P Ternengo

L Bradesi

Berti

E Guinoiseau

A Luciani
email: luciani@univ-corse.fr

P G Rossi

Y Quilichini

S Ternengo

P Bradesi

L Berti

Cellular effects induced by Inula graveolens and Santolina corsica essential oils on Staphylococcus aureus

Cellular effects induced by and essential oils on

Introduction

Staphylococcus aureus is a commensal organism commonly cited as a major hospital-acquired pathogen and the leading cause of nosocomial infections [START_REF]National Nosocomial Infections Surveillance System (2004) National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued[END_REF]. The spectacular adaptive capacity of this Gram-positive pathogen resulted in emergence and worldwide spread of lineages that acquired resistance to the majority of available antimicrobial agents. Among some of the most problematic clinically relevant strains at present, the methicillin resistant S. aureus (MRSA) ranks as one of the most difficult bacteria to treat in patients and eradicate from hospital environments. Recent reports of S. aureus isolates with intermediate or complete resistance to vancomycin portend a chemotherapeutic era in which effective bactericidal antibiotics against this organism may no longer be readily available [START_REF] Cdc | Staphylococcus aureus resistant to vancomycin-United States[END_REF][START_REF] Jevitt | In vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin against a challenge panel of staphylococci and enterococci, including vancomycinintermediate Staphylococcus aureus and vancomycin-resistant Enterococcus faecium[END_REF][START_REF] Tenover | Vancomycin-resistant Staphylococcus aureus isolate from a patient in Pennsylvania[END_REF][START_REF] Jacobson | Daptomycin therapy failure in an adolescent with methicillin resistant Staphylococcus aureus bacteremia[END_REF]. The search for new agents has therefore become indispensable.

Essential oils are complex natural mixtures of volatile secondary metabolites isolated from plants by hydrodistillation. They are known to possess numerous biological properties including antioxidative, antitumoral, anti-inflammatory, antifungal and antibacterial activities [START_REF] Đordević | Antimicrobial, anti-inflammatory, antiulcer and anti-oxidant activities of Carlina acanthifolia root essential oil[END_REF][START_REF] Sousa | Melissa officinalis L. essential oil: antitumoral and antioxidant activities[END_REF][START_REF] Kumar | An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells[END_REF][START_REF] Xiao | Essential oil of Curcuma wenyujin induces apoptosis in human hepatoma cells[END_REF][START_REF] Jasicka-Misiak | Antifungal activity of the carrot seed oil and its major sesquiterpene compounds[END_REF][START_REF] Pinto | Antifungal activity of the essential oil of Thymus puleigoides on Candida, Aspergillus and dermatophyte species[END_REF][START_REF] Tavares | Essential oil of Daucus carota subsp. halophilus: composition, antifungal activity and cytotoxicity[END_REF][START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF]. Although in vitro antibacterial activity and in vivo efficacy of essential oils have been reported [START_REF] Carson | Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties[END_REF], less is known about their mechanism of action. Nevertheless, potential sites of their antibacterial action have been defined, e.g. they can act by damaging the cell wall, the plasma membrane, the membrane proteins or decreasing the proton motrice force. They can also cause an aggregation of the cytoplasm or a leakage of the ions and the cell contents [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF].

In an ongoing project, which aims to identify new antibacterials from plants growing in Corsica, effectiveness of various essential oils and their main components has been demonstrated [START_REF] Liu | Composition, irregular terpenoids, chemical variability and antibacterial activity of the essential oil from Santolina corsica Jordan et Fourr[END_REF][START_REF] Rossi | E)-Methylisoeugenol and elemicin: antibacterial components of Daucus carota L. essential oil against Campylobacter jejuni[END_REF][START_REF] Rossi | Antibacterial action of essential oils from Corsica[END_REF]. The specificity of their composition, mainly due to their acute chemical variability, seems to explain their high antibacterial activity [START_REF] Liu | Composition, irregular terpenoids, chemical variability and antibacterial activity of the essential oil from Santolina corsica Jordan et Fourr[END_REF][START_REF] Rossi | E)-Methylisoeugenol and elemicin: antibacterial components of Daucus carota L. essential oil against Campylobacter jejuni[END_REF][START_REF] Rossi | Antibacterial action of essential oils from Corsica[END_REF][START_REF] Bianchini | Composition of Helichrysum italicum (Roth) G. Don fil. subsp. italicum essential oils from Corsica (France)[END_REF][START_REF] Blanc | Chemical composition and variability of the essential oil of Inula graveolens from Corsica[END_REF]. The objective of this study was to go further into the effects of Corsican essential oils on bacterial cells. In this context, we evaluated the antibacterial activity of Inula graveolens and Santolina corsica essential oils against S. aureus and provided insights into their primary mode of action.

Material and methods

Bacterial strain and growth conditions

The test strain used in this work was Staphylococcus aureus ATCC 6538P (CIP 53.156). S. aureus ATCC 6538P was shown to be the most appropriate test microorganism because of its susceptibility to all β-lactams antibiotics, including methicillin. The cultures of S. aureus ATCC 6538P were obtained from the "Institut Pasteur Collection" (Paris, France), preserved frozen in cryovials at -80°C and routinely grown at 37°C on Mueller-Hinton agar (MHA, bioMérieux).

Essential oils

Inula graveolens essential oil was purchased at "Huiles essentielles et Hydrolats de Corse" (Mandriolu, Corsica). The main components quantified by GC (RI), GC-MS and 13 C NMR were as previously described [START_REF] Blanc | Chemical composition and variability of the essential oil of Inula graveolens from Corsica[END_REF]: bornyl acetate (43.3%), borneol (26.2%) and camphene (7%). Santolina corsica essential oil was isolated from the aerial parts of plants by hydrodistillation. The main components quantified by GC (RI), GC-MS and 13 C NMR were as previously described [START_REF] Liu | Composition, irregular terpenoids, chemical variability and antibacterial activity of the essential oil from Santolina corsica Jordan et Fourr[END_REF]: myrcene (34.6%), santolina triene (13.5%) β-phellandrene (11.7%) and lyratol (6.1%).

Susceptibility testing

The MIC and MBC assays were performed by standard broth dilution methods [START_REF] Carson | Broth micro-dilution method for determining the susceptibility of Escherichia coli and Staphylococcus aureus to the essential oil of Melaleuca alternifolia (tea tree oil)[END_REF][START_REF] Cha | Chemical composition and antibacterial activity of essential oil from Artemisia feddei[END_REF]. In brief, serial twofold dilutions of each essential oil in Mueller-Hinton broth (MHB, Oxoid) were prepared in sterilized test tubes over the range 0.03-8% (v/v). To enhance oil distribution, Tween 80 detergent (Polyoxyethylene sorbitan monoflaurate, Sigma) was included in all assays at a final concentration of 0.01% (v/v). The inoculum to be tested (13 µL, 10 8 CFU/ml) was added to each of the test tubes, which were incubated at 37°C for 24 h and finally centrifuged at 5000 g for 5 min at 20°C. The MIC was then determined from the first test tube devoid of bacterial growth. All determinations were performed in triplicate and a control consisting of MHB with 0.01% (v/v) Tween 80 was included. After determination of the MIC, the samples showing no bacterial growth were streaked on MHA agar plates, which were incubated for 24 h at 37°C and finally examined for 99.9% killing.

Time-kill studies

Time-kill procedures were performed according to the method described by Klepser et al. [START_REF] Klepser | Influence of test conditions on antifungal time-kill curves results: proposal for standardized methods[END_REF] and modified by Viljoen et al. [START_REF] Viljoen | Osmitopsis asteriscoides (Asteraceae)the antimicrobial activity and essential oil composition of a Cape-Dutch remedy[END_REF]. Activities of essential oils against S. aureus were evaluated by measuring the reduction in the number of CFU per milliliter over 2 h. An initial inoculum of 5 x 10 6 CFU•ml -1 was prepared and 1 ml was added to 9 mL of MHB containing either the essential oils and Tween 80 (tests) or Tween 80 only (control). Essential oils were used at a final concentration equal to their MIC. Tween 80 was added to both tests and control at a final concentration of 0.01% (v/v). The test tubes were incubated with agitation at 37°C and samples (100 μl), taken in duplicate at 0, 10, 30, 60, 90 and 120 min, were serially 10-fold diluted, plated onto MHA and the total viable counts were determined after overnight incubation at 37°C. The limit of quantification by this method is 10 2 CFU•ml -1 .

Bacteriolysis

The bacteriolysis assays were carried out according to the standard method described by Carson et al. [START_REF] Carson | Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined par time-kill, lysis, leakage and salt tolerance assays and electron microscopy[END_REF]. A bacterial suspension was prepared by inoculating two colonies of S. aureus from overnight cultures on blood agar into 400 ml of MHB, which was incubated at 37°C for 18 h with shaking. After incubation, the bacteria were separated from the growth medium by centrifugation at 10,000 g for 12 min at 4°C, washed twice with phosphate-buffered saline (PBS, pH 7.4), and resuspended in PBS supplemented with 0.01% Tween 80 (PBS-T, v/v). The bacterial suspension was adjusted so that the optical density at 620 nm (OD 620 ) of a 1 in 100 dilution was 0.310 (∼3×10 10 CFU•ml -1 ). Essential oils were added to the bacterial suspension at concentrations equivalent to the MIC and eight times the MIC at a ratio of 1:9. PBS-T was added to the control suspension. The suspensions obtained were mixed for 20 s with a Vortex mixer. Samples (10 µl) were taken in duplicate at 0, 0.5, 30, 60, 90 and 120 min, serially 10-fold diluted, and the OD 620 was measured immediately (Analityk Jena Specord 205). Each test was performed on three independent experiments. The results were expressed as a ratio of the OD 620 at each time point versus the OD 620 at 0 min (in percent).

Transmission Electron Microscopy (TEM)

A suspension of S. aureus in exponential phase of growth was prepared by inoculating and incubating 80 ml of MHB at 37°C for 24 h with shaking. The bacterial suspension was adjusted so that the optical density at 620 nm (OD 620 ) of a 1 in 100 dilution in MHB was 0.200 (10 7 CFU•ml -1 ). S. aureus cells were treated with the MIC of each essential oil for 5 min. Control stood for 5 min in MHB-Tween 80 (0.1 %, v/v). After centrifugation at 3,000 rpm for 10 min, the pellets were first fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature and then post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature. The postfixed microbial pellets were processed in graded ethyl alcohol, propylene oxide, Spurr resin and cured for 24 h at 45°C. Ultrathin sections were stained with uranyl acetate followed by lead citrate and were examined with a transmission electron microscope (HITACHI H-600 TEM) at an accelerating voltage of 75 kV.

Results

The antibacterial activities of I. graveolens and S. corsica essential oils on S. aureus are presented in Table 1. The MIC values of both essential oils are 5 mg.ml -1 . The MBCs of I. graveolens and S. corsica essential oils are, respectively, 10 mg•ml -1 and 8 mg•ml -1 . The MBC/MIC ratios of both essential oils are lower than 4.

Killing profiles of the two essential oils against S. aureus are presented in Fig. 1. Preliminary treatments of S. aureus with I. graveolens and S. corsica essential oils, used at a concentration equivalent to their MIC (5 mg•ml -1 ), have shown that the bactericidal end-point (99.9% or ≥ 3log10 of inhibition [START_REF]Methods for determining bactericidal activity of antimicrobial agents. Approved guideline M26-A[END_REF]) was obtained after 2 h and 4 h, respectively (data not shown). The 2-h time-kill curves show a detailed progression of the bactericidal activities. The I. graveolens essential oil at the MIC acts at once. During the first 10 min, the number of CFUs fell by 1log10 and the detection threshold (10 2 CFU•ml -1 ) is reached in 1 h. The bactericidal effect of S. corsica essential oil at the MIC is slower, i.e. the number of CFUs slows down gradually by 2log10 in 120 min.

S. aureus cells untreated by essential oils correspond to the negative control of bacteriolysis. The absorbance at 620 nm (OD 620 ) of negative control does not vary significantly (mean OD 620 after 120 min corresponds to 93% of the initial absorbance). Figure 2 shows the OD 620 of S. aureus cells treated with I. graveolens and S. corsica essential oils. Treatment with I. graveolens essential oil at Transmission electron micrographies of S. aureus cells untreated (control) and treated with I. graveolens and S. corsica essential oils at their MIC are presented in Fig. 3.

The cell wall and the plasmic membrane of S. aureus control cells are regular. The cytoplasmic contents are homogeneous. The start of cellular division is also visible in Fig. 3a where the septum of division begins to form. The plasmic membrane of S. aureus cells treated with I. graveolens essential oil appears to have one invagination (Fig. 3b) at the level of which the cell wall presents an unusual thickening. The same modifications are observed in S. aureus cells treated with S. corsica essential oil (Fig. 3d). Under the action of both essential oils, cytoplasm of some cells also seem to be less dense and altered. In Fig. 3c, the cytoplasm seems to have lost its even distribution and intracellular materials are aggregated near the cell wall.

Discussion

In this study, the in vitro antibacterial activity of I. graveolens and S. corsica essential oils on S. aureus are reported. MIC values are close to MBC values, indicating that both essential oils display a bactericidal activity [START_REF] Hammer | Susceptibility of transient and skin flora to the essential oil of Melaleuca alternifolia (tea tree oil)[END_REF]. The oils considerably reduce the viability of S. aureus cells at their MIC and produce a lethal effect at concentrations higher than their MIC. Their bactericidal activity depends Plants of Inula genus belong to the family Compositae [START_REF] Garnier | Ressources médicinales de la flore Française[END_REF] and are widely distributed in the Mediterranean area. The chemical composition of various Inula species, including I. viscosa and I. graveolens, has been described by Bohlmann et al. [START_REF] Bolhmann | Naturally occurring terpene derivatives. 142. New sesquiterpene lactones from Inula species[END_REF]. The essential oil of I. graveolens, used in this study, is constituted by a complex mixture of volatile molecules dominated by two oxygenated so that the activity of the whole oil is related to the presence of these two compounds, recognized as antibacterial agents [START_REF] Cha | Chemical composition and antibacterial activity of essential oil from Artemisia feddei[END_REF][START_REF] Bolhmann | Naturally occurring terpene derivatives. 142. New sesquiterpene lactones from Inula species[END_REF][START_REF] Tabanca | Composition and antimicrobial activity of the essential oil of Micromeria cristata subsp. phrygia and the enantiomeric distribution of borneol[END_REF]. Alcohols are known to possess bactericidal rather than bacteriostatic activity [START_REF] Dorman | Antimicrobial agents from plants: antibacterial activity of plant volatile oils[END_REF]. So, the high amount of borneol could explain the bactericidal effect induced by the whole oil on S. aureus cells. A bacteriostatic activity has however been mentioned by Debat et al. [START_REF] Debat | Inula extract, its method of preparation and its use as pharmaceutical[END_REF] for S. aureus cells treated with various I. graveolens extracts. In another study, Caldes et al. [START_REF] Caldes | Potential antileukemic substance present globularia alypum[END_REF] reported that I. graveolens extracts, obtained by extraction with hot and cold water respectively and then lyophilization, were inactive against S. aureus and S. faecium cells. The process of extraction of plant products influences their chemical composition and this may also influence their antibacterial activity [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF]. This could be an explanation for the different activities observed between the different extracts and the essential oil of I. graveolens.

The genus Santolina is represented by more than ten species in the Mediterranean area. Santolina corsica is an endemic species to Corsica and Sardignia [START_REF] Arrigoni | Le genre Santolina L. en Italie[END_REF]. The composition of essential oil from various species of Santolina has been widely studied [START_REF] Liu | Composition, irregular terpenoids, chemical variability and antibacterial activity of the essential oil from Santolina corsica Jordan et Fourr[END_REF]. The essential oil of S. Corsica, used in this work, is dominated by three monoterpenes hydrocarbons: myrcene (34.6%), santolina triene (13.5%) and β-phellandrene (11.7%). Since hydrocarbons are usually weakly active [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF], the involvement of less abundant constituents in the antibacterial effect of the whole oil should be considered. Indeed, minor constituents might be involved in some type of antibacterial synergism with other active components of essential oils as evident by the work of Marino et al. [START_REF] Marino | Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae[END_REF].

Some antimicrobial agents induce severe membrane damage and provoke whole cell lysis [START_REF] Denyer | Biocide-induced damage to the bacterial cytoplasmic membrane[END_REF][START_REF] Hamouda | Antimicrobial mechanism of action of surfactant lipid preparations in enteric Gram-negative bacilli[END_REF][START_REF] Song | Antimicrobial action of phydroxyphenyl acrylate[END_REF][START_REF] Razzaghi-Abyaneh | Ultrastructural evidences of growth inhibitory effects of a novelbiocide, Akacids®plus, on an aflatoxigenic Aspergillus parasiticus[END_REF] and this has been previously reported for oregano, rosewood and thyme essential oils [START_REF] Horne | Antimicrobial effects of essential oils on Streptococcus pneumoniae[END_REF]. However, the main components of these essential oils, including carvacrol, citronellol, geraniol and thymol [START_REF] Windholz | The Merck index[END_REF], are not found in I. graveolens and S. corsica essential oils. Also, cell death induced by both essential oils does not seem to be caused by a lytic effect. This is also supported by our electron micrographies on which S. aureus cells remain unlysed. The structure of the plasmic membrane is however altered. Cells treated with both oils exhibit abnormal invaginations of their membrane. Carson et al. [START_REF] Carson | Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined par time-kill, lysis, leakage and salt tolerance assays and electron microscopy[END_REF] reported similar effects for S. aureus cells treated with Melaleuca alternifolia essential oil (tea tree oil [TTO]) and/or its main constituents. These agents do not provoke whole cell lysis but compromise the structural integrity of the plasmic membrane and induce a loss of the cytoplasmic contents [START_REF] Carson | Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined par time-kill, lysis, leakage and salt tolerance assays and electron microscopy[END_REF]41].

Cytoplasmic changes also occur under the action of I. graveolens and S. corsica essential oils. The cytoplasm of some cells is less dense and shows clumping of intracellular contents. Areas with aggregated material are observed close to the cell wall, which loses its uniformity and tends to become rough. Unusual thickenings are observed in the wall of S. aureus treated with both oils. This thickened appearance is more pronounced near the membrane invaginations. Similar damage has been previously observed on the wall of Listeria innocua cells treated with the antibacterial polypeptide nisin [START_REF] Maisner-Patin | Cell wall changes in nisinresistant variants of Listeria monocytogenes grown in the presence of high nisin concentrations[END_REF]. Calderόn-Miranda et al. [START_REF] Calderόn-Miranda | Transmission electron microscopy of Listeria monocytogenes treated by pulsed electric fields and nisin in skimmed milk[END_REF] also reported that L. innocua cells, exposed to 32 pulses of electric fields intensity of 30 kV/cm, exhibit an increase of the cell wall roughness. No disruption in either the cell wall or the plasmic membrane of L. innocua occurs. However, the cytoplasm is clumped and not evenly distributed as observed in our work.

The mechanism of essential oil action towards microorganisms is complex and not yet fully explained. The available literature data show that the primary site of the toxic action of essential oils is generally the plasmic membrane. This is directly related to the hydrophobicity of the terpenoid constituents of essential oils, which enables them to partition into the bacterial lipid bilayer, disturbing its structure and increasing its permeability to protons, ions and other cell constituents [START_REF] Carson | Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties[END_REF][START_REF] Sikkema | Interactions of cyclic hydrocarbons with biological membranes[END_REF][START_REF] Sikkema | Mechanism of membrane toxicity of hydrocarbons[END_REF][START_REF] Cox | The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil)[END_REF][START_REF] Ultee | The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogene Bacillus cereus[END_REF][START_REF] Kalemba | Antibacterial and antifungal properties of essential oils[END_REF]. In addition to membrane alterations, terpenoids might also cross the cell membrane, thus penetrating in the cell and interacting with critical intracellular sites [START_REF] Cristani | Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity[END_REF]. They might have several invasive targets which could lead to the inhibition of bacterial pathogens such as S. aureus.

Taken together, our findings suggest that the bactericidal activity of I. graveolens and S. corsica essential oils resides in their ability to detrimentally affect the integrity of the plasmic membrane and the cell wall of S. aureus. Considering the multiple alterations produced and the large number of chemical constituents of essential oils, it is most likely that the effects produced by I. graveolens and S. corsica oils are not attributable to one specific mechanism. For this reason, an in-depth study is required to fully understand the way by which the two oils and their active constituents interact with the bacterial membrane and the consequences they bring out at the cellular level.

Fig. 1 Fig. 2

 12 Fig. 1 Effects of S. corsica and I. graveolens essential oils (MIC, 5 mg•ml -1 ) on growth of S. aureus over 2 hours

Fig. 3

 3 Fig. 3 Transmission electron micrographies of S. aureus cells stained with uranyl acetate and lead citrate. a Control. b and c S. aureus cells treated with I. graveolens essential oil at the MIC. d S. aureus cells treated with S. corsica essential oil at the MIC. PM plasmic membrane, CW cell wall, S septum

Table 1

 1 MICs, MBCs and MBC/MIC ratios of I. graveolens and S. corsica essential oils on S. aureus strain

	Essential oils	MIC	MBC	MBC / MIC
		(mg•ml -1 )	(mg•ml -1 )
	Inula graveolens	5	1 0	2
	Santolina corsica	5	8	1.6
	MIC minimum inhibitory concentration, MBC minimum bactericidal
	concentration

Acknowledgement We would like to thank Pr. B. Marchand from the University of Corsica for the supply of the transmission electron microscopy equipment, and Mrs. A. Cornillac from the CMEABG of C. Bernard University (Lyon) for her expertise on the TEM experiments.


MD Annette J Vangsted

Tobias Wirenfeldt Klausen

Peter Gimsing

Niels Abildgaard

Niels F Andersen

Bjørn Henrik Gregersen

Andersen Nexø

Ulla Birgitte Vogel

Bjørn A Nexø

The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in and

Keywords: multiple myeloma, SNP, chromosome 19q13.3, outcome, interferon-α, thalidomide, bortezomib MM, multiple myeloma, OS, overall survival, TTF, time-to-treatment failure, HDT, high-dose treatment, SNP, single nucleotide polymorphism, LD, linkage disequilibrium (LD), CI, confidence interval, RR, relative

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

Introduction

Multiple myeloma (MM) is an incurable B-cell malignancy. Younger patients are treated with high-dose treatment (HDT) and stem cells support. Despite this intensive treatment strategy, huge variation in treatment outcome is seen, with some patients suffering from early and aggressive recurrence of the disease. Understanding the mechanism of these differences may help us to establish new and better treatment strategies. Response to chemotherapy may reflect the effect of genetic variation related to differences in DNA repair, apoptosis and immune response.

A small region on the human chromosome 19q13.3 may be important for the development of cancers of the breast, lung and skin as well as outcome of chemotherapy (figure 1) [START_REF] Rockenbauer | Association of chromosome 19q13.2-3 haplotypes with basal cell carcinoma: tentative delineation of an involved region using data for single nucleotide polymorphisms in two cohorts[END_REF][START_REF] Nexo | A specific haplotype of single nucleotide polymorphisms on chromosome 19q13.2-3 encompassing the gene RAI is indicative of postmenopausal breast cancer before age 55[END_REF][START_REF] Vogel | Two regions in chromosome 19q13.2-3 are associated with risk of lung cancer[END_REF][START_REF] Vangsted | Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation[END_REF]. The region encompasses 4 genes. Two genes, related to nucleotide excision repair (NER), the excision-repair cross-complementing 1 (ERCC1) and ERCC2 (XDP), are involved in repair of bulky DNA damage caused by chemotherapy [START_REF] De Boer | Nucleotide excision repair and human syndromes[END_REF]. The gene product of the third gene RAI (RelA inhibitor; also named iASPP or protein phosphatase 1, regulatory (inhibitor) subunit 13 like (PPP1R13L)) [6;7] was identified as a protein that binds to the NF-κB subunit p65/RelA and inhibits its transcriptional activity [START_REF] Yang | Identification of a novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor[END_REF]. The inhibition of NF-κB transcription induced apoptosis [START_REF] Yang | Identification of a novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor[END_REF]. Later studies revealed that the RAI protein also inhibits apoptosis by binding to p53 [START_REF] Bergamaschi | iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human[END_REF]. Recently, over expression of RAI has been shown to increase tumor formation in primary mouse embryonic fibroblasts [START_REF] Laska | Enforced expression of PPP1R13L increases tumorigenesis and invasion through p53dependent and p53-independent mechanisms[END_REF]. These data support the hypothesis that RAI may function as an oncogene. The fourth gene in the chromosome region 19q13.3 is named CD3EAP (CD3e molecule epsilon associated protein; alias ASE1(anti-sense to ERCC1); alias CAST)) [START_REF] De Boer | Nucleotide excision repair and human syndromes[END_REF]. The gene was cloned by use of autoimmune serum from a patient with interstitial cystitis [START_REF] Ochs | cDNA cloning and characterization of a novel nucleolar protein[END_REF]. It is orientated anti-sense to and overlaps with the ERCC1 gene and it has therefore been suggested, that the mRNA of CD3EAP may bind the transcript of ERCC1 and inhibit translation [START_REF] Whitehead | ASE-1: a novel protein of the fibrillar centres of the nucleolus and nucleolus organizer region of mitotic chromosomes[END_REF]. This hypothesis was not supported by scientific results. The protein is localized in the fibrillar centers of the nucleus, which may indicate a function in ribosomal RNA transcription [START_REF] Whitehead | ASE-1: a novel protein of the fibrillar centres of the nucleolus and nucleolus organizer region of mitotic chromosomes[END_REF]. Another study by Yamazaki et al. suggests that the gene product of CD3EAP mediates activation signals, important for IL-2 production [START_REF] Yamazaki | CAST, a novel CD3epsilon-binding protein transducing activation signal for interleukin-2 production in T cells[END_REF]. In a recent study of fifty-nine NCI60 human tumour cell lines, sensitivity to anticancer drugs correlated positively with the variant A-allele of the polymorphism CD3EAP G-21A [START_REF] Nissen | Association of a single nucleotide polymorphic variation in the human chromosome 19q13.3 with drug responses in the NCI60 cell lines[END_REF]. At present, the function of the gene is not clearly established. We have earlier shown that genetic variation in CD3EAP is associated with outcome in patients with multiple myeloma treated with HDT [START_REF] Vangsted | Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation[END_REF]. Patients carrying the variant A-allele of CD3EAP G-21A had a significant longer TTF and OS after HDT [START_REF] Vangsted | Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation[END_REF].

These results support the notion that the genomic region influence outcome of chemotherapy.

The gene NFКB1 encodes the p50 subunit and its precursor p105. We have previously shown, that the polymorphism NFКB1 -94 ins/del is associated with outcome of interferon-α (INF-α) maintenance treatment after HDT in patients with MM [START_REF] Vangsted | A polymorphism in NFKB1 is associated with improved effect of interferon-{alpha} maintenance treatment of patients with multiple myeloma after high-dose treatment with stem cell support[END_REF].

The aim of the present study was to gain further knowledge of impact of genetic variation in the human chromosome region 19q13.3, by fine mapping associations between many single nucleotide polymorphisms (SNPs) with outcome of 348 myeloma patients treated with HDT. Furthermore, we analysed if genes in the chromosome sub-region19q13.3 interplay with the polymorphism in NFКB1 in relation to outcome of multiple myeloma treated with HDT and maintenance treatment with interferon-α.

Materials and methods

Subjects, clinical data, response criteria, eligibility criteria, and treatment for patients included in SNP analysis.

Subjects, clinical data, response criteria, and eligibility criteria have previously been described in detail [4;14;15]. Briefly, patients diagnosed with MM and treated with HDT and stem cell support from August 1994 to August 2004

were recruited from four participating centres in Denmark. Three hundred forty-eight patients were included in the study. Staging was according to Durie and Salmon and the International Staging System (ISS). Time-to-treatment failure (TTF) and overall survival (OS) were calculated from date of stem cell infusion to date of progression or death.

Progression was defined by a more than 25% increase in serum M-protein or 25% increase in immunoglobulin levels above upper normal levels, confirmed by 2 separate measurements within a 1-month interval. Increase in bone marrow infiltration of plasma cells by 25%, and increase of Bence-Jones proteinuria to more than 1.0g/24 hr or other signs of progression such as hypercalcaemia, progressive skeletal disease or soft-tissue plasmacytoma were also considered as progression. The occurrence of other malignancies and death without progression was regarded as events not related to progression. These patients were included in the analysis of OS, but they were excluded at time of death in the analysis of TTF. Complete response (CR) after HDT was defined by the disappearance of M-protein form serum and urine, as detected by agarose gel electrophoresis and less than 5% plasma cells in the bone marrow aspirate. Partial response (PR) was defined by a reduction of at least 50% in the initial serum M-protein concentration and a reduction of light chain proteinuria to less than 0.2g/24 hours. Time-to-next treatment (TNT) was used as follow-up after relapse treatment and was defined as the period from start of relapse treatment to start of new relapse treatment, progression without new treatment or death.

Induction therapy was 3 series VAD (vincristine, doxorubicin and dexamethasone) or 2-3 series of cyclophosphamide 1g/m 2 i.v. on day 1 combined with dexamethasone 40 mg daily p.o., on days 1-4 and days 9-11 (total dose 320 mg for each series) repeated every 3 weeks. Peripheral blood stem cells were harvested at regeneration after cyclophosphamide priming, and the patients thereafter underwent high-dose chemotherapy with melphalan (200 mg/m 2 ) followed by stem cell support. Fifty-three patients received a second HDT up-front. Maintenance therapy with IFN-α was implemented 3 months after HDT according to standard treatment practise in the particular region (median 105 days) [START_REF] Vangsted | A polymorphism in NFKB1 is associated with improved effect of interferon-{alpha} maintenance treatment of patients with multiple myeloma after high-dose treatment with stem cell support[END_REF]. Two-hundred and forty-tree patients experienced relapse which required treatment. Among these patients 177 were treated with thalidomide and 74 were treated with bortezomib [START_REF] Vangsted | Improved survival of multiple myeloma patients with late relapse after high-dose treatment and stem cell support; A population-based study of 348 patients in Denmark in 1994-2004[END_REF].

PBMC was purified from 292 leukapheresis products by buffy coat preparation. From 56 patients 10 times 10 μm sections were collected from paraffin embedded bone marrow samples. Material was not available for 19 patients undergoing HDT and these patients were not included in the study.

The study was approved by the Danish Ethical Committee (01-158/03).

DNA purification

DNA for analysis was purified from PBMC by the salting out method [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] or from paraffin embedded tissue by phenol extraction as described elsewhere [START_REF] Saber | K-ras mutations in sinonasal adenocarcinomas in patients occupationally exposed to wood or leather dust[END_REF].

Genotyping of single nucleotide polymorphisms

The XPD (ERCC2) SNP rs238406, rs238407, rs2097215, the RAI SNPs rs10422489, rs4544343, rs6966, rs12986272, rs10402584, rs10402393, rs4572514, rs4803816, rs95947, rs928911, rs4803817, nr.106, rs10412761 and the CD3EAP (ASE1) SNPs rs8113779, rs2013521, rs735482, rs762562, rs2336219, rs3212986, rs3212985, rs1007616, and the ERCC1 SNPs rs3212969, rs3212961, rs2298881, and rs1319052 were tested using a Sequenom® platform (San Diego, California) and the iPLEX gold reaction. Each PCR reaction contained 10 ng of genomic DNA, 0.5 U HotStar Taq from Qiagen (Hilden, Germany), 1.25 x Enzyme Buffer (Qiagen), 3.5 mM MgCl 2 . and 1 mM of each deoxynucleotide. The primers were added to a final concentration of 500 nM each. The total reaction volume was 4 μl. PCR reactions with primers of each plex were performed in 384 well plates with V-shape by performing a 15 min preheat to 94 C followed by cycling the samples 45 times, 94 C for 20 sec, 56 C for 30 sec and 72 C for 1 min, followed by 3 min at 72 C. The plates were stored at -20 C. Treatment with Shrimp Alkaline Phosphatase and extension with molecular weightmodified nucleotides was performed according to the manufacturer's recommendations. The reactions were cleaned with resin and spotted on SpectroChip arrays according to the manufacturer's recommendations. The reactions were analyzed by MALDITOF mass spectrometry on the Sequenom equipment, and the results were analyzed using the MassARRAY Type 4.0 (Sequenom).The primers and probes are presented in supplement Table 1. For XPD-exon23 (rs13181); XPD-exon10 (rs1799793), RAI-intron8-1 (rs1970764), ASE1-exon1 (rs967591), ERCC1-exon4 (rs317770), and NFКB1 ATTG (rs28362491) the method has been previously reported [4;14;19] Statistical methods SPSS statistical software was used for all calculations (SPSS for Windows, Rel. 14.0.0. 2005, Chicago: SPSS Inc.). All tests were two-sided and P-values < 0.05 were regarded as statistically significant. Fisher´s exact test was used to compare categorical variables and the Mann-Whitney test was used to compare continuous and categorical variables.

Kaplan-Meier method and the log rank test were used to compare TTF and OS between groups. The Cox proportional hazard model and log-likelihood statistics were applied for univariate analyses and covariates and for multivariate analysis.

Results

Study population

Three-hundred and forty-eight myeloma patients were treated with HDT and stem cell support. The patient characteristics are presented in Table 1. The median age of the patients was 56 years and the study included 59% males.

The median follow-up of all patients still alive was 93.4 months (range, 54.6-174.2 months). At follow up 208 patients had died. The median OS was 69.8 months (60.5-81.6 months). The median TTF was 27.7 months (23.4-30.8 months).

One-hundred and forty-six patients were treated with INF-α maintenance treatment.

The genotype frequencies of XPD, RAI, CD3EAP and ERCC1 and their influence on TTF, OS and response to treatment

All genetic variation in the chromosomal region was previously identified by sequencing of breast cancer patients and healthy controls [START_REF] Nexo | Linkage disequilibrium mapping of a breast cancer susceptibility locus near RAI/PPP1R13L/iASPP[END_REF]. The genotype frequencies and association of minor allele frequency with TTF and OS after HDT are summarized in supplementary Table 2. For completeness of the investigation, we have also included previously reported SNPs in the region [START_REF] Vangsted | Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation[END_REF]. The percentage of successful genotype determinations was low for a number of polymorphisms, reflecting that the mass-spectrometry-based genotyping was unsuccessful for DNA isolated from paraffin embedded tissues, which constituted 16% of the samples. The outcome for homozygous wild type carriers versus variant carriers association of single polymorphisms in XPD, RAI, CD3EAP and ERCC1 is illustrated in Figure 2 A sub-region of 19q13.3 on the chromosome spanning the regions RAI-intron1-1 to RAI-intron1-3 and the region exon1 to exon3-6 in CD3EAP, was significantly associated with TTF and OS in myeloma patients treated with HDT, and carriers of the variant allele of the SNPs had prolonged TTF and OS. There was no difference in the allele frequencies among patients dependent on the participating centres, and there was no difference in genotype distribution in relation to age, β2-mcroglobulin, creatinine, albumin, Durie-Salmon stage, ISS stage, gender, tandem HDT upfront, INF-α maintenance treatment, second HDT at relapse, treatment with thalidomide or bortezomib (supplementary table 3).

There was no association between allele frequencies and response to treatment.

Haplotype analysis of XPD, RAI, CD3EAP and ERCC1 and the influence on TTF and OS in myeloma patients treated with HDT.

Pair-wise linkage disequilibrium and block structure of 33 SNP´ in the genes XPD, RAI, CD3EAP and ERCC1, using the haplo-view software program, are illustrated in supplementary Figure 1. Two haplotype analyses were generated for the sub-region 19q13.3. One haplotype analysis covered the genes RAI, CD3EAP and ERCC1 (rs4803816, rs967591, rs3212985 and rs1319052), and the other haplotype covered the gene CD3EAP (rs8113779, rs735482, rs3212986 and rs1007616). The two haplotype analyses included 97.6% and 85% of all patients with available genotype data in the study, respectively. None of the haplotype analyses revealed stronger association to TTF or OS than the two most significant SNP´s; RAI-intron1-1 (rs4572514) and ASE1-exon1 (rs967591).

Combination of SNPs

Supplementary Table 4 and Figure 3 show the combination analysis of NFKB1 -94ins/del, CD3EAP G-21A and RAI-intron1-1. When genotypes of CD3EAP or RAI were combined with genotypes of NFKB1 -94ins/del, carriers of the variant A-allele of CD3EAP G-21A or the variant C-allele of RAI-intron1-1, a prolonged TTF was seen independently of the NFKB1-94ins/del genotype, in contrast to OS, where an increase in median survival to more than 10 years, only was seen among carriers of the variants alleles of either CD3EAP G-21A or RAI-intron1-1, when combined with the wild type ins-allele of NFKB1 -94ins/del. treatment. From previous results we know that TTF and OS are not significantly affected by the polymorphism NFKB1 -94ins/del [START_REF] Vangsted | The polymorphism IL-1beta T-31C is associated with a longer overall survival in patients with multiple myeloma undergoing auto-SCT[END_REF]. The deletion produces lower transcripts levels in luciferase reporter systems, and thus results in lower levels of NF-κB p50 [START_REF] Karban | Functional annotation of a novel NFKB1 promoter polymorphism that increases risk for ulcerative colitis[END_REF]. In myeloma patients treated with HDT, the effect on TTF for the polymorphism RAI-intron1-1 was independent of the polymorphism NFKB1 -94ins/del. Assuming that RAI is indeed an inhibitor of NF-kB p65, this would indicate that NF-B p65 levels influence TTF, and that this effect is independent from differences in NF-B p50 levels. For OS, the effect of RAI-intron1-1 was in favour of patients also carrying the wild type ins-allele of NFKB1 -94ins/del, and these findings support that the classical NF-κB pathway (p65/p50 dimer) is involved in survival of myeloma patients after HDT.

In this population of patients a subgroup of 146 patients were treated with INF-α as maintenance treatment after HDT.

The decision of starting INF-α treatment was solely based on different treatment guidelines on starting post-HDT maintenance treatment at the participating departments and therefore not connected with selection bias. Interferon and NF-кB are central for the regulation of apoptosis, cell growth and for modulation of the immune system [22;23]. From previous results, we know that the polymorphism NFKB1 -94ins/del influences outcome for patients treated with INF-α as maintenance treatment [START_REF] Vangsted | A polymorphism in NFKB1 is associated with improved effect of interferon-{alpha} maintenance treatment of patients with multiple myeloma after high-dose treatment with stem cell support[END_REF]. Among patients treated with INF-α, carriers of the variant del-allele carriers of NFKB1 -94ins/del and the wild type T-allele of RAI-intron1-1 had a significant shorter TTF and OS survival as compared to double homozygous wild type carriers. The effect of INF-α may therefore depend on a normal transcription level of the NF-κB p50 subunit, and the availability of the p65 subunit of NF-κB for transcription activity. In the combination analyses CD3EAP G-21A AG+GG was similar to RAI-intron1-1 TC+CC, probably due to their strong linkage (supplementary figure 1). In general, RAI-intron1-1 had the stronger effect, perhaps indicating that RAI-intron1-1 is the biologically relevant polymorphism. In carriers of the variant A-allele of CD3EAP -G-21A, a significantly prolonged OS was seen for patients treated with bortezomib. If true, this result could be interesting because Hideshima et al recently found that bortezomib treatment increases NF-κB dependent transcription in myeloma cells in vitro via activation of two upstream NF-κB-activating kinases RIP2 and IККβ [START_REF] Hideshima | Bortezomib induces canonical nuclear factor-kappaB activation in multiple myeloma cells[END_REF]. However, in our cohort of patients only few patients were treated with bortezomib, and consequently the statistical power to detect interaction with bortezomib was limited. No effect on TNT was seen, and combination analysis of RAI-intron1-1 did not reveal the same statistically significant results (table 2). We conclude that the polymorphisms RAI-intron1-1 and CD3EAP-exon3-6 are associated with outcome of myeloma patients treated with HDT. The effects of the polymorphisms on gene function are unknown, as well as the function of the genes RAI and in particular CD3EAP. The findings should be sought replicated in an independent study group of similarly treated multiple myeloma patients. Combination analyses with the functional polymorphism in NFKB1 suggest that a possibly functional effect of RAI or CD3EAP could be related to NF-B availability. This hypothesis should be tested at the molecular level.

Legends to figures

Figure1.

  Figure1.Schematic presentation of the sub-region 19q13.3 investigated in the current study.

Figure2.

  Figure2.Association of single polymorphisms with TTF (A) and OS (B) in multiple myeloma patients treated with high-dose treatment and stem cell support. The p value (logarithmic scale) for association of homozygous wild type carriers compared to all variant carriers of each marker with TTF and OS, are depicted against the position of the marker.

Figure 3 .

 3 Figure 3. Kaplan-Meier plots of TTF and OS. Combination analysis of CD3EAP G-21A or RAI-intron1-1 with NFKB1 -94ins/del. The numbers at risk at 24, 48, 72, 96 and 120 months are presented below the figure. Wild type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with wild type carriers of NFKB1 -94ins/del (solid blue line), variant type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with wild type carriers of NFKB1 -94ins/del (dashed blue line), wild type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with variant type carriers of NFKB1 -94ins/del (solid red line), variant type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with variant type carriers of NFKB1 -94ins/del (dashed red line).

Figure 4 .

 4 Figure 4. Kaplan-Meier plots of A: TTF and B: OS. Patients treated with interferon-α in relation to genotypes of CD3EAP G-21A or RAI-intron1-1 in combination with genotypes ofNFKB1 -94ins/del. The numbers of patients at risk at 0, 24, 48, 72, 96 and 120 months are presented below the figure. Wild type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with wild type ins-allele carriers of NFKB1 -94ins/del (solid blue line), variant type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with wild type ins-allele carriers of NFKB1 -94ins/del (dashed blue line), wild type carriers of either CD3EAP G-21A or RAI-intron1-1 in combination with variant-type del-allele carriers

Figure 2 .

 2 Figure 2.

Table 1 .

 1 Patient characteristics at the time of diagnosis of 348 patients.

		No of patients Median	Range
	Sex
	Male	204 (59%)
	Female	144 (41%)
	Age		56	(28-69)
	<60	246 (71%)
	>= 60	101 (29%)
	β2-microglobulin (mg/dL)	249	3.9	(1.2 -57)
	Creatinine (µmol/L)	332	98	(47 -833)
	Albumin (g/dL)	297	3.5	(0.25 -5.3)
	ISS
	I	56 (23%)
	II	94 (39%)
	III	89 (37%)
	Durie-Salmon stage
	I	35 (10%)
	II	77 (23%)
	III	228 (67%)
	TTF (months)		27.7	(23.4-30.8)
	OS (months)		69.8	(60.5-81.6)
	Treated with thalidomide at	177
	relapse
	Treatment with bortezomib at	74
	relapse

Table 2

 2 Univariate analysis of the effect of genotypes on treatment outcome of INF-α, thalidomide and bortezomib.

	+ Bortezomib		AG + AA	22	13.3	0.6 (0.3-1.1)	0.06
	NFКB1-94 INS/DEL combined with CD3EAP G-21A
	+ IFN-α	ins	GG	27	37.6	1
	+ IFN-α	ins	AG + AA	22	56.4	0.6 (0.3-1.3)
	+ IFN-α	Ins/del + del	GG	64	26.0	1.2 (0.7-2.0)
	+ IFN-α	Ins/del + del	AG + AA	27	56.3	0.7 (0.4-1.4)	0.1
	NFКB1-94 INS/DEL combined with CE3EAP G-21A
		ins	GG	33	17.7	1
		ins	AG + AA	23	22.3	0.7 (0.4-1.2)
		Ins/del + del	GG	86	26.2	0.6 (0.4-0.9)
		Ins/del + del	AG + AA	27	28.7	0.6 (0.3-1.0)	0.1
	NFКB1-94 INS/DEL combined with RAI-intron1-1
	+ IFN-α	ins	TT	23	39.8	1
	+ IFN-α	ins	TC + CC	19	112.4	0.6 (0.3-1.3)
	+ IFN-α	Ins/del + del	TT	56	27.8	1.4 (0.8-2.4)
	+ IFN-α	Ins/del + del	TC + CC	26	52.5	0.8 (0.4-1.5)	0.04
	NFКB1-94 INS/DEL combined with RAI-intron1-1
		ins	TT	34	15.6	1
		ins	TC + CC	18	32.2	0.6 (0.3-1.1)
		Ins/del + del	TT	79	24.3	0.6 (0.4-1.0)
		Ins/del + del	TC + CC	24	28.7	0.6 (0.3-1.0)	0.12

Abbreviations: CI= confidence interval; HR= hazard ratio; TTF= time-to-treatment failure; TNT: time-to-next treatment; OS= overall survival; mo = months; ∞ : indicates that median survival was not reached

Acknowledgement:

We thank Bettina Hansen and Lars Bentzen for expert technical assistance. Participants from the Nordic Myeloma Study Group (NMSG) are thanked. This work was supported by The Research Fund at Region Sjaelland.

Analysis of the effect of genotypes on TTF and OS in patients treated with INF-α, thalidomide and bortezomib.

TTF and OS were compared in groups subdivided by genotype and INF-α treatment. Among patients treated with INFα, carriers of the variant C-allele or RAI-intron1-1 had a significant prolonged TTF and OS (56.3 months and not reached, respectively) as compared to homozygous T-allele carriers (30.5 months and 74.4 months, respectively). However, when tested in a multivariate Cox analysis, the analysis showed that the difference in TTF was not related to treatment with INF-α and only a trend was found for OS (p=0.06). No association between treatment outcome of thalidomide and bortezomib with genotype was found, except for carriers of the variant A-allele of CD3EAP -G-21A, where a significantly prolonged TNT and OS was seen. We explored combination analyses of NFKB1 -94ins/del, CD3EAP G-21A and RAI-intron1-1 in relation to treatment with INF-α, and the results are shown in Table 2. Patients treated with INF-α, who carried two copies of the T-allele of RAI-intron1-1 and the variant del-allele of NFKB1 -94ins/del had a significantly shorter TTF, and patients who carried the variant del-allele of NFKB1 -94ins/del and either of the wild type alleles of CD3EAP G-21A or RAI-intron1-1 had a significant shorter OS survival as compared to carriers of the wild type ins-allele of NFKB1 -94ins/del in combination with the variant alleles of either CD3EAP G-21A or RAI-intron1-1 (figure 4).

Discussion

We have explored a sub-region in 19q13.3 by fine mapping associations between many single nucleotide polymorphisms (SNPs) and treatment outcome in 348 myeloma patients treated with HDT. We found that several polymorphisms in a region spanning the genes RAI-intron1-1 and CD3EAP-exon3-6 were significantly associated with TTF and OS in myeloma patients. The effects of the polymorphisms RAI-intron1-1 and CD3EAP G-21A on gene functions are not known. Both SNPs are located in the 5'region of their respective genes, the first intron and the 5'UTR, respectively, but none of them are located in the reading frame, and one may speculate that they affect transcription efficiency or gene regulation. In the studied population of patients, the effects of polymorphisms in RAI and CD3EAP

were similar, and we found a pair wise linkage disequilibrium between RAI-intron1-1 (rs4572514) and CD3EAP G-21A (rs967591) of D´= 0.94, suggesting that they detect the same biological effect.

The gene RAI was initially identified as an inhibitor of the NF-κB subunit p65. The pro-inflammatory NF-B consists of the dimer p50/p65. The NFКB1 -94ins/del ATTG polymorphism is an insertion/deletion of four bases in the promoter region of the NFКB1 gene encoding both of the NF-κB transcription factors, p50 and p105. We therefore combined the polymorphism NFKB1 -94ins/del with polymorphism in RAI-intron1-1 in relation to outcome of


MD Elisa Tinazzi

MD Ernesto Amelio

MD Elettra Marangoni

MD Claudio Guerra

MD Antonio Puccetti

MD Orazio Michele Codella

MD Sara Simeoni

PhD Elisabetta Cavalieri

MD Martina Montagnana

MD Roberto Adani

MD Roberto Corrocher

Professor Claudio Lunardi
email: claudio.lunardi@univr.it

Effects of shock wave therapy in the skin of patients with progressive systemic sclerosis: a pilot study

Keywords: ESWT: extracorporeal shock wave therapy, SSc: progressive systemic sclerosis, skin fibrosis, endothelial cell damage

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

Introduction

Progressive systemic sclerosis (SSc) is an autoimmune disease characterized by excessive deposition of collagen in the skin and visceral organs. Pathogenesis of the disease is complex: the pivotal steps are endothelial cell damage, immune activation and collagen production by fibroblasts [START_REF] Abraham | Scleroderma: from cell and molecular mechanisms to disease models[END_REF]. Endothelial involvement is associated with the increase of some circulating markers including von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), intracellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-1) [START_REF] Cerinic | Blood coagulation, fibrinolysis and markers of endothelial dysfunction in systemic sclerosis[END_REF][START_REF] Carvalho | Vascular endothelial growth factor (VEGF) in autoimmune disease[END_REF].

The treatment of SSc is still disappointing since it is not able to modify the course of the disease. Conventional therapies are directed to improve peripheral blood circulation, to prevent the synthesis and release of harmful cytochines and possibly to inhibit or reduce fibrosis [START_REF] Denton | Targeted therapy comes of age in scleroderma[END_REF].

Extracorporeal shock waves (ESW) are defined as a sequence of sonic pulses characterized by high peak pressure (up to 100 MPa), fast pressure rise (10-100 ns) and short lifecycle. First applied in 1980 for the treatment of kidney stones, during the last 10 years this tecnique was found to induce an immediate anthalgic and antiinflammatory effect and a long-term tissue regeneration together with increase of angiogenesis [START_REF] Meier | Shock wave therapy: an innovative treatment method for partial thickness burns[END_REF][START_REF] Mariotto | Extracorporeal shock waves: from lithotripsy to anti-inflammatory action by NO production[END_REF][START_REF] Ciampa | Nitric oxide mediates anti-inflammatory action of extracorporeal shock waves[END_REF][START_REF] Aicher | Low-energy shock wave for enhancing recruitment of endothelial progenitor cells. A new modality to increase efficacy of cell therapy in chronic hind limb ischemia[END_REF][START_REF] Schaden | Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study[END_REF].

ESW have found widespread use in orthopaedics. ESW have a positive influence on both calcifying tendonitis of the shoulder and fracture healing [START_REF] Rompe | Extracorporeal shock wave therapy for calcifying tendonitis of the shoulder[END_REF][START_REF] Haupt | Influence of shock waves on fracture healing[END_REF]. Moreover, low-energy shock waves therapy is used for persistent tennis elbow syndrome and painful heel with significant positive clinical results [START_REF] Rompe | Low energy extracorporeal shock wave therapy for persistent tennis elbow[END_REF][START_REF] Rompe | Low-energy extracorporeal shock wave therapy for painful heel: a prospective controlled single-blind study[END_REF].

Recently Nishida et al performed extracorporeal shock wave application on the ischemic myocardial region (200 shots/spot for 9 spots at 0,09 mJ/mm 2 ) in a porcine model of chronic myocardial ischemia [START_REF] Nishida | Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo[END_REF]. The cardiac shock wave therapy intervention improved global and regional myocardial functions in the treated animals as well as regional perfusion measured as myocardial blood flow of the chronic ischemic region without any adverse effects. No rise in CK, CkmB or Troponin was observed in that study. Vascular density increased in the shock wave treated area and VEGF production was enhanced in the ischemic myocardium in vivo [START_REF] Nishida | Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo[END_REF].

The aim of this work was to investigate whether ESWT applied to limited skin area might reduce fibrosis and increase vascularization in patients affected by systemic sclerosis.

Matherials and methods

Patients selection

From January to April 2008 we enrolled 30 patients affected by systemic sclerosis, 29 female and 1 male, aged 27-76 years old (mean age 55.9 yrs), 19 with limited cutaneous disease, 10 with diffuse cutaneous disease and 1 with overlap syndrome polymiositis/systemic sclerosis; disease duration varied from 11 months to 23 years (mean duration 6,7 yrs).

All patients were treated with calcium channel blockers and antiplatelet aggregation drugs (acetyl-salicylic acid) and monthly infusion of prostanoids for 8 hours.

Written informed consent was obtained from all patients before entering the study.

Treatment regimen

An electromagnetic lithotriptor (DUOLITH SD1 device; Storz Medical AG, Switzerland) was used for ESWT. The electromagnetic generator of the device consists of a cylindrical wire wound coil, a metallic membrane and a concentric paraboloid reflector. Swithching an electrical pulse in the kilo amperes range to a cylindrical coil which is surrounded by the metallic membrane, strong eddy currents are induced and the membrane is elongated, emitting a cylindrical acoustic wave in water. This wave is not yet a shock wave. It is focused by the reflector and it is steepening on its way to the focus.

Treatment protocol consisted of three sittings. The pressure pulses were focused on the volar and dorsal side of the forearm and on the hand and the fingers of one upper arm, following this protocol: 2000 shots along the dorsal forearm, 2000 shots along the volar forearm, 1000 shots along the dorsal side of the hand and fingers, 1000 shots along the volar side of the hand and fingers. The other upper arm and hand were used as controls. Defocused energy applied was 0.20/0.25 mJ/mm 2 with a repetition frequency of 4Hz. Treatment did not require any kind of anaesthetic.

Twenty-eight patients completed the entire treatment, while two patients received only two sittings.

Clinical evaluation

Skin involvement was determined by the modified Rodnan skin score (mRSS) [with palpation of 17 anatomical sites and scoring on a 0-3 scale, where 0= normal skin, 1= slight thickening, 2= moderate thickening, 3= hidebound skin sclerosis].

A visual-analogic scale (VAS) was used to asses skin wellness; it assessed skin elasticity and softness and oedema, sensitivity and pain of the hand scoring on a 0-100 scale (0= the best possible condition, 100= the worst possible condition) RSS and VAS were performed before, immediately after the first ESWT and then at 7, 30, 60, 90 days after the end of the treatment.

Ultrasonographic evaluation

Skin thickness and vascularity were measured with a high frequency ultrasound scanner (LOGIQ Book XP ultrasound machine; GE Healthcare, UK) using a 12 MHz transducer (I12L) and an ultrasound pad to increase ultrasonographic signal. Scans were obtained form volar and dorsal side of upper harm 10 cm distal from elbow; total skin thickness (expressed in centimetres) was calculated as mean of these two measures. Skin vascularity was obtained by colorDoppler analysis and represented on an arbitrary scale ranging from 1 (scarse) to 3 (elevate); total vascular score was the mean of values obtained for dorsal and volar side of upper harm.

Sonographic examination was performed before ESWT and at 7, 30, 60 and 90 days after the end of the treatment.

Biochemicals markers and circulating endothelial cells

Blood samples were obtained before and 30, 60 days after the end of ESWT. Among the markers of endothelial cell disfunction we have tested vWF, VEGF, ICAM-1 and MCP-1 which is also a marker of fibrosis. vWF-Ag was measured by an automated quantitative enzyme linked immunosorbent assay (ELISA) on the mini Vidas (BioMeriuex, Marcy L'Etoile, France). Determination of VEGF (R&D System Quantikine ®; Minneapolis, MN, USA), MCP-1 and ICAM-1 (Endogen Thermo Scientific®; Rockford, Illinois, USA) were carried out using commercially available kits following the manufacturer's instructions.

The number of endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) was determined at the same time points. CECs and EPCs were detected by flow-cytometry by lyse-no-wash method. Two hundred μL of each sample were incubated with a panel of monoclonal antibodies for 20 minutes at room temperature.

Fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (10 μl), R-Phycoerythrin (PE)-conjugated anti-CD34,-CD31 and -CD146 (10 μl) or isotype-matched control (IgG 1 ), allophyco-cyanine (APC) anti-CD3, -CD16, -CD19 and -CD33 (5 μl) were used. 7-amino-actinomycin (7-AAD) was added for dead cells exclusion. Samples were also stained with anti-CD45 FITC, anti-CD34, -CD31 and -CD146 PE, anti-CD106 or anti-VEGFR2 APC and peridin chlorophill protein (PerCP)-conjugated anti-CD3, -CD16, -CD19 and -CD33. All reagents were purchased from Becton Dickinson (San Jose, CA, USA), except for anti-CD16 (Caltag, Burlingame, CA, USA), anti-CD106 (Biolegend, San Diego, CA, USA) and anti-VEGFR2-APC (R&D System, Minneapolis, MN, USA). After labeling, red blood cells were lysed by incubation with 2 ml of Ammonium Chloride lysis solution and then the sample was analysed on a FACS Calibur cytometer (Becton Dickinson). The sensitivity of fluorence detectors was set and monitored using Calibrite Beads (Becton Dickinson) according to the manufacturer's recomendations; 500.000 cells per sample were acquired in live gating. Data were analyzed with CellQuest software (Becton Dickinson). Appropriate analysis gates, designed to remove dead cells, platelet aggregates and debris, and to exclude CD45+ and CD3+/CD16+/CD19+/CD33+ hematopoietic cells (dump channel), were used to enumerate total CECs and EPCs .

Nitric Oxide evaluation

In 3/30 patients levels of nitric oxide were measured before and during ESWT at time 2 and 4 minutes after beginning of treatment. Enzymatic Griess assay on deproteinated serum was used to determine nitric oxide values (kit Cayman Chemical; Ann Arbor, Michigan, USA) [START_REF] Romitelli | Comparison of nitrite/nitrate concentration in human plasma and serum samples measured by enzymatic batch Griess assay, ion-pairing HPLC and ion-trap GC-MS: the importance of correct removal of proteins in Griess assay[END_REF].

Statystical analysis

Calculations were performed with the SPSS16 statistical package. For statistical analysis Student's t test was used and a p<0,05 was considered statistically significant.

Results

Clinical evaluation

Rodnan Skin Score showed a statistically significant reduction (p<0,001) immediately after the first sitting of ESWT and 7 and 30 days after treatment compared to the basal value, while VAS showed a statistically significant decrease at all time points (p= 0,03 90 days after the end of treatment), as shown in table 1.

Ultrasonographic evaluation

No significant changes were observed in both skin thickness and vascularity at time 7, 30, 60 and 90 days after ESW therapy compared to the basal scores. However we observed a more regular skin structure, as shown in figure 1. Moreover, the vascular score 90 days after treatment was increased compared to the basal score nearly reaching a statistical significant difference (p=0.06). Table 2 shows the results of ultrasonographic examination.

Biochemical markers and circulating endothelial cells

The baseline evaluation of biochemical parameters gave the following results: vWF 103.3 ± 54.9 mg/L, VEGF 710.4 ± 592.1 pg/mL, ICAM-1 291±139.1 ng/mL, MCP-1 323.7 ± 213 pg/mL. No significant changes were observed at time 30 and 60 days after the end of treatment (table 3).

Both EPCs and CECs showed a significant increase 30 and 60 days after the end of treatment compared to the basal (p< 0,05) as shown in table 4.

Nitric Oxide evaluation

In 2 out of the 3 patients examined nitric oxide dosage showed an increase after 2 minutes from the beginning of ESW therapy (data not shown).

Discussion

The present study demonstrates that, in patients affected by SSc, skin application of extracorporeal shock waves causes a rapid and persistent improvement of clinical parameters (RSS and VAS for skin wellness) and a late increase in skin vascularization and in number of EPCs and CECs. Shock waves were used since 1980's to treat kidney stones. During the last 10 years this tecnique was found to induce an immediate anthalgic and anti-inflammatory effect and a long-term tissue regeneration together with increase of angiogenesis [START_REF] Meier | Shock wave therapy: an innovative treatment method for partial thickness burns[END_REF][START_REF] Mariotto | Extracorporeal shock waves: from lithotripsy to anti-inflammatory action by NO production[END_REF][START_REF] Ciampa | Nitric oxide mediates anti-inflammatory action of extracorporeal shock waves[END_REF][START_REF] Aicher | Low-energy shock wave for enhancing recruitment of endothelial progenitor cells. A new modality to increase efficacy of cell therapy in chronic hind limb ischemia[END_REF][START_REF] Schaden | Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study[END_REF][START_REF] Wang | Ras induction of superoxide activates ERK-dependent angiogenic transcriptional factor HIF-1 and VEGF-A expression in shock wave-stimulated osteoblasts[END_REF]. One of the possible mechanisms of action of the anti-inflammatory effect of ESWT may be related to the ability of ESW to keep local NO contents at a physiological level in the early phase of inflammatory response, enhancing either a non-enzymatic or enzymatic production of NO [START_REF] Romitelli | Comparison of nitrite/nitrate concentration in human plasma and serum samples measured by enzymatic batch Griess assay, ion-pairing HPLC and ion-trap GC-MS: the importance of correct removal of proteins in Griess assay[END_REF]. Therefore induction of NO synthesis has been suggested to be one of the most important mechanism implicated in the anti-inflammatory effect of ESWT, while increased expression of VEGF and the consequent mobilization of endothelial progenitor cells can explain the proangiogenic action [START_REF] Mariotto | Extracorporeal shock waves: from lithotripsy to anti-inflammatory action by NO production[END_REF][START_REF] Ciampa | Nitric oxide mediates anti-inflammatory action of extracorporeal shock waves[END_REF][START_REF] Aicher | Low-energy shock wave for enhancing recruitment of endothelial progenitor cells. A new modality to increase efficacy of cell therapy in chronic hind limb ischemia[END_REF][START_REF] Mariotto | Extracorporeal shock wave therapy in inflammatory diseases: molecular mechanism that triggers anti-inflammatory action[END_REF]. It has been recently demonstrated that, in addition to angiogenesis due to migration and proliferation of endothelial cells in situ, EPCs contribute to neovascularization in ischemic tissue through a vasculogenetic mechanism and through secretion of a variety of angiogenic factors [START_REF] Urbich | Soluble factors released by endothelial progenitors cells promote migration of endothelial cells and cardiac resident progenitor cells[END_REF][START_REF] Yan | Improvement of blood flow, expression of nitric oxide, and vascular endothelial growth factor by lowenergy shock wave therapy in random-pattern skin flap model[END_REF]. Finally, recent studies have marked out that ESW are able to recruit stem cells and to stimulate their differentiation in various damaged tissues inducing reparative phenomena [START_REF] Chen | Recruitment of mesenchimal cells and expression of TGF-1 and VEGF in the early stage of shock wave[END_REF][21][22].

On these scientific bases, we studied the effect of low-energy ESWT on skin and serological biomarkers in patients affected by SSc.

Our results showed both a short-term and a long-term effect of ESW: we observed a rapid improvement of skin elasticity as measured by RSS and VAS, with a persistent effect during the time (until 30 days after the treatment for RSS and until 90 days for VAS).

Ultrasonographic evaluation of the skin showed no significant difference of skin thickness before and after ESWT, and after ESWT compared to the untreated controlateral arm. However we observed a regularization of skin structure with more defined skin layers; the lack of statistical significance is probably due to the limited number of patients analyzed. Moreover the modification of the skin structure may be responsible for the improvement of RSS and VAS. Finally we are evaluating whether a modification in skin thickness is detectable by ultrasonography in a longer period of observation.

The increasing of vascular score 90 days after the end of treatment is concordant with the hypothesis of ESW-induced neoangiogenesis. However, our study did not demonstrate an increase in serological levels of VEGF or a decrease of other biomarkers (vWF, MCP-1, ICAM-1) indicative of endothelial cell damage; this is possibly related to the application of ESW to a very limited skin area. On the contrary the pro-angiogenetic and reparative effect of ESW is demonstrated by the persistent increase of CECs and EPCs, which remains elevated until 60 days from the end of treatment.

Nitric oxide measurement was limited to only three patients because of difficulties in maintaining a venous access during ESW sitting. The increased values found in 2/3 patients is in accordance with literature [START_REF] Mariotto | Extracorporeal shock waves: from lithotripsy to anti-inflammatory action by NO production[END_REF][START_REF] Ciampa | Nitric oxide mediates anti-inflammatory action of extracorporeal shock waves[END_REF][START_REF] Mariotto | Extracorporeal shock wave therapy in inflammatory diseases: molecular mechanism that triggers anti-inflammatory action[END_REF], but further studies are needed.

For all paramethers considered, we found no significant difference both between patients with limited and diffuse skin involvement or between patients with or without digital ulcerations, although the absence of differences may depend on the limited number of patients enrolled in each subgroup.

In conclusion, the results of our study suggest that ESWT is a novel and efficacious treatment that can be added to the pharmacological therapy in order to decrease endothelial cell damage and skin fibrosis in patients affected by SSc. This treatment is well tolerated and can be repeated without side effects; in the majority of cases it determines a rapid improvement of skin elasticity and skin wellness, even if the effects tend to reduce during the time.

We are now planning to evaluate the time of retreatment and the effects of ESWT applied to more extensive skin areas, such as face and neck, to ameliorate both functional and aesthetic aspects. Moreover we are performing skin biopsy with immunohistochemical analysis prior to and after ESWT. ESWT in one of the treated patients: similar findings were found in the other patients.

A more regular skin structure is clearly present in panel B.

  promoted bone regeneration of segmental defects in rats. J Orthop Res 22: 526-534. 21. Wang SF, Yang KD, Chen RF et al (2002) Extracorporael shock-wave promotes growth and differentiation of bone marrow stromal cells towards osteoprogenitors associated with induction of TGF-1. J Bone Joint Surg Br 84: 457-461. 22. Nurzynska D, Di Meglio F, Castaldo C et al (2008) Shock waves activate in vitro cultured progenitors and precursors of cardiac cell lineages from the human heart. Ultrasound Med Biol 34: 334-342.

Figure 1 .

 1 Figure 1. Ultrasonographic examination of foreharm skin before (A) and after (B)

Table 1 .

 1 Rodnan Skin Score (RSS) and Visual-analogic Scale (VAS) before and after

	ESW therapy.
	Measure	RSS	VAS (0-100)
	Pre-ESWT	28.62 ± 7.56	47.2 ± 14.9
	After Ist sitting	27.14 ± 8.24 *	27.2 ± 13.3 *
	After 7 days	27.98 ± 6.29 *	31.4 ± 16.8 *
	After 30 days	28.09 ± 6.30 *	31.4 ± 17.3 *
	After 60 days	28.21 ± 6.14	40.7 ± 18.1 *
	After 90 days	27.96 ± 7.25	42.8 ± 16.2 *
	* p <0,05

Table 2 .

 2 Skin thickness and vascular score measured by ultrasound.

	Measure	Skin thickness (cm)	Vascular score
	Pre-ESWT and	2.7 ± 0.3	1.78 ± 0.65
	controlateral untreated
	arm (any time point)
	After 7 days	2.6 ± 0.3	1.76 ± 0.51
	After 30 days	2.6 ± 0.2	1.62 ± 0.58
	After 60 days	2.5 ± 0.3	1.55 ± 0.47
	After 90 days	2.6 ± 0.3	1.42 ± 0.37*
	*p =0,06

Table 3 .

 3 Biochemical markers measured before and after ESWT.

	Measure	VWF	sVEGF	sICAM-1	sMCP-1
		(mg/L)	(pg/mL)	(ng/mL)	(pg/mL)
	Pre-ESWT	103.3 ± 54.9 710.4 ± 592.1	291 ± 139.1	323.7 ± 213
	After 30 days 121.6 ± 111 698.6 ± 382.2 279.4 ± 116.8 408.1 ± 185.4
	After 60 days 98.7 ± 65.3	702 ± 370.5	290.9 ± 129.7 396.7 ± 196.4

Table 4 .

 4 Number of Endothelial Progenitors Cells (EPCs) and Circulating Endothelial Cells (CECs) before and after ESW therapy.

	Measure	CECs/mm 3	EPCs/mm 3
	Pre-ESWT	586 ± 356	121 ± 86
	After 30 days	798 ± 452	168 ± 100
	After 60 days	775 ± 382	186 ± 104
	* p <0,05

Acknowledgment

We thanks Dr. Ernst Marlinghaus for his invaluable technical support.