann | Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation[END_REF][START_REF] Taylor | The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality[END_REF].

Initial promising results from a clinical study investigating the adoptive transfer of donor

Tregs in an HLA haploidentical setting suggest that human Tregs provide beneficial effects against the development of GvHD [START_REF] Di Ianni | Adoptive Immunotherapy with Tregs prevents GvHD and favours immune reconstitution after HLA haploidentical transplants for hematological malignancies[END_REF]. Human studies investigating the impact of Tregs on GvHD following allogeneic SCT are conflicting, possibly due to the heterogeneity of study populations. In acute GvHD, reduced frequencies of Tregs have been shown independently of the stem cell source [START_REF] Rezvani | High donor FOXP3-positive regulatory T-cell (T-reg) content is associated with a low risk of GVHD following HLAmatched allogeneic SCT[END_REF]. A study in chronic GvHD has observed increased CD4 + CD25 high Treg cell numbers including a high number of peripheral blood stem cell transplantations (PBSCT) [START_REF] Clark | Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4(+)CD25(high) regulatory T cells[END_REF]. In contrast, others identified decreased frequencies of circulating Tregs in chronic GvHD patients [START_REF] Li | Decrease of CD4(+)CD25(+) regulatory T cells and TGF-beta at early immune reconstitution is associated to the onset and severity of graft-versus-host disease following allogeneic haematogenesis stem cell transplantation[END_REF][START_REF] Zorn | Reduced frequency of FOXP3(+) CD4(+)CD25(+) regulatory T cells in patients with chronic graft-versus-host disease[END_REF]. However, these studies included a higher number of bone marrow (BMT) and cord blood transplantations. Here, we present the kinetic reconstitution of CD4 + CD25 high CD127 low/-Tregs during the first six months following allogeneic PBSCT in correlation to the clinical manifestation of acute and/ or chronic GvHD.

Materials and Methods

Patients

Prior to sample collection appropriate permission was given from the institutional ethics committee. Between 2007 to 2009 87 patients from the stem cell transplantation unit of Hannover Medical School were enrolled into the study. The patients donated peripheral blood samples every 4 weeks within the first 6 months following allogeneic SCT. Exclusion criteria for the immune reconstitution analysis were: missing blood samples resulting from an indisposition of the patient, withdrawal from study or death, bone marrow transplantation, incomplete donor cell chimerism and/ or signs for acute infection. Hence, presented data result from 29 patients whose characteristics are summarized in Table 1. All patients received cyclosporine (CSA) for GvHD prophylaxis. The majority of the patients developed an acute GvHD (n=18) with a high proportion of a subsequent chronic GvHD manifestation, whereas 5 patients had only signs of a chronic GvHD. Most of the GvHD patients suffered from mild to moderate acute GvHD (grade I-II) and limited chronic GvHD. Seven patients developed a severe acute GvHD (> grade II). Half of the patients who did not develop an acute and/ or chronic GvHD (n=6) received preemptive therapy with 2 mg/kg body weight prednisolone based on urine proteomic patterns highly predictive for acute GVHD [START_REF] Weissinger | Proteomic patterns predict acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation[END_REF]. Acute GvHD was staged by modified Glucksberg criteria [START_REF] Przepiorka | Consensus Conference on Acute Gvhd Grading[END_REF] and was histologically confirmed by organ biopsies in some cases. Chronic GvHD was diagnosed by dyspigmentation and poikiloderma, lichen planus-like eruptions or sclerotic features of the skin, ulcerations and lichen-type features of the mouth as well as sicca-syndrome of the eyes and eosinophilia.

Collection of blood samples and data analysis

Peripheral blood samples (80ml) were obtained from patients every 30 days (± 5 days) after transplantation for six months (d30, d60, d90, d120, d150, d180). Mononucleated cells were isolated from fresh blood samples by Ficoll density gradient centrifugation. Subsequently, cells were stained with anti-human CD25 PE (Miltenyi Biotech, Germany), anti-human CD4 FITC and anti-human CD127 Alexa647 antibodies (all BD Biosciences, Germany) and analyzed by flowcytometry (FACS Aria; BD Biosciences, Germany). Samples were first gated for lymphocytes and then gated for CD4 + CD25 high and CD25 high+ CD127 low//-Tregs, which resulted in a purity of > 99% as shown in Figure 1. Reconstitution of CD4 + T cells and CD4 + CD25 high+ CD127 low/-Tregs following PBSCT was calculated using Excel 2000. The frequency of Tregs was expressed as percentage of CD4 + T cells and total lymphocytes, respectively, by sequential gating on lymphocytes and CD4 + T cells. Statistical analysis was performed using SigmaPlot 2000. Student´s t-test was used to identify significant expression changes. P values < 0.05 were regarded statistically significant.

Results

CD4 + T cell and Treg cell recovery after PBSCT

In the first six months following allogeneic PBSCT, a continuously improving recovery of CD4 + T cells and Tregs was observed for all 29 patients (Table 2). Quantitative differences between the clinical subgroups regarding the GvHD manifestation became statistically significant for CD4 + T cell and Treg cell counts from day 90 to day 180. The lowest CD4 + T cell numbers were found early after PBSCT at day 30. In this lymphopenic situation, only a very small Treg cell population circulates in the periphery. The median proportion of CD4 + T cells within the total lymphocyte population increased within the six months following PBSCT, whereas the highest median percentage of Treg cells within CD4 + T cells in the study population was reached at day 150. The Treg cell proportion within increasing numbers of CD4 + T cells remains relatively stable with a remarkable median proportion of up to approximately 25% early after transplantation in some patients.

Kinetic reconstitution of CD4 + T cells and Tregs in immune tolerant and GvHD patients

Results from the comparative analysis of Treg cell recovery in patients with and without

GvHD are presented in Table 3. In patients developing an acute GvHD, the CD4 + T cell counts and their proportion within the total lymphocyte population very early after PBSCT (day 30) were higher than in immune tolerant patients. At later timepoints, the median CD4 + T cell numbers were lower in the acute/ chronic GvHD patients and their proportion within the total lymphocyte population was clearly lower from day 120 onward. Continuous improvement of CD4 + T cell reconstitution was only observed in immune tolerant patients within the first six months following PBSCT. In contrast, the cell numbers of circulating Tregs were continuously lower in the GvHD group in the first half year after PBSCT, whereas their proportion within the CD4 + T cell population was only significantly reduced at day 30 in comparison to immune tolerant patients. Moreover, referring to the total lymphocyte population, the absolute Treg cell numbers were also clearly reduced in patients with acute GvHD at this very early timepoint, while the relative Treg cell numbers were similar in the two groups (data not shown). In contrast, GvHD patients displayed an increased Treg cell proportion within the decreasing CD4 + T cell population from day 60 onward (Table 3). Chronic GvHD patients not developing an acute manifestation after PBSCT showed nearly equal or even higher circulating Treg cell numbers than immune tolerant patients (Table 3) except at day 180, the latest timepoint evaluated. However, the clinical signs of chronic GvHD were apparent at an earlier timepoint. Interestingly, in these patients at day 30 the proportion of Tregs within the CD4 + T cell population was comparable to acute/ chronic GvHD patients and lower than in immune tolerant patients, whereas later on their percentage was significantly higher. In contrast to the acute/ chronic GvHD group, chronic GvHD patients without an acute manifestation displayed higher peripheral CD4 + T cell counts. Moreover, the proportion of Tregs within this numerically improving CD4 + T cell population also increased (Table 3).

Steroid therapy increases the percentage of Tregs

In order to investigate the effect of steroid therapy on Treg recovery after PBSCT, we compared within the group of patients without clinical GvHD manifestation those patients who received preemptive prednisolone therapy (n=3) very early after SCT (< day 30) with patients that did not receive steroids (n=3). Preemptive application of prednisolone in these patients was performed because a specific proteomic expression pattern which is highly predictive for the development of GvHD was observed (see also Materials and Methods) [START_REF] Weissinger | Proteomic patterns predict acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation[END_REF]. Except for days 90 and 180, median CD4 + T cell numbers were lower in the steroid treated patients compared to the untreated immune tolerant patients (Table 4). Circulating Treg cell counts were slightly lower in the steroid treated patients, whereas their median proportion within the CD4 + T cell population was predominantly higher within the group of patients receiving steroids. However, probably due to the small sample size, the relative quantitative difference of Tregs within the CD4 + T cell population was only statistically significant at day 30.

Discussion

Recent data from murine models implicate that regulatory T cells play a pivotal role in the maintenance of immune tolerance and may suppress graft-versus-host disease in humans following allogeneic SCT. To determine whether Tregs ameliorate GvHD, several studies were performed to quantify this regulatory T cell subpopulation after SCT in correlation to the incidence of GvHD. Currently, the results on Treg reconstitution are conflicting, probably due to heterogeneous study populations with respect to the stem cell source, application of T cell depletion strategies of the graft, other variations of the immunosuppression, donor cell chimerism and/ or active infections. Therefore, only patients without any variations of these parameters have been included in the present study. Furthermore, the recent studies varied regarding the phenotyping of Tregs: For example, Clark et al. quantified CD4 + CD25 high T cells [START_REF] Clark | Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4(+)CD25(high) regulatory T cells[END_REF], thus probably including activated T cells. The intracellular marker and master transcription factor for Tregs, FoxP3, which was used in later studies [START_REF] Rezvani | High donor FOXP3-positive regulatory T-cell (T-reg) content is associated with a low risk of GVHD following HLAmatched allogeneic SCT[END_REF][START_REF] Zorn | Reduced frequency of FOXP3(+) CD4(+)CD25(+) regulatory T cells in patients with chronic graft-versus-host disease[END_REF], was meanwhile also identified in human non-regulatory CD4 + Foxp3 + T cells [START_REF] Wang | Transient expression of FOXP3 in human activated nonregulatory CD4(+) T cells[END_REF]. However, expression of IL-7Rα (CD127) has been established as a useful marker for discriminating CD4 + CD25 + Treg cells from activated T cells [START_REF] Liu | CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4(+) T reg cells[END_REF]. Therefore, we monitored the kinetic reconstitution of Tregs by phenotyping CD4 + CD25 high CD127 low/-T cells every 4 weeks during the first six months after allogeneic PBSCT.

We observed a reduction of absolute and relative Treg cell numbers in acute GvHD patients as compared to patients without clinical signs of GvHD. Thus, our results confirm former reports in which different Treg phenotyping strategies were applied, including T cell depleted SCT regimens as well [START_REF] Rezvani | High donor FOXP3-positive regulatory T-cell (T-reg) content is associated with a low risk of GVHD following HLAmatched allogeneic SCT[END_REF][START_REF] Li | Decrease of CD4(+)CD25(+) regulatory T cells and TGF-beta at early immune reconstitution is associated to the onset and severity of graft-versus-host disease following allogeneic haematogenesis stem cell transplantation[END_REF][START_REF] Mielke | Reconstitution of FOXP3(+) regulatory T cells (T-regs) after CD25-depleted allotransplantion in elderly patients and association with acute graft-versus-host disease[END_REF]. In our study, the proportion of Tregs in the decreasing CD4 + T cell population improved markedly in GvHD patients during the first six months after allogeneic SCT. In contrast, chronic GvHD patients, who exhibited only a limited clinical manifestation in our analysis, had not only a relative increase within the CD4 + T cell population, but also significantly higher circulating Treg cell numbers around day 100 in comparison to the no GvHD group. This numerical induction of Tregs could only be monitored in chronic GvHD patients, who did not previously develop an acute GvHD. However, chronic inflammation involves a complex interplay between T and non T cell effectors, humoral responses and changes in chemokine and cytokine expression in target tissues. Therefore, although Tregs are numerically elevated in chronic GvHD, this immune regulatory T cell population might not be efficient enough to suppress this chronic inflammatory process.

Consistent with our data, Clark et al. described elevated Treg cell levels in chronic GvHD patients [START_REF] Clark | Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4(+)CD25(high) regulatory T cells[END_REF]. This is the only available study which is comparable to our analysis regarding the stem cell source for transplantation as PBSCT was performed in the majority of the patients. However, whereas Clark et al. included mostly patients suffering from extensive chronic GvHD in their study [START_REF] Clark | Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4(+)CD25(high) regulatory T cells[END_REF], the patients in our study predominantly presented a limited manifestation. In another study that included a high number of bone marrow and cord blood transplantations, extensive chronic GvHD was associated with a reduction of CD4 + CD25 high CD127 low/-Tregs [START_REF] Li | Decrease of CD4(+)CD25(+) regulatory T cells and TGF-beta at early immune reconstitution is associated to the onset and severity of graft-versus-host disease following allogeneic haematogenesis stem cell transplantation[END_REF]. Zorn et al. also included mainly BM transplanted patients (n=26 of 30 patients) and also observed a reduced frequency of FoxP3 + CD4 + CD25 + Tregs in patients with predominantly mild chronic GvHD [START_REF] Zorn | Reduced frequency of FOXP3(+) CD4(+)CD25(+) regulatory T cells in patients with chronic graft-versus-host disease[END_REF].

Our analysis provides very preliminary data that suggest a possible role of glucocorticoid therapy in the numerical induction of regulatory T cells. Recently, we have shown that specific urine proteomic patterns are highly predictive for the development of GvHD [START_REF] Weissinger | Proteomic patterns predict acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation[END_REF]. The kinetic analysis of Treg reconstitution in a small cohort of these patients without clinical signs of GvHD indicates an induction of Tregs at day 30, probably resulting from preemptive steroid therapy. Consequently, conclusions regarding Treg recovery in patients with acute and chronic GvHD should be handled with care. Future research should clarify whether changes in Treg frequencies in GvHD patients are drug induced or disease related. Unfractionated peripheral blood mononuclear cells (PBMCs) were stained with anti-human CD25 PE (Miltenyi Biotech), anti-human CD4 FITC and anti-human CD127 AlexaFluor 647 (BD Biosciences) and have been analyzed by FACS. Cells were gated on lymphocytes via their forward-and side-scatter properties. The resulting CD4 + CD25 high regulatory T cell population was expressing CD127 low/-and showed a purity >99%. Representive data of one individual are presented here.

Table 1. Patients´ characteristics

Pts indicates patients, AML acute myeloid leukemia, ALL acute lymphatic leukemia, MDS myeloid dysplastic sydrome, MPS myeloproliferative syndrome, CML chronic myeloid leukemia, TBI total body irradiation, Flu fludarabine, BU busulfan, Cy cyclophosphamide, ATG antithymocytes globuline, ClaraC clofarabine/ Ara-C, Flamsa fludarabine AraC amsacrin, Melphalan, VP-16 etoposide, MRD matched related donor, MUD matched unrelated donor, MMUD mismatched unrelated donor, n.a not applicable, * patients that received preemptive steroid therapy with 2mg/kg body weight prednisolone, GvHD graft-versus-host disease.

Pts

Figure 1 .

 1 Figure 1. Phenotypical analysis of CD4 + CD25 high CD127 low/-regulatory T cells.

Figure1

  Figure 1

Table 2 . Absolute and relative T cell reconstitution following allogeneic PBSCT.

 2

	Table
	no.	age sex diagnosis	conditioning protocol	donor age	donor sex	donor type	CD34 + T cells kg/ bw [x10 6 ]	CD3 + T cells [ x 10 8 ]	nuclear cells [ x 10 8 ]	GvHD
		54	m MDS	Flamsa/TBI/ATG	20	m	MUD	4.20	3.9	9.60	acute
		55	f MDS	ClAraC/TBI/ATG	44	m	MUD	5.90	3.17	16.80	acute
		56	m AML	Bu/Cy/ATG	46	m	MMUD	10.60	3.51	6.80	acute
		47	m AML	Bu/Cy	41	m	MRD	3.88	n.a.	8.74	acute
		36	m AML	Flamsa/TBI/ATG	38	m	MMUD	12.70	298	7.65	acute/chronic
		31	m ALL	TBI/VP-16/ATG	28	f	MRD	4.70	n.a.	5.64	acute/chronic
		45	m MPS	Flu/Bu/ATG	48	f	MRD	10.40	n.a.	5.85	acute/chronic
		27	f CML	Bu/Cy/ATG	26	f	MUD	9.30	3.38	10.99	acute/chronic
		46	m MPS	Flu/Bu/ATG	37	m	MUD	5.50	4.09	10.30	acute/chronic
		61	m AML	Flamsa/TBI/ATG	27	m	MMUD	9.20	6.00	14.00	acute/chronic
		50	m MDS	Flamsa/TBI/ATG	45	f	MRD	2.50	n.a.	3.10	acute/chronic
		58	m MDS	ClAraC/TBI/ATG	46	m	MRD	4.12	2.51	8.32	acute/chronic
		21	m AML	Flu/TBI/ATG	21	m	MUD	10.50	2.41	8.57	acute/chronic
		32	f ALL	TBI/VP-16	29	f	MRD	7.40	3.02	6.75	acute/chronic
		62	m AML	Flamsa/TBI/ATG	25	f	MMUD	5.35	6.07	12.30	acute/chronic
		51	m MDS/MPS	Flu/Bu/ATG	47	f	MRD	11.40	n.a.	15.32	acute/chronic
		50	m AML	Bu/Cy/ATG	42	m	MUD	5.19	3.01	8.14	acute/chronic
		39	m MDS	ClAraC/TBI/ATG	42	m	MUD	4.12	5.57	9.05	acute/chronic
		42	m AML	ClAraC/TBI/ATG	27	f	MMUD	5.10	3.88	13.02	chronic
		54	m AML	Flu/TBI	53	m	MRD	7.20	4.63	10.30	chronic
		52	m MDS	ClAraC/TBI/ATG	39	m	MUD	8.10	n.a.	9.70	chronic
		61	m MDS	ClAraC/TBI/ATG	35	m	MMUD	7.00	5.93	11.20	chronic
		30	m ALL	TBI/Cy/ATG	37	m	MMUD	8.16	6.03	14.00	chronic
		58	f Plasmozytom Flu/Melph/ATG	60	m	MRD	5.90	2.97	7.50	no GvHD
		58	f MDS	Flamsa/TBI/ATG	44	m	MUD	5.90	4.05	12.48	no GvHD
		65	f MDS/MPS	Flamsa/TBI/ATG	61	f	MRD	4.20	7.23	16.73	no GvHD
		66	m MDS	ClaraC/TBI/ATG	68	m	MRD	5.70	2.61	8.76	no GvHD*
		35	f AML	Flu/TBI/ATG	20	f	MUD	13.00	3.45	8.43	no GvHD*
		44	f MPS	Flu/Bu/ATG	55	f	MUD	6.00	2.27	15.90	no GvHD*

D indicates days after PBSCT; * p value < 0.05 compared to d30.

Table 3 . Absolute and relative T cell reconstitution in acute/ chronic and chronic GvHD.

 3 indicates days after PBSCT; a/cGvHD acute/ chronic GvHD; cGvHD chronic GvHD; * p value < 0.05 compared to no GvHD at the respective timepoint.

			CD4 + /µl	CD4 + CD25 high /µl	CD4 + [%]	CD4 + CD25 high [%]
	d30	no GvHD	4.8 (1.8-59.8)	0.5 (0.4-1.2)	1.8 (0.7-22.8)	6.1 (2.0-26.7)
		a/cGvHD	10.3	0.2 *	6.1	5.7 *
			(0.8-73.2)	(0.05-0.64)	(0.3-25.2)	(1.1-15.7)
		cGvHD	30.1	0.6	6.3	2.0
			(6.4-65.3)	(0.1-1.4)	(2.3-26.0)	(0.8-11.1)
	d60	no GvHD	25.9 (4.3-57.5)	1.0 (0.2-1.6)	4.7 (0.7-29.4)	2.3 (0.6-20.2)
		a/cGvHD	15.9	0.8	9.3	3.7
			(2.7-103.7)	(0.1-1.6)	(1.8-24.8)	(0.9-22.0)
		cGvHD	28.7	1.4	9.4	7.2
			(8.1-398.8)	(0.6-18.5)	(3.5-17.6)	(1.4-8.4)
	d90	no GvHD	35.8 (3.1-112.2)	1.0 (0.3-4.6)	7.0 (3.3-16.8)	4.6 (1.9-9.6)
		a/cGvHD	19.2	1.1	6.8	6.4
			(0.8-124.8)	(0.1-3.4)	(0.9-21.0)	(0.7-34.0)
		cGvHD	76.3	7.4 *	11.7	9.8 *
			(17.8-250.1)	(1.8-14.8)	(6.0-22.3)	(4.6-18.9)
	d120 no GvHD	37.6 (7.3-65.2)	1.4 (0.3-2.9)	6.5 (3.3-15.9)	3.9 (1.7-9.3)
		a/cGvHD	31.2	2.0	6.0
			(2.8-150.2)	(0.2-6.2)	(1.3-16.1)

D

Acknowledgement

This work was supported by a grant of the DFG (SFB738). We thank the clinical co-workers from the BMT unit for patient caring and their support in probe sampling. We are very thankful to Michael Morgan carefully reading the manuscript.

Competing interests

The author(s) declare having no competing interests.