Tatyana Karabencheva

Rossen Donev

Kia Balali-Mood

Christo Christov

Individual Contributions of the Aromatic Chromophores to the Near-UV Circular Dichroism in Class A β-lactamases: A Comparative Computational Analysis

Keywords: class β-lactamase, rotational strength, excited states, aromatic chromophores, tyrosine and tryptophan chromophores, matrix method

Class A β-lactamases are enzymes which are responsible for the bacterial resistance against antibiotics and therefore are of great importance in rational inhibitor design. In this Paper we comparatively analyze all the individual contributions of the aromatic chromophores in three classA β-lactamases (from Staphylococcus aureus, Streptomyces albus and Bacillus licheniformis) to their near-UV Circular Dichroism. The analysis is performed using recently developed procedure based on established theoretical method We found that in β-lactamase from Str. albus the most significant contributions to the total near-UV CD intensity exhibit Y251 and Y229. In the tryptophan-containing βlactamases from B.licheniformis and Str albus, W229 and W251 express the strongest individual contributions. A comparative analysis of the individual contributions of conservative chromophores in class A enzymes namely W165, W210, W229, W251, Y97 and Y105 is presented.

Introduction

β-Lactamases are of great interest to the pharmaceutical and biotechnology industries as they are responsible for bacterial resistance against antibiotics. [START_REF] Walsh | Introduction: Antibiotic resistance[END_REF], As a result intensive experimental and computational investigations have been reported. [START_REF] Sauvage | Structural Basis of the Inhibition of Class beta-Lactamases and Penicillin-Binding Proteins by 6-beta-Iodopenicillanate[END_REF][START_REF] Dc Marciano | Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 beta-lactamase[END_REF][START_REF] Christov | Relationship between chiroptical properties, structural changes and interactions in enzymes: A computational study on beta-lactamases from class A[END_REF][START_REF] Jc Hermann | Molecular mechanisms of antibiotic resistance: QM/MM modelling of deacylation in a class A betalactamase[END_REF]. ß-Lactamases are separated in four classes (A, B, C and D). The A class ß-Lactamases contain an active site serine residue which acts as a nucleophilic agent that attacks the lactam bond {Majiduddin, 2002 #147. This class of ß-Lactamase is the most extensively investigated one. The enzymes from this class are approximately 30 kDa proteins, consisting of one polypeptide chain organized into two domains: a-helical and mixed aß {Matagne, 1995 #148).

Circular Dichroism (CD) ( 6)is a powerful tool for the elucidation of structural information for proteins. The far UV CD spectra is informative for secondary structure changes, whereas the near-UV the method is sensitive to changes in protein tertiary structure resulting from ligand binding, mutations effects and catalysis [START_REF] Pc Kahn | The interpretaion of Near-Ultraviolet Circular Dichroism[END_REF][START_REF] Woody | Circular Dichroism and the Conformational Analysis of Biomolecules[END_REF].

A Combination of CD measurements and computational approaches leads to a fast and accurate understanding of the absolute configuration of small molecules [START_REF] Worch | Synthesis of enantiopure sulfonimidamides and elucidation of their absolute configuration by comparison of measured and calculated CD spectra and X-ray crystal structure determination[END_REF][START_REF] Atodiresei | Quantumchemical calculations on the electronic circular dichroism of (-)dibromophakellin and (-)-dibromophakellstatin[END_REF][START_REF] Berova | Application of electronic circular dichroism in configurational and conformational analysis of organic compounds[END_REF] In addition, deeper atomistic insights into specific interactions between protein chromophores and the influence of structural and electrostatic effects can be attained via this dual approach [START_REF] Christov | Relationship between chiroptical properties, structural changes and interactions in enzymes: A computational study on beta-lactamases from class A[END_REF][START_REF] Woody | Individual tyrosine side-chain contributions to circular dichroism of ribonuclease[END_REF][START_REF] Kurapkat | Calculations of the CD spectrum of bovine pancreatic ribonuclease[END_REF](14)[START_REF] Christov | Calculation of the CD spectrum of class A beta-lactamase from Escherichia coli (TEM-1)[END_REF][START_REF] Christov | Mechanisms of Generation of the Rotational Strengths in TEM-1 ß-Lactamase Part II: Theoretical Study of the Effects of the Electrostatic Interactions in the Near-UV[END_REF][START_REF] Christov | Mechanisms of generation of rotational strengths in TEM-1 beta-lactamase.Part I: Theoretical analysis of the influences of conformational chenges in the near-UV[END_REF][START_REF] Christov | Modeling study of the influences of the aromatic transitions and the local environment on the far-UV rotational strengths in TEM-1 beta-lactamase[END_REF][START_REF] Christov | Aromatic interactions and rotational strengths within protein environment: An electronic structural study on betalactamases from class A[END_REF]. The CD spectrum of TEM-1 β-lactamase was calculated in good agreement with the experimental one [START_REF] Christov | Mechanisms of generation of rotational strengths in TEM-1 beta-lactamase.Part I: Theoretical analysis of the influences of conformational chenges in the near-UV[END_REF]. TEM -1 β-lactamase as the enzymes which are object of this study belongs to class A which common structural feature is that the central section of the protein sequence folds into a globular unit which contains mainly of the α-helices and the N-and C-terminals form a five-stranded β-sheet which is surrounded by the the helices [START_REF] Vanhove | RH Pain: beta-lactamases as models for protein-folding studies[END_REF]. The Root Mean Squere deviation superposing the α-carbon atoms in respect to TEM-1 structure is as follows: for the enzyme from St aureus -2.47 Å, for the enzyme from Str albus-2.21 Å and for B licheniformis 2.10 Å.

On the basis of the agreement between the predicted and computed spectrum of TEM-1 enzyme, the theoretical spectra of the β-lactamases from Staphylococcus aureus, Streptomyces albus and Bacillus licheniformis were computed and analyzed in terms of mechanisms of generations of rotational strengths [START_REF] Christov | Aromatic interactions and rotational strengths within protein environment: An electronic structural study on betalactamases from class A[END_REF][START_REF] Karabencheva | Comparative theoretical study of the mechanisms of generation of rotational strengths in the near-UV in from class A[END_REF]. However, the CD method The methodology for calculating the above contributions is described in details in [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF] and is presented briefly here:

The individual net rotational strength of a chromophore Rn was calculated as a sum of the absolute values of the rotational strengths for all transitions of a particular chromophore:

A

The individual percentage contribution to the total one electron rotational strength Rroe was calculated as a percentage from the quotient between the individual absolute contribution of a chromophore transitions contributing to the one electron mechanism and the total one electron rotational strength:

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

5

Theoretical Background

The basic molecular unit of the CD phenomena is the Rotational Strength (RS) which represents the imaginary part from the scalar product between the electric transition moment and magnetic transition moment of a particular transition (29). In the proteins most of the choromophores (disulfide group and non-planar peptide group are the only exceptions) are achiral, however, become secondary chiral by the effects of the protein environment [START_REF] Woody | Circular Dichroism and the Conformational Analysis of Biomolecules[END_REF]. Three mechanisms may be followed in this (achiral to chiral) process:

i) one electron mechanism or static field effect(31), which represents coupling between electrically and magnetically allowed transitions within the same chromophore; ii) μ-μ mechanism or the coupling between electrically allowed transitions in different chromophores [START_REF] Kuhn | The physical significance of optical rotatory power[END_REF][START_REF] Kirkwood | On the theory of optical rotatory power[END_REF](this mechanism includes the so-called exciton effect (34)); iii) μm mechanism, which is realized through the coupling between magnetically and electrically allowed transitions in two distinct chromophores [START_REF] Schellman | Symmetry rules for optical rotation[END_REF]. Mechanisms (ii) and

(iii) are also called coupled oscillators type.

Computational Method

Calculation of rotational strengths in complex systems as proteins which contain hundreds or thousands of chromophores can not be cone directly even using powerful supercomputers, therefore we applied the well-established matrix method of Baylay, Nielsen and Schellman [START_REF] Bayley | Rotatory properties of molecules containing two peptide groups: theory[END_REF]. The method is implemented in the program MATMAC, which was kindly provided by Prof. Joerg Fleischhauer RWTH-Aachen, Germany) [START_REF] Fleischhauer | 0 Matrix and Tinoco method Program for the calculation of Rotational Stengths of Biopolymers[END_REF].

It utilizes matrix diagonalization, which makes it easy to implement in computer programs and also includes a modification of Goux and Hooker which include the matrix elements of the momentum in order to avoid the origin dependence of the magnetic transition moment [START_REF] Wj Goux | Chiroptical Properties of Proteins .1. Near-Ultraviolet Circular-Dichroism of Ribonuclease-S[END_REF]. The method for calculations of monopoles is the same as described in ( 13) and ( 39) and utilizes a semi-empirical method (CNDO/S).

Experimental excitation energies and electric transition dipole moments were used for the excited states of the model chromophores as described in [START_REF] Kurapkat | Calculations of the CD spectrum of bovine pancreatic ribonuclease[END_REF](14)[START_REF] Christov | Calculation of the CD spectrum of class A beta-lactamase from Escherichia coli (TEM-1)[END_REF]. The method uses the protein X-ray structures thus allowing the orientation and distances between the chromophores within the protein to be taken into account. The matrix method implemented in the program MATMAC was successfully implemented to investigate mechanisms of CD spectra in ribonuclease A and ribonuclease S. The programme predicts rotational strengths in close agreement to experiment and revealed the important role of the coupling between Y73 and Y115 to tyrosine Lb rotational strengths which was experimentally confirmed [START_REF] Kurapkat | Calculations of the CD spectrum of bovine pancreatic ribonuclease[END_REF]. The method is also used to calculate CD spectra of myoglobin, lactate dehydrogenase and other proteins [START_REF] Woody | Individual tyrosine side-chain contributions to circular dichroism of ribonuclease[END_REF][START_REF] Sreerama | Tyrosine, phenylalanine, and disulfide contributions to the circular dichroism of proteins: Circular dichroism spectra of wild-type and mutant bovine pancreatic trypsin inhibitor[END_REF](41)[START_REF] Na Besley | Theoretical studies toward quantitative protein circular dichroism calculations[END_REF][START_REF] Bhattacharjee | Influence of tyrosine on the electronic circular dichroism of helical peptides[END_REF][START_REF] Oakley | First-principles calculations of protein circular dichroism in the far-ultraviolet and beyond[END_REF].

All rotational strength contributions of the chromophores were calculated in the interval 240-300nm.The rotational strengths are presented in Debye-Bohr Magnetons (DBM).

More details about the matrix method are presented in Ref. [START_REF] Christov | Relationship between chiroptical properties, structural changes and interactions in enzymes: A computational study on beta-lactamases from class A[END_REF][START_REF] Kurapkat | Calculations of the CD spectrum of bovine pancreatic ribonuclease[END_REF][START_REF] Christov | Mechanisms of Generation of the Rotational Strengths in TEM-1 ß-Lactamase Part II: Theoretical Study of the Effects of the Electrostatic Interactions in the Near-UV[END_REF][START_REF] Christov | Mechanisms of generation of rotational strengths in TEM-1 beta-lactamase.Part I: Theoretical analysis of the influences of conformational chenges in the near-UV[END_REF][START_REF] Woody | Circular Dichroism and the Conformational Analysis of Biomolecules[END_REF][START_REF] Bayley | Rotatory properties of molecules containing two peptide groups: theory[END_REF][START_REF] Bm Bulheller | Circular and linear dichroism of proteins[END_REF].To generate a CD-spectrum, the calculated rotational strengths were combined with Gaussian band shape functions as was reported by Kurapkat and co-workers [START_REF] Kurapkat | Calculations of the CD spectrum of bovine pancreatic ribonuclease[END_REF]. The visualization of the enzyme structures was done with VMD 1.8.4 [START_REF] Humphrey | VMD: Visual molecular dynamics[END_REF].

Results and Discussion

A. Individual contributions of tyrosine chromophores in β-lactamase from St. aureus

This enzyme contains thirteen tyrosine chromophores (Figure 1). The analysis of the individual chromophore roles is presented in Table 1. The most significant individual contribution is exhibited by Y251, which generates 21.4% from the total near-UV CD.

It follows the one electron mechanism and generates also the most significant part (32%) from the total one electron rotational strength. This residue is located on the solvent exposed area of β-sheet. Y229 (analogous to W229 in TEM1, B.licheniformis and Str.albus) has the second strongest relative contribution to the total RS (20%).

However, it follows coupled oscillators mechanism (interacting with Y47 as described in [START_REF] Karabencheva | Comparative theoretical study of the mechanisms of generation of rotational strengths in the near-UV in from class A[END_REF] and provides 67% of it. The tyrosine Y40 which is located on a short α-helix at the top of the αβ domain provides 12% from the total RS and 17% from the total one electron (generated by the static field effect) RS. Y105 is a conserved tyrosine which occurs also in β-lactamases from E.coli (TEM-1) and B.licheniformis. It is located in a loop structure in all-α domain and generates around 10% from the total CD intensity and 14% from the total one electron RS. Y68 contributes 8% to the total near UV intensity. It is also "one electron chromophore" and provides 12% of the total near-UV CD which is generated by the one-electron mechanism. Y165 (analogous to W165 in TEM-1 and Str.albus β-lactamases) is located in a solvent exposed loop of the all-α domain. It contributes 7% from the total CD and 10 % from the one electron intensity.

Y129 which is situated in a solvent exposed part of an α-hellix in the all α-domain generates 5.3% from the total RS and 8% from the one electron RS, respectively. Y172 is located in a loop in the αβ-domain. It generates 3% from the total coupled oscillators RS and 5% from the total RS. Y88 is positioned within a 3-10 helix in an all α-domain.

It is a one electron chromophore which provides 6.6% from the total one electron and 4.5% from the total near-UV RS. The "coupled oscillators" Y171 and Y47 provides lower than 1% than the total RS and Y112 seems not to contribute. The CD spectrum of this enzyme is predominantly generated by the one electron mechanism, which provides 67% from the total intensity. Coupled oscillators mechanisms still make a considerable contribution, providing around 32% from the total RS.

B. Tryptophan and tyrosine chromophores individual roles in β-lactamase from Str. albus

This enzyme contains four tryptophans and six tyrosine chromophores (Figre 2). The most contributive are two tryptophans W229 and W251 which both are "coupled oscillator" chromophores and interact each other (Table 2). They are closely spaced to each other and located at the end of β-sheet within the αβ domain. W229 and W251 contribute 45% and 46% respectively from the total rotational strength and 48% and 50% from the total coupled oscillators RS. The other two tryptophans which are also conserved in other class A β-lactamase, namely W165 and W210 act as "one electron" chromophores which have minor contributions to the total RS -1.5% and 3.7 %. The strongest contributor within the tyrosines was Y190 which is a "one electron" residue providing 1.2% from the total and 14% from the total one "electron" RS. The other three "one electron" tyrosines have minor roles as follows: Y59 -0.05%, Y97 (also conserved in some class A β-lactamases), -0.75% and Y190 -1.2%. Two tyrosine chromophores which are "coupled oscillators" -Y47 and Y49 have small contributions to the total intensity 0.2% and 0.6% respectively. This spectrum is a strongly "coupled oscillators" type at 92% and 96% dominated by tryptophans. It is interesting to be note that in the case of tryptophans stronger influences have "coupled oscillator" chromophores. However, from the tyrosine chromophores the "one electron" ones have a stronger more pronounced effect.

C. Individual roles of the chromophores and mechanistic type of the spectrum in B. licheniformis

The protein contains three tryptophans and five tyrosines (Figure 3). The strongest individual contribution were W229 and W251 which were "coupled oscillator" type chromophores and generated 47% from the total RS and 48% from the total "coupled oscillators" RS (Table 3). The third tryptophan, W210 provides a relatively insignificant

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT 8 contribution from the total RS -only 2%. However it has the major contribution to the total "one electron" RS (88%). It's worth mentioning that both W229 and W251 also make very slight "one electron" contributions in addition to their major "coupled oscillators" one. The tyrosine chromophores here also can be divided to "one electron" (Y105, Y129, Y241) and "coupled oscillators": Y60 and Y97. The strongest contribution to the total RS here as a "couple oscillator" was Y129 (2.3%) which contributes very weakly to the total "coupled oscillator" spectrum. The other "coupled oscillators" tyrosines Y60, Y97 and Y274 have a minor influence on the total RS resp. 0.2%, 0.5% and 0.02%, respectively. The one electron tyrosines Y241 and the Y105 also contribute weakly to the total RS (0.8%, and 0.2%). This spectrum is almost entirely (97.3%) "coupled oscillators" type. Again the "coupled oscillators" mechanism is the favourable one for the tryptophans and the "one electron" mechanism is the more effective mechanism in the case of the tyrosines.

D. Comparative systematic analysis of the individual contributions of conservative chromophores in class-A enzymes

There are conserved residues which are also near-UV CD chromophores which are encountered in several class A β-lactamases. The analysis of their individual contributions provides the opportunity to monitor how these contributions evaluate between the different enzymes. All individual contributions in different class A enzymes analysed below can be seen in Figure 5.

W165

W165 is encountered in TEM1-β-lactamase (Figure 4) as well in β-lactamase from Str.

albus. It provides relatively small contributions to the total RSs in the both enzymes, however it's contribution in TEM-1 is at least twice as large (3.7%) [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF] than in the Str.

albus (1.5%). In both cases it act as "one electron" chromophore, however in the case of TEM-1 it generates 4% from the total one electron RS whether in the β-lactamase from Str. albus it generates almost 20% from the total one electron RS. The influence of catalytically important structural changes and electrostatic effects on the contributions of W165 were investigated in detail [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF]. It is important to underline that the structural changes of the same enzyme (TEM-1) β-lactamase can provoke stronger changes in it's W165 contribution than the structural differences between free enzymes from TEM-1 and Str. albus.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

W210

This chromophore occurs in TEM-1 as well as in the β-lactamases from Str.albus and B. licheniformis. Whereas in the enzymes from Str.albus and B. licheniformis. it provides the lowest contribution to the total RS (3.7% and 2.3%, respectively) in TEM-1 it is the most significant chromophore and generates 39% from the total RS [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF]. In all enzymes it participates in the one electron chromophore, however its contribution in respect to the total one electron RS is 88% in B. licheniformis, 44% in TEM-1 and 20 % in Str.

albus enzymes.

W229

TEM-1, Str. albus and B.licheniformis contain this chromophore. In the last two enzymes it behaves in quite a similar manner and provides around 45-47% from the total RS. In these enzymes it is also a strong "coupled oscillators" type chromophore which couples strongly with W251. It also accounts for approximately 50% from the total "coupled oscillators" RS in both enzymes. In the free enzyme from TEM-1 βlactamase it acts as a one electron chromophore and provides 34% from the total RS and 39% from the total "one electron "RS [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF]. As a result of important structural changes due to interaction with the transition state analogous inhibitor it (W229) transforms into a strong "coupled oscillators" chromophore.

W251

Β-lactamases from Str albus and B. licheniformis contain this chromophore. In both enzymes it acts as a "coupled oscillator" type chromophore interacting with W229 and providing a matching contribution to the W229 chromophore.

Y97

This chromophore is found in β-lactamases isolated from Str. albus, B. lichenoformis and TEM-1. In Str.albus it generates only 0.1% from the total RS, in B. licheniformis it provides 0.5% in TEM-1. It exhibits the most significant contribution of 2.8% [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF]in what??? In Str. albus it realizes "one electron" mechanism whereas in B. licheniformis and the free enzyme from TEM-1 it behaves as "coupled oscillators" type chromophore.

It is important to note that the conformational changes in TEM-1 enzyme induced by interactions with ligands may alter more considerably with the contribution of Y97 [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF] than the structural differences between different enzymes from class A.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Y105

This tyrosine residue is conserved in TEM-1 β-lactamase as well in β-lactamases from

St. aureus and B.licheniformis. In the last enzyme it has a very small contribution to the total RS of 0.2%. In TEM-1 it provides 1.5% [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF] and in β-lactamase from Str.albus it raises up to 10%. In the enzyme from St. aureus it realizes the "one electron" mechanism whereas in B.licheniformis and TEM-1 β-lactamase it follows "coupled oscillators" mechanism. In this case again the conformational changes induced by ligands in TEM-1 β-lactamase induces stronger perturbations of the relative individual contribution of Y105 to the total RS than the structural differences between different enzymes from class A (B. licheniformis, St. aureus, and TEM-1).

E. Overall mechanistic nature of the modelled spectrum

Finally we are going to compare the total relative contributions of the two types of mechanisms: one electron and coupled oscillators in the four enzymes from class A.

In the case of TEM-1 the spectrum is generated 88% by the one electron mechanism and 11% by the coupled oscillators mechanism [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF]. In the case of β-lactamase from St.

aureus (which contains only tyrosines) the spectrum is again mostly dominated by the one electron effect with 67%), however the relative part of the coupled oscillators is increased to 32%. It is worth mentioning that in TEM-1 structural changes perturbate this proportion within 10% [START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF]. However this ratio dramatically increases in βlactamases fron Str. albus and B.licheniformis where the contribution of the coupled clusters mechanism is above 90% (92% and 98% respectively). From this point of view the four enzymes can be divided in two subgroups based on their "mechanistic type" of spectrum: "one electron" enzymes -TEM-1 and St. aureus and "coupled oscillator" enzymes -β-lactamases from Str. albus and B. licheniformis.

The analysis improves our understanding of structure-spectra relationships in proteins.

In particular the role of individual chromophores is revealed from both quantitative and mechanistic aspect which is gained without structural perturbations to the proteins. The

Conclusions

In this Paper we described the novel application of methodology for assignment of the individual chromophore contributions in complex heterogeneous multichromophore protein systems, namely class-A beta-lactamases. We found that in β-lactamase from

TABLES

  has low signal resolution, therefore it is difficult to analyze experimentally the individual contributions of protein chromophores. The problem is accentuated by the structural complexity, the large number of chromophores and conformational flexibility of proteins. The absence of such atomistic information leads to numerous limitations in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 3 it's application and prevents the elucidation of the role of protein chromophores, especially in the near-UV spectra, whereby the aromatic residues are major contributors. CD experiments on mutated proteins may facilitate the understanding of chromophore contributions. However, mutagenesis leads to delicate structural effects which influence the orientations between the chromophores leading to inaccurate information. Aromatic residues play an important role ligand orientation, substrate activation, and reaction mechanisms (22,23) therefore detailed atomistic insight about their spectral contributions and mechanisms are important for deeper exploration of enzyme functions. In our recent paper we presented a novel computational strategy to calculate individual chromophore contributions and linked them to structural and environmental changes in TEM-1 β-lactamase (24). This methodology allowed for systematic computational analysis of different but structurally similar β-lactamases from class A. In this Paper we apply our recently developed approach to investigate how β-lactamases from class A differ in respect to their chiroptical properties. We demonstrate the potential of computational methods to reveal this relationship from a unique and unusual point of view: namely how the contributions of the individual chromophores differ within three β-lactamases from class A (from Staphylococcus aureus (containing no tryptophans), Streptomyces albus (containing both tryptophans and tyrosines) and Bacillus licheniformis (containing tyrosines and tryptophans). In particular, the following key findings will be presented: i) The Extraction of individual contributions of the thirteen tyrosines in β-lactamase from Staphylococcus aureus ii) Revealing the individual contributions of the four tryptophans and six tyrosines in βlactamase from Streptomyces albus iii) Differentiating between the individual contributions of the three tryptophans and six tyrosines in the β-lactamase from Bacillus licheniformis iv) Systematic comparative analysis of the evolution of the individual contributions of chromophores which are conservative in class A β-lactamases (W165, W210, W229, W251, Y97 and Y105) iv) Evolution of the overall mechanistic type of the modelled spectra between the above three enzymes and TEM-1 β-lactamase Methodology The individual contributions of the aromatic chromophores will be characterized in the following directions: i) Relative individual contribution of each chomophore in respect to the total CD generated by the one electron mechanism ii) Relative individual contribution of each chromophore in respect to the total CD generated by coupled oscillators mechanism iii) Relative individual contribution of each near-UV chromophore in respect to the total CD spectral intensity

  contribution to the total coupled oscillators rotational strength Rrco was calculated in the same manner.The individual percentage contribution in respect to the total rotational strength was calculated as a percentage from the quotient between the individual net rotational strength and the total rotational strengths over all chromophores and transitions Rtot,all: the crystal structures of the enzymes taken from Protein Data Bank (25) as initial coordinates. In the case of β-lactamase from St. aureus PC1 -the structure (PDB code: 3blm) (26); for the enzyme from B. licheniformis 749/C -the structure with PDB code 4blm(27) and for the β-lactamase from Str. albus G (28) the PDB structure 1bsg. The calculation details of TEM-1 β-lactamase which is used for comparisons are presented in[START_REF] Christov | Atomistic Insight in Protein Circular Dichroism: Computational Dissection of Contributions of Individual Chromophores in TEM-1 ß-Lactamase[END_REF].

  application of the method to structurally similar proteins (like β-lactamases from class A) provides not only information about the evolution of the conservative chromophores contributions but also uncovers the structural similarity between proteins from other unusual sides which could be linked to experimental science and industrial applications. The potential to differentiate contributions between chromophores fromA C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT11 the same type (e.g. tryptopthans or tyrosines) without structural perturbations would be a useful computational tool complementing experimental techniques in biophysical chemistry and protein science but also in biotechnological and pharmaceutical technology.

  Str. albus the most significant contributions to the total near-UV CD intensity are exhibited by Y251 and Y229. In the tryptophan-containing β-lactamases from B.licheniformis and Str albus, W229 and W251 express the strongest individual contributions. We performed systematic analysis of the individual contributions of conservative chromophores in class A enzymes, namely W165, W210, W229, W251, Y97 and Y105.The analysis provides a unique opportunity to investigate protein structural similarity and evolution from another perspective. A better understanding of the trends in the evolution of class A β-lactamases from a unique mechanistic point of view and an insight into how the individual chromophore contributions are altered between proteins with similar structure was presented. Finally, this study demonstrated the power of computational methods to provide insight which can not be attained experimentally but nevertheless successfully complemented experimental studies. This study provided atomistic dimension to the CD phenomena which may have important applications in biomedicine and the biotechnology and pharmaceutical sectors.

Figure 1 .

 1 Figure 1. Structure of β-lactamase from St. aureus. Tyrosines are shown in red

Figure 2 .

 2 Figure 2. Structure of β-lactamase from Str.albus. Tryptophans are shown in green and tyrosines in red.

Figure 3 .

 3 Figure 3. Structure of β-lactamase from B. licheniformis. Tryptophans are shown in green and tyrosines in red.

Figure 4 .Figure 5 .

 45 Figure 4. Structure of β-lactamase from E.coli (TEM-1). Tryptophans are shown in green and tyrosines in red.

Table 1 .

 1 Individual contributions of all tyrosine chromophores in β-lactamase from St. aureus

	Chromophore	Relative %	Relative %	Relative %
		contribution to the	contribution to	contribution
		total one electron	the total coupled	to the total RS
		RS	oscillators
			RS
	W165	19.8	0	1.54
	W210	45.1	0	3.66
	W229	7.41	47.91	44.62
	W251	7.49	50.31	46.31
	Y47	0	0.16	0.15
	Y49	0	0.60	0.55
	Y59	0.62	0	0.05
	Y97	1.00	0	0.08
	Y190	14.20	0	1.15
	Y241	9.26	0	0.75

Table 2 .

 2 Individual contributions of all tyrosine chromophores in β-lactamase from Str. albus

	Chromophore	Relative %	Relative %	Relative %

Table 3 .

 3 Individual contributions of all tyrosine chromophores in β-lactamase from B. licheniformis

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Acknowledgements

The authors are many thankful to Prof. Joerg Fleischhauer and Prof Gerhard Raabe from the Institute of Organic Chemistry, RWTH-Aachen, Germany for kindly providing MATMAC code and Prof Juan Andres (University Jaume I, Castellon, Spain) for the useful discussions. We would like also to acknowledge the support of HPC-Europe.

Reference


Dr Arun Nair
email: a.nair@dundee.ac.uk

Dr Lorna Mckinlay

Dr Peter Williamson

Dr Philip Short

Patricia Mrs

BSc Burns

Professor Brian J Lipworth
email: brianlipworth@googlemail.com

Comparative lung bioavailability of fluticasone/salmeterol via breath actuated spacer and conventional plastic spacers. Running Head: Lung bioavailability of Seretide via breath actuated and plastic spacers

Keywords: Asthma, Lung Bioavailability, Fluticasone, Salmeterol, Seretide, Spacer

Purpose

This study compares the in vivo relative lung bioavailability of Hydrofluoroalkane (HFA) Seretide™ delivered via unprimed and unwashed Aerochamber™ Plus(AP) or Volumatic™(VM) spacers; a integrated breath actuated vortex Synchro-Breathe (SB); and Evohaler™ pMDI(EH) using adrenal suppression and early fall in serum potassium as surrogates for respirable dose.

Methods

Seventeen Healthy Volunteers completed this randomised double blind double dummy crossover study. Single doses of placebo/Seretide250 (total dose ex-valve Fluticasone 2000 mcg/ Salmeterol 200mcg) were administered via the devices. Overnight urinary cortisol/ creatinine (OUCC), serum potassium (K) were measured at baseline and after each dose.

Results

Significant suppression of OUCC & K occurred from baseline with SB, AP and VM but not EH (geom. mean fold suppression, 95%CI, p); EH: 1.59(0.80 to 3.14, p=0.40); AP: 4.26(3.01 to 6.02, p<0.001); VM: 3.11(1.99 to 4.78, p<0. 001); SB: 3.29(2.04 to 5.24, p<0.001). For K, the arithmetic mean falls (mmol/l), 95%CI for EH were -0.10 (-.25 to 0.05, p=0.18); AP: -.23(-.41 to -.04, p=0.02); VM: -0.22(-.44 to -.01, p=0.04); SB: -0.28(-.42 to -.13, p=0.001) respectively.

Conclusions

The breath actuated SB device was comparable to "out of the box" small and large volume spacers and produced similar improvements in relative systemic lung bioavailability for Fluticasone and Salmeterol.

Introduction

Valved Holding Chambers (VHC) or spacer devices are recommended for individuals who have problems coordinating actuation and inhalation of metered dose inhaler devices (pMDI) [START_REF]British Guideline on the Management of Asthma[END_REF][START_REF]Expert Panel Report 3 (EPR-3): Guidelines for the Diagnosis and Management of Asthma-Summary Report 2007[END_REF]. They increase the fine respirable dose fraction by causing impaction of larger particles within the walls of the holding chamber, reduce aerosol velocity and consequently reduce oropharyngeal deposition and local adverse effects. This leads to increased systemic drug absorption from the lung and therefore increased systemic bioavailability which can be measured by using sensitive surrogates of systemic bioactivity [START_REF] Wilson | Importance of drug-device interaction in determining systemic effects of inhaled corticosteroids[END_REF][START_REF] Martin | Systemic Effect Comparisons of Six Inhaled Corticosteroid Preparations[END_REF][START_REF] Lipworth | Measures for detecting systemic bioactivity with inhaled and intranasal corticosteroids[END_REF][START_REF] Guhan | Systemic effects of formoterol and salmeterol: a dose-response comparison in healthy subjects[END_REF][START_REF] Nair | A novel breath-actuated integrated vortex spacer device increases relative lung bioavailability of fluticasone/salmeterol in combination[END_REF]. The propensity for dose related systemic and local adverse effects with ICS is often dictated by the choice of inhaled steroid, its pharmacokinetic and pharmacodynamic properties, its respirable particle size and site of lung deposition, and type of drug delivery device used.

Fluticasone Propionate (FP) and Salmeterol (SM) are both highly lipophilic drugs that are commonly used in asthma. FP has negligible oral bioavailability on account of its near complete hepatic first pass metabolism [START_REF] Harding | The human pharmacology of fluticasone propionate[END_REF][START_REF] Thorsson | Pharmacokinetics and systemic effects of inhaled fluticasone propionate in healthy subjects[END_REF]. Therefore the systemic bioavailability of FP is dependant entirely on lung absorption and therefore the delivered lung dose. Suppression of overnight urinary cortisol creatinine ratio (OUCC) following inhaled steroid therapy is a sensitive surrogate of relative lung dose delivery [START_REF] Wilson | Importance of drug-device interaction in determining systemic effects of inhaled corticosteroids[END_REF][START_REF] Lipworth | Measures for detecting systemic bioactivity with inhaled and intranasal corticosteroids[END_REF][START_REF] Martin | Systemic Effect Comparisons of Six Inhaled Corticosteroid Preparations[END_REF][START_REF] Nair | Respirable dose delivery of fluticasone propionate from a small valved holding chamber, a compact breath actuated integrated vortex device and a metered dose inhaler[END_REF]. Thus comparisons of the relative systemic bioavailability to the lung via different spacer devices can be reliably measured using OUCC. Similarly salmeterol, which is nearly 8 times as potent as salbutamol, has a high hepatic first pass metabolism and exhibits dose dependent systemic beta-2-adrenoreceptor pharmacodynamic and pharmacokinetic effects which are predominantly due to the inhaled fraction [START_REF] Bennett | The contribution of the swallowed fraction of an inhaled dose of salmeterol to it systemic effects[END_REF][START_REF] Kempsford | Comparison of the systemic pharmacodynamic effects and pharmacokinetics of salmeterol delivered by CFC propellant and non-CFC propellant metered dose inhalers in healthy subjects[END_REF]. Maximal salmeterol plasma concentration (Cmax) following inhalation predicts maximal K fall [START_REF] Kempsford | Comparison of the systemic pharmacodynamic effects and pharmacokinetics of salmeterol delivered by CFC propellant and non-CFC propellant metered dose inhalers in healthy subjects[END_REF] and changes in serum K in particular, are not significantly influenced by the swallowed salmeterol fraction which has been estimated to contribute 28-36% of systemic effects [START_REF] Bennett | The contribution of the swallowed fraction of an inhaled dose of salmeterol to it systemic effects[END_REF][START_REF] Kempsford | Comparison of the systemic pharmacodynamic effects and pharmacokinetics of salmeterol delivered by CFC propellant and non-CFC propellant metered dose inhalers in healthy subjects[END_REF]. Spacer devices further reduce the effect of the swallowed gut component to overall systemic bioavailability. Therefore the early maximal falls in serum K occurs primarily due to lung absorption.

Thus relative lung bioavailability of salmeterol via different devices can be reliably performed by measuring differences in early fall in serum potassium (K).

Conventional VHC/spacer devices are bulky, cumbersome and therefore not attractive to most patients (Figure 1). They are also influenced by the occurrence of electrostatic charge which can reduce its respirable dose delivery [START_REF] Clark | Effect of multiple actuations, delayed inhalation and antistatic treatment on the lung bioavailability of salbutamol via a spacer device[END_REF][START_REF] Anhoj | Effect of electrostatic charge in plastic spacers on the lung delivery of HFA-salbutamol in children[END_REF]. In real life, patients would be unlikely to optimally prepare a new plastic spacer out of the box by performing either washing or priming manoeuvres and so one might expect their performance to be initially suboptimal. We have previously evaluated a novel palm sized antistatic breath actuated vortex spacer device (Synchro-Breathe, Vortran Medical Technology Inc, Sacramento, California, USA) [START_REF] Nair | A novel breath-actuated integrated vortex spacer device increases relative lung bioavailability of fluticasone/salmeterol in combination[END_REF][START_REF] Nair | Respirable dose delivery of fluticasone propionate from a small valved holding chamber, a compact breath actuated integrated vortex device and a metered dose inhaler[END_REF]. This breath actuated device has a reduced actuator orifice and unique integrated vortex extension which creates turbulent ex-valve flow and a slower plume velocity when compared to pMDI. This alleviates any gagging due to throat impaction and could potentially improve compliance and drug delivery to the lung.

Having compared the relative systemic bioavailability to the lung of FP via an optimally prepared (washed and primed) AP spacer; and of combined FP/SM via washed but unprimed Volumatic spacer vs. pMDI and SB respectively, we wanted to study the electrostatic impact of using conventional spacers when used brand new out of the box (i.e. without pre-washing or priming) on relative systemic bioavailability, and therefore respirable dose delivery. We have therefore evaluated the relative systemic bioavailability of Seretide™250 pMDI delivered to the lung via the conventional Evohaler(EH) pMDI alone, via Synchro-Breathe(SB), an unprimed and unwashed 149 ml small volume Aerochamber™ Plus(AP), an unprimed and unwashed 750 ml large volume plastic spacer, Volumatic™ (VM): using OUCC and K as surrogates of relative lung delivery.

Methods

Patients:

Nineteen non smoking healthy participants between the ages of 18 to 65 years were recruited from our existing database of research volunteers. The Tayside Committee on Medical Research Ethics reviewed and approved the study protocol. The trial was registered on the European Clinical Trials Database.

Study Design:

This was a single centre, randomised double blind, double dummy study using a 4 way crossover design. The details of the randomization and blinding process are given below. Each subject had 3 placebo pMDI canisters and 1 active Seretide pMDI canister which were independently labelled, blinded, and packed under supervision of the clinical trials pharmacist into separate treatment packs based on the computer generated randomization sequence available online at www. randomization.com. Evohaler actuators were used throughout except when using the Synchro-Breathe. The placebo/active canisters were independently inserted into the Evohaler actuator and the Synchro-Breathe device prior to use. As the Evohaler, Aerochamber Plus, Volumatic and Synchro-Breathe devices are visually different, further blinding of the trial via the double dummy technique by using the pMDI via each of the four devices at each study visit. Thus the participants and the researchers had no way of identifying which device was delivering the active drug even though they were all being used at each study visit.

At the initial screening visit, suitability was checked and informed consent was obtained. The healthy volunteers then had a full physical examination, baseline spirometry, and evaluation of routine biochemistry, full blood count and electrocardiogram. If deemed suitable, they were then randomized into the study and were provided a pre-labelled container with written instructions on overnight urine collection. This was brought in on the morning of the first study visit from the volunteers" residence by a taxi service, so as to minimise patient visits and encourage compliance.

There were 4 treatment visits following screening and were held 5-7 days apart (Figure 2) to enable a steroid free washout period with FP [START_REF] Wilson | Dose response with fluticasone propionate on adrenocortical activity and recovery of basal and stimulated responses after stopping treatment[END_REF]. The study visits took place between 3-4 pm. At each of the study visits, the use of any concomitant medications and adverse events were recorded. Optimal inhaler technique was demonstrated by the researcher and technique reviewed at each study visit. An intravenous canula was inserted into the forearm ante-cubital vein and flushed with 10 ml of normal saline to maintain patency. An automated sphygmomanometer (Dynamap®), which records heart rate and blood pressure, was attached to the opposite arm. After 30 minutes of rest, a baseline sample (5ml) of blood was collected, and three consecutive readings of heart rate and blood pressure recorded.

Thereafter, the following treatments were administered under direct supervision in a randomized double blind, double dummy fashion: (1) Eight puffs of HFA placebo or active FP/SM combination (Seretide™ 250: FP 250 mcg ex-valve and SM 25 mcg ex-valve per actuation (total dose ex-valve FP 2000 mcg & SM 200 mcg) via the Evohaler pMDI, Aerochamber Plus, Volumatic, and Synchro-Breathe spacer devices respectively. We recognize that the dose used above does not reflect standard clinical practice in asthma, but was chosen primarily to ensure that an adequate systemic signal in terms of suppression of OUCC and K was detectable, to compare differences between devices.

The pMDIs used in the study were primed prior to first use by shaking them vigorously and discharging 5 puffs prior to use. Thereafter, the participants were asked to exhale to residual volume, and the inhaler was actuated at the beginning of inhalation and steady slow inhalation was maintained till total lung capacity and followed by breath holding for 10 seconds or for as long as possible. After 30 seconds, the procedure was repeated for the remaining doses. The pMDI canister was inserted in to the SB and the device was used similar to a conventional pMDI. The main difference being that the SB device was breath actuated. The AP and VM spacers were used out of the box, brand new at each visit and were not washed or primed prior to first use to simulate real life patient use. Single puffs were inhaled without delay between actuation and inhalation to optimize respirable dose delivery [START_REF] Clark | Effect of multiple actuations, delayed inhalation and antistatic treatment on the lung bioavailability of salbutamol via a spacer device[END_REF]. There was a 30 second delay between inhalations and the procedure was repeated for each of the eight inhalations.

Measurements:

Assessment of FP lung bioavailability: Assessment of overnight urinary cortisol creatinine ratio was performed prior to the first study visit and following each study visit. All volunteers were given written instructions regarding overnight urine collection and were provided pre-labelled sealed urine containers. The volunteers were asked to empty their bladder at 10:00 PM and to collect all of the voided urine till 8:00 AM. The total volume of urine sample was recorded, and aliquots were taken for urinary cortisol and creatinine for overnight (10 hour) urinary cortisol creatinine measurements. The procedure was repeated on the night following completion of drug inhalation at each of the study visits.

Assessment of SM lung bioavailability: An intravenous canula was inserted into the ante-cubital vein at the start of the study visit and all pharmacodynamic measurements of systemic beta-2adrenoreceptor response were carried out with the participants being semi-recumbent and after 30 minutes of rest. Baseline Serum K was collected and was repeated 1 hour post study drug inhalation to measure the early fall in K. Measurements of heart rate and blood pressure were taken in triplicate at baseline and at 15 minute intervals for 60 minutes with patients in semi-recumbent position.

Assessment for adverse events: A record of palpitations & visual analogue tremor scales was maintained at baseline and at 15 minute intervals post drug inhalation for 1 hour, to document immediate adverse effects to Salmeterol. Patients were also asked to report adverse events between study visits.

Assays:

All assays were performed in duplicate in a blinded fashion. The urinary cortisol was measured using a commercial radioimmunoassay kit which has no cross reactivity for FP (Diasorin Ltd, Wokingham, Berkshire, UK). The intra assay coefficient of variation was 4% and the inter assay coefficient of variation was 8%. Urinary creatinine was measured on a Cobas-Bio auto analyzer (Roche Products, Welwyn Garden City, UK).The inter and intra assay coefficient of variation was 3% and 4.6 % respectively. Serum Potassium was analyzed using the Roche Modular Multi ISE 1800 auto analyzer and the co-efficient of variation of the test was 1.2%

Statistical Analysis:

The primary endpoint was overnight urinary cortisol-creatinine ratio. A sample size of 16 completed patients per protocol was chosen to power the study at 80% to detect a 20% difference in overnight urinary cortisol-creatinine ratio, using data from a previous study which detected a 50% difference between spacer and pMDI with a sample size of 14 completed patients [START_REF] Dempsey | Evaluation of the effect of a large volume spacer on the systemic bioactivity of fluticasone propionate metered-dose inhaler[END_REF].

Data sets were analyzed for patients who completed the crossover study per protocol. All data was tested for normality prior to analysis. The OUCC data was log transformed, and K, Heart rate, Blood Pressure data was analyzed without transformation in view of its normal distribution. Comparisons were made using repeated measures General Linear Model [GLM] Analysis of Variance with Bonferroni correction for multiple comparisons, set with 95% confidence intervals for differences. All effects are reported as being significant <.05 (2 tailed) and violation of sphericity of within subject effects was tested with the Mauchly"s test. The analysis was carried out using SPSS Version 15.

Results

Nineteen healthy volunteers were randomised into the study and 17 completed the study per protocol.

One volunteer withdrew because of prolonged palpitations following first study visit and another was withdrawn due to his personal circumstances. The OUCC samples of two additional subjects were inconsistent on repeated assay testing and hence excluded from analysis and 2 subjects were excluded from K analysis as the samples were haemolysed. Baseline data are detailed in table 1 and 2.

Significant suppression of OUCC (geometric mean fold suppression, 95% CI, P) and serum K occurred from baseline with AP, VM, and SB but not the EH pMDI (Table 3 and4). Following combined FP/SM pMDI inhalation via the AP, the geometric mean fold suppression, 95% CI, p, of OUCC from baseline was 4.26(3.01 to 6.02,p<0.001). This was 3.11 fold (1.99 to 4.78, p<0.001) via the VM, 3.29 fold (2.04 to 5.24, p<0.001) via SB and 1.59 fold (0.80 to 3.14, p=0.4) via the EH pMDI (equating to 76.5%, 67.8%, 69.6% and 37% suppression via the AP, VM, SB and EH respectively).

For Serum K, the mean fall, 95% CI, p, was -0.23 mmol/l (-0.41 to -0.04, p=0.02) via AP; -0.22 mmol/l (-0.44 to -0.01,p =0.04) via VM; -0.28 mmol/l (-0.42 to -0.13,p =0.001) via SB; -0.10 mmol/l(-0.25 to 0.05, p=0.18) via EH (equating to 5.7%, 5.4%,7% and 2.5% fall via AP,VM,SB and EH respectively).

There were significant increases in additional time course markers of systemic beta-2 adrenoreceptor response of maximal heart rate response, maximum increase in systolic BP, and maximal fall in diastolic BP following study drug inhalation via all four devices including the pMDI (Table 4).

Significant differences in OUCC suppression were demonstrated between the EH pMDI and AP, VM, SB devices (Table 5). When compared to the EH pMDI, the AP device resulted in 2.6 fold greater suppression of OUCC 95%CI (1.50 to 4.80), p<0.001. Similarly when compared to EH pMDI the VM device resulted in 1.95 fold greater OUCC suppression 95% CI( 1.09 to 3.46), p=0.01 and the SB resulted in 2.06 fold greater suppression,95% CI (1.15 to 3.63), p =0.007( Equating to 62%, 49%,52% greater suppression compared to EH pMDI by AP,VM, and SB respectively). There were no significant differences in OUCC or K between spacer devices. The maximal heart rate response did however indicate a significant difference between SB & EH, and SB and VM and there were no significant differences between devices for changes in systolic and diastolic BP.

The following adverse events were reported by the study participants. Nine participants developed mild tremors as observed on the visual analogue scale and these were observed on 4 occasions via the Aerochamber Plus and Volumatic spacers and on 5 occasions via the breath actuated Synchro-Breathe. Three participants developed palpitations and 1 subject was withdrawn from the study due to discomforting tremors/palpitations.

Discussion

We have shown that the improvement in relative systemic bioavailability of fluticasone/salmeterol via the Synchro-Breathe™ is comparable to conventional small and large volume plastic spacers when used in new condition out of the box. We elected to make comparisons under real life conditions without pre-washing or priming of the new plastic spacers to assess if there was any effect of electrostatic charge during initial use. All three spacers significantly improved respirable drug dose delivery of FP/ SM from baseline when compared to the metered dose inhaler alone [Figure 3&4].

There are limited data comparing the lung bioavailability of FP/SM pMDI via small and large volume spacers in either new or primed condition. In a previous study where we evaluated the relative lung bioavailability of inhaled FP via washed and primed spacers in mild to moderate asthmatics, we found that the optimally primed and washed Aerochamber Plus spacer resulted in 2.8 fold (65%) greater lung delivery when compared to the pMDI and the breath actuated Synchro-Breathe device resulted in 1.59 fold (37%) greater lung delivery, again in terms of OUCC suppression [START_REF] Nair | Respirable dose delivery of fluticasone propionate from a small valved holding chamber, a compact breath actuated integrated vortex device and a metered dose inhaler[END_REF]. To help tease out the effect of pre washing on relative systemic drug bioavailability, we carried out a further study in healthy volunteers where in we compared the relative systemic bioavailability of FP/SM in combination via unwashed but primed Volumatic spacer, Synchro-Breathe, and pMDI [START_REF] Nair | A novel breath-actuated integrated vortex spacer device increases relative lung bioavailability of fluticasone/salmeterol in combination[END_REF]. The unwashed but primed Volumatic spacer resulted in 1.66 fold (40%) and the breath actuated Synchro-Breathe device resulted in 1.75 fold (43%) greater suppression of OUCC, when compared to Seretide pMDI. As regards the SM moiety, the Volumatic and Synchro-Breathe resulted in 4.7% and 6% greater fall in K compared to pMDI respectively.

Comparing previous results with our current HV study would suggest that when initially using new conventional spacers, the presence of electrostatic charge does not exhibit a significant negative impact on lung delivery of FP/SM. We believe that the use of 8 serial puffs of Seretide pMDI in our study resulted in progressive priming of the spacer device over a short period of time and hence the observed improvements in lung delivery of each active moiety. Therefore if this observation is translated into day to day clinical practice, the use of 2 puffs twice a day for 2 days (i.e. 8 puffs) via a spacer would adequately negate the effects of electrostatic charge on respirable dose delivery. In this regard, it is important to recognise that FP exhibits unique lung absorption characteristics that are influenced by the effects of airway calibre and its high lipophilicity. The systemic lung absorption of inhaled FP is therefore higher in healthy volunteers and mild asthmatics who have normal or near normal airway calibre, as compared to more severe asthmatics, who have attenuated systemic effects due to reduced lung function and possibly increased mucociliary clearance from central airways [START_REF] Lee | Effects of high-dose inhaled fluticasone propionate on the hypothalamicpituitary-adrenal axis in asthmatic patients with severely impaired lung function[END_REF].

This protective effect of impaired lung function on FP systemic bioavailability has been previously

studied by Lee et al [START_REF] Lee | Effects of high-dose inhaled fluticasone propionate on the hypothalamicpituitary-adrenal axis in asthmatic patients with severely impaired lung function[END_REF]. They demonstrated that in severe asthmatics mean FEV1 47%, 2000 mcg of FP delivered via a primed Volumatic spacer for 2 weeks did not lead to suppression of plasma cortisol levels or OUCC in contrast to normal controls thereby demonstrating the protective effects of reduced airway calibre and increased severity on systemic hypothalamic pituitary axis (HPA). We believe that comparisons of FP lung bioavailability between healthy volunteers and asthmatics are valid across a range of severities when comparing different spacer devices as the relative ratio of OUCC suppression will be similar across the two groups, despite there being less absolute OUCC suppression in severe asthmatics. Time course data over 1 hour evaluating systemic beta-2 adrenoreceptor response for the SM moiety indicated that there were significant early increases in heart rate, fall in diastolic blood pressure and increase in systolic blood pressure with all spacers including the pMDI, with a numerical trend favouring Synchro-Breathe[Figure 5]. This would suggest that there is rapid drug absorption across the alveolar capillary barrier and fits previous studies that have confirmed that most of the systemic effects of inhaled SM are due to lung absorption [START_REF] Bennett | The contribution of the swallowed fraction of an inhaled dose of salmeterol to it systemic effects[END_REF].The cardiac effects is presumed to be due to its direct effects on cardiac beta-2-adrernoreceptors and beta-2 mediated dilatation of peripheral arteries.

Indeed SM has a steep dose response curve for its cardiac effects and these effects are therefore highly sensitive but not necessarily specific enough to enable comparisons between devices [START_REF] Fowler | Pharmacokinetics and systemic beta2-adrenoceptor-mediated responses to inhaled salbutamol[END_REF][START_REF] Bennett | Time course and relative dose potency of systemic effects from salmeterol and salbutamol in healthy subjects[END_REF].

The metabolic responses of early fall in serum K has been shown to be a more reliable surrogate and permitted relevant comparisons between devices as the dose given was not on the top of the dose response curve. Indeed room for greater falls in serum K with higher doses has been demonstrated by Kempsford et al [START_REF] Kempsford | Comparison of the systemic pharmacodynamic effects and pharmacokinetics of salmeterol delivered by CFC propellant and non-CFC propellant metered dose inhalers in healthy subjects[END_REF]. Our results indicated that only the Aerochamber Plus, Volumatic, and Synchro-Breathe resulted in statistically significant fall in K from baseline with a numerical non significant trend in favour of Synchro-Breathe. We were however not able to replicate a significant difference in SM systemic bioavailability between the spacers and pMDI which we had demonstrated earlier. In this regard the wider confidence intervals indicate that larger studies should be carried out before any definite deductions can be made.

It is important to clarify that we did not attempt to evaluate the anti-asthmatic efficacy of FP/SM combination via the different delivery devices, as we only gave single doses of FP/SM. We recognise that the dose of FP/SM used were significantly higher than normal, but this was essential to demonstrate assay sensitivity as we were able to separate out spacers from the pMDI and at lower doses, the signal from FP/SM would have been too small to make meaningful comparisons. Further when interpreting lung bioavailability studies it is also important to recognise that there are several factors which influence systemic lung bioavailability. These include the type of inhaled steroid/LABA being used, the lipophilicity of the compound, the formulation, the type of actuator, and the use of adjunctive devices such as spacers and more importantly the technique of drug inhalation. We have used the spacers brand new with optimal inhalation technique and hence the observed differences are likely to be a more relevant in day to day clinical practice. We recognise that increased lung dose delivery with spacer devices is a double edged sword as it can on one hand lead to greater clinical efficacy at perhaps lower nominal doses with fewer local adverse effects but on the other hand potentially increase the systemic adverse effect burden with chronic dosing particularly with FP. This is however less likely to be a problem with SM due to the tachyphylaxis of response due to beta-2adrenoreceptor down regulation and sub-sensitivity with chronic dosing [START_REF] Grove | Bronchodilator subsensitivity to salbutamol after twice daily salmeterol in asthmatic patients[END_REF].

Conclusion

In conclusion, we have demonstrated that the novel breath actuated Synchro-Breathe® device is comparable to new small volume Aerochamber™ Plus and large volume Volumatic™ spacers in terms of improvements in relative lung delivery of combined FP/SM. The use of serial puffs as used in real life, counteracts the effects of electrostatic charge on the respirable drug delivery of FP/SM (as early as day 2 of using 2 puffs FP/SM pMDI bid), by effecting progressive priming and suggests that conventional plastic spacer devices can be used out of the box without need for prewashing or priming.

Figure Captions:Fig 1 Fig 2 Fig 3 Fig 4 Fig 5

 12345 Figure Captions:

Table 1 :

 1 Baseline Demographics:

	Subject No:	Age	Sex	FeV1 %	FVC%	OUCC
	1	19
			F	92	96	9.24
	15	21	F	84	91	3.97
	16	63	F	109	127	12.2
	17	20	F	125	118	4.61
	19	23	M	99	93	5.24
	21	34	F	98	93	14.71
	23	26	M	102	111	17.11
	Mean(SEM)			106(3.1)	105(2.6)	4.84*(.83)

FeV1%: percentage predicted Forced expiratory volume in 1 second.

FVC%: percentage predicted forced vital capacity

All data expressed as mean (SE) except Overnight cortisol creatinine ratio (OUCC) which is expressed as Geometric mean (SEGM)

Table 2 :

 2 Mean (SE) Baseline values of systemic beta-2 response outcomes

	Device	Serum Potassium	Heart rate	Systolic BP	Diastolic BP
		mmol/l	(bpm)	(mm Hg)	(mmHg)
	Evohaler	4.04 (.05)	69(2.5)	127(4.0)	75(3.3)
	Aerochamber	4.03(.06)	68(2.9)	122(2.7)	69(3.4)
	Plus
	Volumatic	4.09(.04)	69(2.5)	128(3.6)	71(2.8)
	Synchro-Breathe	3.98(.05)	71(2.4)	130(2.6)	72(2.9)

Table 3 :

 3 Overnight urinary cortisol creatinine (OUCC) ratio pre and post treatment with geometric mean fold changes from baseline following inhalation of Seretide pMDI (2000mcg FP & 200 mcg SM combination) via Evohaler, Aerochamber Plus, Volumatic and Synchro-Breathe devices *Overnight Urinary Cortisol Creatinine (OUCC) values at baseline and following treatment. Data presented as Geometric mean (SEGM) † Geometric mean fold change in OUCC from baseline

	Device	OUCC ratio*	Geometric mean	95% CI	Significance
			fold suppression †		P
	Pre treatment	4.43 (0.77)	_	_	_
	Baseline
	Evohaler pMDI	2.79 (0.40)	1.59	(0.80 to 3.14)	0.4
	Aerochamber	1.04 (0.63)	4.26	(3.01 to 6.02),	<0.001
	Plus
	Volumatic	1.42 (0.73)	3.11	(1.99 to 4.78)	<0.001
	Synchro-Breathe	1.34 (0.68)	3.29	(2.04 to 5.24)	<0.001

Table 4 :

 4 Mean maximal systemic beta-2 adrenoreceptor responses over 60 minutes following inhalation of Seretide pMDI (2000mcg FP& 200 mcg SM combination) via Evohaler, Aerochamber Plus, Volumatic, and Synchro-Breathe devices recorded as arithmetic mean fall in K & diastolic blood pressure, Maximal increase in Heart rate and systolic blood pressure.

	Device		Serum K	Maximal Heart rate	Max SBP		Min DBP
			(mmol/l)		(bpm)		(mmHg)		(mmHg)
		Mean	95% CI,	Mean	95% CI,	Mean	95% CI,	Mean	95% CI,
		fall	p		p		p		p
	Evohaler	-0.10	-0.25 to 0.05,	8.3	5.8 to 10.9,
			p=0.18		p<0.001

K: Serum Potassium; SBP: Systolic blood pressure; DBP: Diastolic blood pressure

Table 5 :

 5 Between device comparisons of geometric mean fold differences in Overnight Urinary cortisol creatinine ratio(OUCC), arithmetic mean differences in serum potassium(K) , and heart rate after inhalation of 2000 mcg of FP and 200mcg of SM combination pMDI via the Evohaler, Aerochamber Plus, Volumatic, and Synchro-Breathe devices. Geometric mean fold difference, 95% CI, P.Data expressed as arithmetic mean differences except when stated otherwise will be made prior to Visit V1, and post V1, V2, V3 and V4. Each Randomised crossover treatment arm incorporates 8 puffs of HFA Seretide 250/25 µg or HFA Placebo via Evohaler, Aerochamber Plus, Volumatic and Synchro-Breathe delivery devices.

	Device	OUCC*	K	Heart rate
			(mmol/l)	(bpm)
	Aerochamber vs.	2.63(1.50 to 4.80),	-.13(-.39 to .14),	3.5(-2.7 to 9.6),
	Evohaler	p<0.001	P=1	P=0.66
	Volumatic vs. Evohaler 1.95(1.09 to3.46),	-.12(-.49 to .24),	1.5(-3.3 to 6.2),
		p=.01	P=1	P=1
	Synchro-Breathe vs.	2.06(1.15 to3.63),	-.18(-.46 to.09),	7.3(0.8 to 13.7),
	Evohaler	p.007	P=0.39	P=0.02
	Aerochamber vs.	1.37(0.79 to 2.45),	-.01(-.35 to .34),	2(-5.8 to 9.8),
	Volumatic	p=.86	P=1	P=1
	Aerochamber vs.	1.30(0.72 to 2.32),	.05(-.19 to .30),	-3.8(-10.2 to 2.7),
	Synchro-Breathe	p=1	P=1	P=0.57
	Synchro-Breathe vs.	1.05(0.59 to1.89),	-.06(-.42 to .31),	5.8(-0.3 to 11.8),
	Volumatic	p=1	P=16	P=.07
	*

Acknowledgements

We are grateful to the University of Dundee for having institutionally sponsored and funded this study.

We wish to thank the Tayside Committee for Medical Research Ethics for having approved the conduct of this study. Arun Nair and Brian Lipworth had full access to all data in the study and take responsibility for the integrity of the data and data analysis. Study concept and design were made by AN, BL. Acquisition of data was made by AN, LM, PW, PS, PB. Analysis and interpretation was done by AN, BL. Critical review of the manuscript was performed by AN, BL, LM, PW, PS, PB. Statistical Analysis was performed by AN, LM. We would like to thank Mrs. Lesley McFarlane and Ms. Kara Robertson for technical and administrative support. Study supervision was performed by BL and none of the authors have any conflicts of interest to disclose.


Johan Rasmuson
email: johan.rasmuson@climi.umu.se

Charlotta Andersson

Eva Norrman

Michael Haney

Magnus Evander

Clas Ahlm

Time to revise the paradigm of hantavirus syndromes? Hantavirus pulmonary syndrome caused by European hantavirus

Keywords: Hantavirus, hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, acute respiratory distress syndrome, Puumala virus, pathology

Hantaviruses have previously been recognised to cause two separate syndromes; hemorrhagic fever with renal syndrome in the Eurasia, and hantavirus pulmonary syndrome (HPS) in the Americas. However, increasing evidence suggest that this dichotomy is no longer fruitful when recognising human hantavirus disease and understanding the pathogenesis. Herein are presented three cases of severe European Puumala hantavirus infection that meet the HPS case definition. The clinical and pathological findings were similar to those found in American hantavirus patients. Consequently, hantavirus infection should be considered as a cause of acute respiratory distress in all endemic areas worldwide.

Introduction

The present paradigm is that hantaviruses in the Americas cause hantavirus pulmonary syndrome (HPS), while hemorrhagic fever with renal syndrome (HFRS) is caused by hantaviruses present in Eurasia [START_REF] Duchin | Hantavirus pulmonary syndrome: A clinical description of 17 patients with a newly recognized disease. The hantavirus study group[END_REF][START_REF] Nolte | Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent[END_REF][START_REF] Vapalahti | Hantavirus infections in Europe[END_REF]. Hantaviruses associated with HFRS and HPS are primarily transmitted by inhalation of viral particles shed by infected rodents [START_REF] Botten | Shedding and intracage transmission of Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus) model[END_REF][START_REF] Hardestam | Puumala hantavirus excretion kinetics in bank voles (Myodes glareolus)[END_REF]. In both syndromes there is a local immune reaction in the lungs, mainly in terms of a CD8 + T lymphocyte response [START_REF] Nolte | Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent[END_REF][START_REF] Linderholm | Local host response in the lower respiratory tract in nephropathia epidemica[END_REF][START_REF] Zaki | Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease[END_REF]. Another important feature is endothelial dysfunction and capillary leakage [START_REF] Duchin | Hantavirus pulmonary syndrome: A clinical description of 17 patients with a newly recognized disease. The hantavirus study group[END_REF][START_REF] Nolte | Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent[END_REF][START_REF] Vapalahti | Hantavirus infections in Europe[END_REF][START_REF] Mackow | Hantavirus regulation of endothelial cell functions[END_REF]. After the prodromal phase, including fever, nausea, myalgia and headache, patients with HFRS commonly develop renal failure whereas in HPS the kidneys are often spared and instead the patient frequently present with severe cardiopulmonary dysfunction [START_REF] Duchin | Hantavirus pulmonary syndrome: A clinical description of 17 patients with a newly recognized disease. The hantavirus study group[END_REF][START_REF] Vapalahti | Hantavirus infections in Europe[END_REF][START_REF] Saggioro | Hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hantavirus pulmonary syndrome[END_REF].

Although usually less severe and sometimes overlooked, pulmonary symptoms are common in European HFRS caused by Puumala virus (PUUV). Frequent clinical findings include cough, dyspnoea, interstitial lung infiltrates, pleural effusion and impaired pulmonary function [START_REF] Linderholm | Pulmonary involvement in nephropathia epidemica as demonstrated by computed tomography[END_REF][START_REF] Linderholm | Impaired pulmonary function in patients with hemorrhagic fever with renal syndrome[END_REF][START_REF] Kanerva | Pulmonary involvement in nephropathia epidemica: Radiological findings and their clinical correlations[END_REF]. Pronounced lung involvement in HFRS has previously been reported [START_REF] Clement | Hantavirus pulmonary syndrome in New England and Europe[END_REF][START_REF] Caramello | Puumala virus pulmonary syndrome in a Romanian immigrant[END_REF][START_REF] Hoier | Puumala virus RNA in patient with multiorgan failure[END_REF][START_REF] Seitsonen | Corticosteroids combined with continuous veno-venous hemodiafiltration for treatment of hantavirus pulmonary syndrome caused by Puumala virus infection[END_REF]. However, none of those reported patients had a fatal outcome that could be attributed to the acute infection, and there is no description of histopathological findings.

Our hypothesis is that also European hantaviruses can cause HPS. The aim of the present study was to investigate whether HFRS patients with severe cardiopulmonary distress fulfil HPS criteria and have similar clinical and pathological features.

Materials and methods

In a prospective study, during the latest hantavirus outbreak 2007 in the county of Västerbotten, Sweden [START_REF] Pettersson | Outbreak of Puumala virus infection, Sweden[END_REF], HFRS patients with cardiopulmonary failure and need of invasive assisted ventilation, i.e. intubation and mechanical ventilation, were selected. We identified three patients, whereof two had fatal outcome and one survived. The two patients who died both underwent autopsy three days post-mortem. Organ samples were investigated using immunohistochemistry to describe the immunological response and detect presence of viral antigen. Quantitative real-time RT-PCR was used to detect viral RNA in organ samples, plasma and bronchoalveolar lavage [START_REF] Evander | Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome[END_REF].

For defining HPS we used the case definition criteria published by the U.S. Centers for Disease Control and Prevention (CDC) [START_REF]Hantavirus pulmonary syndrome, 2010 case definition[END_REF]. The project was approved by the Research Ethics Committee of Umeå University. Informed consent was obtained either from the patient, or a close relative when not possible.

Results

Patient 1

A 73-year-old woman was admitted to the intensive care unit (ICU) with acute respiratory distress. She was a lifelong non-smoker living with her husband in a rural area, and had a medical history of hypertension and type II diabetes. The day prior to admission she fell ill with malaise and fever. Nausea, vomiting and pronounced shortness of breath ensued. The initial findings at admission included fever, tachycardia, tachypnoea, hypoxia and somnolence. Laboratory findings during hospitalisation indicated coagulopathy, elevated levels of lactate dehydrogenase (LDH), and development of renal failure (Table 1). There was release of cardiac enzyme troponin T (peak value 1.6; normal value < 0.01 µg/L), indicating myocardial tissue damage.

Lung computer tomography (CT) on admission revealed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, dependant atelectasis, and moderate pleural effusions (Fig. 1) which were later drained (>800 ml). Echocardiography showed inferior hypokinesia, moderate mitral valve insufficiency, normal sized left ventricle and atrium, and systolic pulmonary arterial pressure estimated at 55 mm Hg. Despite non-invasive positive pressure respiratory support and furosemide, her respiratory distress progressed and she was intubated and mechanically ventilated on the first hospital day. The preliminary diagnosis was acute respiratory distress syndrome of uncertain cause, and she was treated with broad-spectrum antibiotics and corticosteroids without improvement. Vasopressor and inotropic support was required to maintain adequate circulation. Repeated echocardiogram showed no overt signs of cardiac failure, though several days into her illness the systolic pulmonary arterial pressure increased to >65 mm Hg. Maximal inspiratory pressures (40 cm H 2 O) were required to maintain minimally adequate oxygenation. She suffered a pneumothorax, and received a large bore thoracostomy with negative pressure drainage. The patient developed multiple organ dysfunction engaging the central nervous, respiratory, cardiovascular, renal and coagulatory systems, and she died after 13 days of ICU care.

PUUV serology was initially negative, but seroconversion occurred during the first week with development of positive immunoglobulin M (IgM) and IgG. No PUUV RNA could be detected in serum or bronchoalveolar lavage fluid, sampled two days after onset of disease. At autopsy PUUV RNA was detected in lung tissue, but not in samples from heart, brain, spleen and liver. Sequencing of the PUUV RNA from lung tissue showed that it was homologous to PUUV strains circulating in northern Sweden. No PUUV antigen could be detected in the tissue by immunohistochemistry using PUUV specific monoclonal antibody. Relevant bacterial cultures were all negative. Notably, three weeks prior to ICU admission the patient had sought medical treatment at our clinic for a urinary tract infection caused by E. coli. In the serum collected at that time PUUV RNA (1,700 copies/ml) was later detected.

Autopsy revealed oedematous and atelectatic lungs with no normal aerated tissue. Pulmonary histopathological features consisted of diffusely oedematous parenchyma with interstitial and intraalveolar fibrosis. The alveoli contained exudates of fibrinous fluid, high number of alveolar macrophages (CD68 + ), proliferative epithelial cells and thick septa but without characteristic hyaline membranes. Interstitial mononuclear cell infiltrates were common, consisting mainly of CD3 + T lymphocytes whereof a vast majority was CD8 + . Many mononuclear cells were expressing cytotoxic markers granzyme B and T cell restricted intracellular antigen-1, TIA-1 (Fig. 2). In pulmonary vessels, focal thrombosis was evident. Microscopy of the heart showed thrombosis in small vessels and focal massive infiltrates of granulocytes and macrophages. In the brain there was focal vasculitis, perivascular infiltration of CD8 + T lymphocytes and non-occlusive thrombosis. Infarctions were seen in the brain, lungs and spleen. Notably, the kidneys had no prominent inflammatory infiltrates.

Patient 2

A 65-year-old woman was admitted to the ICU with acute respiratory distress and circulation insufficiency. Besides a history of Waldenstrom's macroglobulinemia that had not required active treatment, she had no history of health problems, infections or recent hospitalisations. She was a lifelong non-smoker, lived in a rural home with her husband, and handled firewood for home heating. Four days prior to admission the patient noted fever, chills, dyspnoea with dry cough and diarrhoea. On the day of admission, these symptoms were more severe, and an ambulance had been called because of syncope. Her ability to oxygenate deteriorated progressively during the first hospital day, and she was intubated and mechanically ventilated.

Chest x-ray on admission showed bilateral diffuse lung infiltrates and signs of interstitial oedema. Large bilateral pleural effusions were noted, and >1000 ml were drained. Echocardiographic examination identified normal left ventricular wall motion, and no signs of structural abnormalities or of pulmonary hypertension. Thincut CT images of the lungs on the third ICU day showed diffuse bilateral alveolar and interstitial infiltrates with dependent consolidation (Fig. 1). The clinical course during the first seven days was dominated by respiratory insufficiency requiring maximal ventilatory support with high levels of inspired oxygen, as well as circulatory shock requiring treatment with vasopressor and inotropic infusions. Other important clinical aspects included coagulopathy with diffusely spread petechiae, progression of renal failure with anuria requiring dialysis, and elevated levels of LDH (Table 1). She was treated presumptively for bacterial pneumonia and sepsis with a series of broad spectrum antibiotics and corticosteroids, without apparent response.

Hantavirus infection was verified with the detection of PUUV RNA in plasma (630,000 copies/ml) on the day of admission, while IgM and IgG were negative. Seroconversion with positive IgM and IgG occurred two and seven days later, respectively. Consecutive plasma samples were analysed for PUUV RNA with declining viral copy numbers until negative 16 days post onset of disease (data not shown). PUUV RNA was found in bronchoalveolar lavage fluid (11,000 copies/ml) nine days after onset of disease. Bacterial cultures were all negative. The patient remained ventilator-dependent for 13 days, but was finally extubated. She developed critical illness myopathy and needed six weeks in-hospital rehabilitation. At follow up six months later, she complained of muscle ache during exercise and lowered general fitness, but was steadily improving.

Patient 3

A 63-year-old male construction worker was admitted to the ICU with acute respiratory distress, confusion and hypotension. He was a current and long time smoker with a medical history of mild chronic obstructive pulmonary disease and hypertension. Three days prior to admission he had fallen ill with fever, chills, diarrhoea, dry cough, and dyspnoea. In the emergency room, physical examination was notable for fever, somnolence, tachycardia and hypotension. Arterial blood gas analysis on room air revealed hypoxia and respiratory alkalosis. The patient was taken to the ICU and therapy was started with broad spectrum antibiotics due to suspicion of pneumonia and sepsis. As with the other patients, coagulopathy, increased LDH levels, and renal failure were detected (Table 1).

Initial bedside chest X-ray showed no findings to explain the respiratory distress. During the two days ICU course, the patient complained mostly of dyspnoea. He received supplemental oxygen and non-invasive ventilator support initially, but was eventually intubated and mechanically ventilated. CT findings which included only the lower lung lobes revealed bilateral pleural effusions and bibasilar atelectasis (data not shown). During the first day blood pressure was maintained with i.v. fluid, but on the second day his circulatory condition rapidly deteriorated. Despite massive efforts with i.v. fluid, corticosteroids and vasoactive drugs, the patient died in refractory circulatory shock.

PUUV serology on the first hospital day was IgM positive, while IgG was negative. The patient had 130,000 copies of PUUV RNA/ml in plasma. Bacterial cultures were all negative. At autopsy, PUUV RNA was detected in samples from lungs, heart, liver, kidney, brain and spleen. The organs were examined for viral antigen, which was found in the vascular endothelium and in mononuclear cells (Fig. 2).

Post mortem examination showed that pulmonary architecture was preserved but with oedema and focal nonocclusive thrombosis. Similar to Patient 1, there were pulmonary infiltrates of lymphocytes with the same immunophenotype as described (data not shown). Kidneys showed no prominent inflammation.

Discussion

Growing evidence show that there are similarities between HFRS and HPS, as both syndromes can give rise to hemorrhagia, renal impairment and cardiopulmonary dysfunction, which have been attributed to thrombocytopenia and capillary leakage. [START_REF] Duchin | Hantavirus pulmonary syndrome: A clinical description of 17 patients with a newly recognized disease. The hantavirus study group[END_REF][START_REF] Vapalahti | Hantavirus infections in Europe[END_REF][START_REF] Mackow | Hantavirus regulation of endothelial cell functions[END_REF][START_REF] Saggioro | Hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hantavirus pulmonary syndrome[END_REF][START_REF] Linderholm | Impaired pulmonary function in patients with hemorrhagic fever with renal syndrome[END_REF][START_REF] Peters | Hantavirus pulmonary syndrome: The new American hemorrhagic fever[END_REF][START_REF] Bayard | Outbreak of hantavirus pulmonary syndrome, Los Santos, Panama, 1999-2000[END_REF] Severe pulmonary involvement has not been generally perceived to be a significant feature of HFRS. Previously, respiratory symptoms were commonly attributed to fluid overload as a result of renal failure. However, increasing evidence show that lung and heart involvement is common during the acute phase of HFRS [START_REF] Linderholm | Pulmonary involvement in nephropathia epidemica as demonstrated by computed tomography[END_REF][START_REF] Linderholm | Impaired pulmonary function in patients with hemorrhagic fever with renal syndrome[END_REF][START_REF] Puljiz | Electrocardiographic changes in patients with haemorrhagic fever with renal syndrome[END_REF][START_REF] Makela | More than half of the patients with acute Puumala hantavirus infection have abnormal cardiac findings[END_REF]. Concerning the cases of European hantavirus infection in our current report, there was only mild or no renal impairment at the time of admission, whereas the respiratory involvement was early and severe, consistent with acute respiratory distress syndrome (ARDS), fulfilling criteria of HPS according to CDC case definition [START_REF]Hantavirus pulmonary syndrome, 2010 case definition[END_REF]. As described in HPS, the three reported patients suffered from cardiovascular dysfunction, requiring treatment with inotropic and vasopressor drugs [START_REF] Saggioro | Hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hantavirus pulmonary syndrome[END_REF]. Autopsy results support the cause of death to be cardiopulmonary distress due to hantavirus infection. The lungs of both the deceased patients were oedematous and contained mononuclear cell infiltrates with predominantly CD8 + T lymphocytes, as described in HPS patients [START_REF] Nolte | Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent[END_REF][START_REF] Zaki | Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease[END_REF]. Many cells expressed granzyme B and TIA-1, indicating a cytotoxic activity, which could further be supported by high levels of LDH. We chose to demonstrate the immune response in a non-smoker's lungs (Patient 1), but a similar picture was seen in Patient 3.

In the first two patients, the IgM response was delayed, while the quantitative real-time RT-PCR for PUUV RNA was positive, suggesting it as a valuable tool to hasten the aetiological diagnosis [START_REF] Evander | Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome[END_REF]. Notably, in Patient 1 PUUV RNA was found in serum from three weeks prior to the acute onset of disease, that is, in incubation phase and consistent with hantavirus incubation time ranging from one to four weeks [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF][START_REF] Ferres | Prospective evaluation of household contacts of persons with hantavirus cardiopulmonary syndrome in Chile[END_REF].

Pulmonary involvement is well documented in European Puumala hantavirus infection. From a single outbreak in our county, we report on three cases of life-threatening hantavirus pulmonary syndrome. Arguably, additional cases are likely to either go undiagnosed or unreported. A clinical implication of this observation is that hantavirus infection should be included as a differential diagnosis for patients with febrile illness and acute respiratory distress of uncertain cause in endemic areas in Eurasia.

Legends to figures

Fig. 1 Chest CT-scans of two European patients with hantavirus pulmonary syndrome. The examination showed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, together with bilateral dependent atelectasis and moderate pleural effusions (a, Patient 1), and diffuse bilateral alveolar and interstitial infiltrates with dependent consolidation (b, Patient 2) Fig. 2 Immunohistochemistry results in lung sections from two fatal cases of hantavirus pulmonary syndrome. The findings were condensed oedematous pulmonary parenchyma with alveolar fibrinous exudate and infiltrates of mononuclear cells (a), whereof a vast majority were CD8 + T lymphocytes (b), and many were holding granules containing granzyme B (c) and T cell restricted antigen-1, TIA-1 (d); this immunophenotype is characteristic of activated cytotoxic T lymphocytes. In contrast, CD4 + helper T lymphocytes (e) were uncommon. Viral antigen was detected in capillary vascular endothelium (f) and in mononuclear cells, here represented by a monocyte (f; inset), using Puumala hantavirus nucleocapsid protein monoclonal antibody (A1C5, Progen Biotechnik GmbH, Heidelberg, Germany). For viral antigen, lung samples from two non-hantavirus patients were used as negative controls (data not shown). Panels (Patient 1 in a-e; original magnification, x 400; and Patient 3 in f; original magnification, x 600) display lung sections from paraffin embedded material. Staining was performed using hematoxylin and eosin (a), and immunoperoxidase technique (b-f)

Table 1

 1 Laboratory findings in three European hantavirus patient suffering from hantavirus pulmonary syndrome

	Patient No. Sex/Age	WBC a	TPC b	aPTT a	INR a	Albumin b Creatinine a	LDH a
	1: F/73 y	6.5/40.9	293/42	26.7/40.9	1.2/1.9	29/28	50/234	11.4/30.8
	2: F/65 y	15.5/50.3	108/17	49.9/>180.0	1.4/1.9	18/18	87/171	5.2/8.7
	3: M/63 y	12.4/23.0	137/9	42.0/80.0	1.5/2.0	ND	124/164	21.2/21.2
	WBC: white blood cell count, normal range 3.5-8.8 x 10 9 /L; TPC: thrombocyte particle count, normal range for women 165-387, and for
	men 145-348 x 10 9 /L; aPTT: activated partial thromboplastin time, normal range 24.0-36.0 seconds; INR: international normalized ratio,
	normal range 0.8-1.2; serum albumin, normal range 36-45 g/L; serum creatinine, normal range for women 50-90 and for men 60-100
	μmol/L; LDH: lactate dehydrogenase, normal value < 3.4 µkat/L; ND: No data
	a Initial and peak laboratory results, respectively
	b Initial and nadir laboratory results, respectively

Acknowledgements

Irene Eriksson and Ingrid Gustafsson are greatly acknowledged for their skilled technical assistance. This work was supported with grants from the Swedish Heart Lung Foundation, the Heart Foundation of Northern Sweden, the County Councils of Northern Sweden, the County Council of Västerbotten, and the Medical Faculty of Umeå University.

Conflict of interest

The authors declare that they have no conflict of interest.