Patrick Declan

Gabor Mckernan

Eamonn Gaszner

John F Quigley

Ted G Cryan

Dinan

Eamonn Gabor Quigley

PhD. Declan P Mckernan
email: d.mckernan@ucc.ie

Gabor Gaszner

Eamonn M Quigley

John F Cryan

Timothy G Dinan

Altered Peripheral Toll-like Receptor Responses in

Keywords: Irritable Bowel Syndrome Irritable bowel syndrome < Disease-based, Immunology < Topics, Inflammation < Topics, Basic science < Topics

REVIEWERS' COMMENTS TO AUTHOR:

Reviewer:

Comments for Transmission to the Authors

On re-analysis the authors found that LPS stimulation (TLR4) induced cytokine released significantly more IL1beta, IL6 and TNFalpha in female IBS compared to male IBS patients. This sentence has been included in the results section. However, no discussion of this finding is made. Was this also observed in their mucosal studies made previously and if so how relevant may be to point out that IBS is much more frequent in females.

This finding has now been included in the discussion. This finding was not observed in the mucosal studies as the number of patients was lower and the study was not powered to detect sex differences in either the control or IBS cohorts.

Where cortisol levels significantly different between males and females with IBS. Would distribution between males and females explain differences previously reported by other authors

No sex differences were found for plasma cortisol levels or cytokine levels measured in this study. This has been mentioned in the results section.

Finally I do not understand their Conclusion in their abstract:

-" Taken together, these data demonstrate the presence of low-grade inflammation in the periphery of IBS patients" This is difficult to conclude and no referenceto this has been made in the discussion. Probably these TLRs agonsit mediate cytokine release does not contribute but is the result of an immune dysfunction in IBS to the intestinal flora.

The abstract conclusion has now been rephrased and now reads -"Taken together, these data demonstrate elevated cytokine levels and TLR activity in the periphery of IBS patients indicating some immune dysregulation in IBS patients." The aim of this study was to characterize TLR activity in IBS.

Methods:

Thirty IBS patients and 30 healthy controls (HC) were recruited. Venous blood was collected, and cultured with a panel of TLR agonists for 24 hours. Cell supernatants were analysed using a multiplex ELISA approach to measure IL1β, IL6, IL8 and TNFα. Plasma was analysed for levels of inflammatory cytokines and cortisol.

Results: TLR agonist-induced cytokine (IL1β, IL6, IL8 and TNFα) release was markedly enhanced in stimulated whole blood from IBS (n=30) patients compared with healthy controls (n=30). An exaggerated response to the TLR8 agonist for all cytokines investigated was seen in IBS patients. In addition, enhanced TLR2-induced TNFα release, TLR3-induced IL-8 release, TLR4-induced IL1β and TNFα release, TLR5-induced IL1β and TNFα release and TLR7-induced IL-8 release were also observed in IBS patients. No differences in TLR1, TLR6 or TLR9 activity were detected. In addition, plasma levels of cortisol, IL-6 and IL-8 were significantly increased in IBS patients.

Conclusion:

Taken together, these data demonstrate elevated cytokine levels and TLR activity in the periphery of IBS patients indicating some immune dysregulation in IBS patients. Key Words: Irritable Bowel Syndrome; Toll-like receptors; stress; cytokine; inflammation. Irritable Bowel Syndrome (IBS) is a highly prevalent functional disorder of the gastrointestinal tract characterized by the presence of abdominal pain, an alteration in bowel habit and the absence of reproducible biomarkers [START_REF] Drossman | AGA technical review on irritable bowel syndrome[END_REF][START_REF] Thompson | Functional bowel disorders and functional abdominal pain[END_REF][START_REF] Clarke | Irritable bowel syndrome: towards biomarker identification[END_REF] . Altered visceral perception and gut dysmotility are important contributors to symptom expression [START_REF] Quigley | Current concepts of the irritable bowel syndrome[END_REF][START_REF] Mayer | Evolving pathophysiologic models of functional gastrointestinal disorders[END_REF] . IBS is a disorder of the brain-gut axis with stress being a frequently invoked precipitant of symptoms among sufferers [START_REF] Mayer | Neuroimaging of the brain-gut axis: from basic understanding to treatment of functional GI disorders[END_REF][START_REF] Cervero | Visceral nociceptors: a new world order?[END_REF] . IBS subjects have been shown to demonstrate exaggerated motor and sensory response to a variety of stressors [START_REF] Quigley | Disturbances of motility and visceral hypersensitivity in irritable bowel syndrome: biological markers or epiphenomenon[END_REF][START_REF] Tache | Stress and the gastrointestinal tract III. Stress-related alterations of gut motor function: role of brain corticotropin-releasing factor receptors[END_REF][START_REF] Tache | CRF1 receptor signaling pathways are involved in stress-related alterations of colonic function and viscerosensitivity: implications for irritable bowel syndrome[END_REF] .

Patients also display an over activation of the hypothalamic-pituitary adrenal (HPA) axis [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Chang | Dysregulation of the hypothalamicpituitary-adrenal (HPA) axis in irritable bowel syndrome[END_REF][START_REF] Videlock | Childhood trauma is associated with hypothalamic-pituitary-adrenal axis responsiveness in irritable bowel syndrome[END_REF] .

Immune dysfunction in IBS patients in both mucosal and systemic compartments has been reported [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Liebregts | Immune activation in patients with irritable bowel syndrome[END_REF][START_REF] Kindt | Immune dysfunction in patients with functional gastrointestinal disorders[END_REF][START_REF] Scully | Plasma cytokine profiles in females with IBS and extra-intestinal co-morbidity[END_REF] . We have shown that plasma levels of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) are elevated in IBS patients [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Scully | Plasma cytokine profiles in females with IBS and extra-intestinal co-morbidity[END_REF] . Others have reported that levels of tumour necrosis factor α (TNFα), interleukin-1β (IL-1β) and IL-6 were elevated in diarrhoea predominant IBS patients (IBS-D) [START_REF] Liebregts | Immune activation in patients with irritable bowel syndrome[END_REF] .

Toll-like receptors (TLRs) are activated by various bacterial and viral cell components [START_REF] Takeuchi | Pattern Recognition Receptors and Inflammation[END_REF] . Receptor binding results in transcription of inflammatory cytokines [START_REF] O'neill | The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling[END_REF][START_REF] Liew | Negative regulation of toll-like receptor-mediated immune responses[END_REF][START_REF] Akira | Toll-like receptor signalling[END_REF] . TLRs are found in high numbers on mucosal surfaces (e.g. the colonic epithelium) and on monocytic cells [START_REF] Nishimura | Tissue-specific mRNA expression profiles of human toll-like receptors and related genes[END_REF] . Recently, we have also shown that the mRNA expression of certain TLRs is increased in the colonic mucosa of rat models of visceral hypersensitivity [START_REF] Mckernan | Toll-like receptor mRNA expression is selectively increased in the colonic mucosa of two animal models relevant to irritable bowel syndrome[END_REF] and have demonstrated increased expression of TLR-4 in mucosal biopsies from IBS patients [START_REF] Brint | Differential Expression of Toll-Like Receptors in patients with Irritable Bowel Syndrome[END_REF] . However, the relevance of these mucosal findings to the immune activation observed in the systemic circulation is unclear. In these studies we sought to characterize the activity of various TLRs on peripheral blood cells to explore the possibility that, as in the colonic mucosa, IBS patients may be primed for an inflammatory response via certain TLR ligands. The study protocol (APC020 2009), was approved by the University College Cork Clinical Research Ethics committee of the Cork University Hospital. IBS patients were recruited from gastroenterology and rheumatology clinics at Cork University Hospital, while healthy controls were recruited from the laboratory, institute or hospital staff. Each healthy control and patient donated 20 ml of venous blood between 11:00 and 13:00 hours to avoid diurnal variations. Thirty IBS patients aged between 18-65 years of age who satisfied Rome II criteria for the diagnosis of IBS were studied. No patient was categorised as post-infectious IBS (PI-IBS). Individuals with inflammatory diseases, celiac disease, lactose intolerance, immunodeficiency, individuals who had undergone any abdominal surgery, with the exception of hernia repair and appendectomy, and those with a psychiatric history were excluded. No attempt was made to select IBS patients on the basis of predominant bowel habit. Each potentially eligible patient was evaluated by a full review of clinical history including current health status, vitals, BMI, family history and current medication. Bowel habit was defined as constipation-dependent (IBS-C), diarrhoea-predominant (IBS-D) or alternating (IBS-A) according to Rome II subclassifications. In addition, a questionnaire was given to participants that assessed IBS severity. The IBS severity scoring system (IBS-SS) [START_REF] Francis | The irritable bowel severity scoring system: a simple method of monitoring irritable bowel syndrome and its progress[END_REF] assesses abdominal pain, abdominal distension (bloating), bowel habit (including frequency and stool consistency) and the effect of IBS on quality of life. A cumulative score (maximum is 500) was calculated and symptom severity was assigned as mild (75-175), moderate (175-300) or severe (>300). Current mood was assessed using the patient health questionnaire (PHQ-9) [START_REF] Spitzer | Validation and utility of a selfreport version of PRIME-MD: the PHQ primary care study. Primary Care Evaluation of Mental Disorders. Patient Health Questionnaire[END_REF] and interview with a psychiatrist. with a final concentration of 1 µg/ml except for HKLM (10 8 cells) and Poly I:C 10 µg/ml. Each treatment was carried out in duplicate. After the indicated culture period, supernatant from both untreated and stimulated cells was aspirated and stored at -80ºC.

Plasma Isolation & Whole Blood

Enzyme-linked Immunosorbent Assay (ELISA):

Measurements of IL-1β, IL-6, IL-8 and TNFα in both plasma and following TLR agonist stimulation of whole blood was carried out using an electrochemiluminescence multiplex system with the Mesoscale Discovery (MSD) 4-plex Human Proinflammatory Kit II. Measurements of IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IFN-γ in plasma were carried out in duplicate using the MSD 7-plex Human Th1/Th2 kit. ELISA plates were analysed using the Sector 2400 imager from Mesoscale Discovery (Gaithersburg, MD, USA). This is an ultra-sensitive method which has a detection limit for IL-1β of 0.3pg/ml, IL-6 of 0.3pg/ml, IL-8 of 1.0pg/ml, IL-10 of 1.9pg/ml, IL-12p70 of 1.1pg/ml, IL-13 of 2.8pg/ml, TNFα of 0.3pg/ml, and IFNγ of 0.8pg/ml. Plasma cortisol

Statistical Analysis:

The sample size was determined by a power calculation based on of our previous data and aimed at detecting differences between IBS patients and healthy controls at the 0.05 level. ELISA data was expressed as mean +/-SEM. All statistical analysis was carried out using GraphPad Prism for Windows (Version 4). Plasma cortisol cytokine levels were compared using an unpaired twotailed Student's t-test, differences were considered significant at P < 0.05. Differences between the IBS and control groups for TLR-agonist induced release of cytokines were determined using two-way ANOVA with a Bonferroni post-hoc test. Demographic data for IBS patients and control subjects are presented on Table 1. There were no significant differences between the groups in terms of age, gender distribution or body mass index (BMI). According to Rome II subclassification in terms of predominant bowel habit, this cohort had 11 alternating (IBS-A), 10 constipation-predominant (IBS-C) and 9 diarrhoea-predominant (IBS-D) patients. IBS symptoms were assessed using the IBS-SS, it was found that the average cumulative score for the IBS patients was 258.8 (moderate) compared to 4.83 (out of 500) for the control subjects; as expected, the difference was highly significant (p < 0.0001) (Table 1).

Additionally, 2 of the control group had a family history of IBS compared to 13 of the IBS group (Table 1).

Plasma cytokine and cortisol levels:

Plasma from both healthy controls and IBS patients was analyzed for eleven cytokines and cortisol. Student's t-tests (with correction for multiple comparisons) confirmed that there were significantly elevated levels of IL-6 (p = 0.008), IL-8 (p = 0.028) and cortisol (p = 0.048) in the plasma of IBS patients compared to healthy controls (Figure 1). Levels of other cytokines tested (IL-1β, TNFα, IL-2, IL-4, IL5, IL-10, IL-12, IL-13 and IFNγ) were not found to be significantly different between groups (Table 2). No sex differences were found for plasma cortisol or cytokines measured.

TLR-agonist cytokine release:

Supernatants from whole blood treatments with TLR agonists of both healthy controls and IBS patients were analysed for levels of four different pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNFα. Two-way ANOVA demonstrated a significant effect of both disease state (F 1, 1684 = 38.10, p < 0.001) and TLR agonist treatment (F 8, 1684 = 115.7, p < 0.001) with a significant interaction (F 8, 1684 = 5.98, p < 0.001) between the two factors for IL-1β levels. A significant effect of both disease state (F 1, 1717 = 10.41, p = 0.0013) and treatment (F 8, 1717 = 119.7, p < 0.001) with a significant interaction (F 8, 1717 = 2.142, p = 0.0293) between the two factors was found for IL-6 levels. Two-way ANOVA demonstrated a significant effect of both disease state (F 1, 1725 = 66.93, p < 0.001) and treatment (F 8, 1725 = 31.50, p < 0.001) with a significant interaction (F 8, 1725 = 8.321, p < 0.001) between the two factors for IL-8 levels. A significant effect of both disease state (F 1, 1709 = 51.71, p < 0.001) and treatment (F 8, 1709 = 124.3, p < 0.001) with a significant interaction (F 8, 1709 = 6.515, p < 0.001) between the two factors for TNFα levels also. When age was entered as a co-variate the differences between the groups are still significant.

Post-hoc analysis of individual treatments revealed significant differences between IBS patients and healthy controls. In the untreated blood, levels of IL8 were found to be significantly higher in IBS patients compared to healthy controls (HC) (1287 +/-119.7 vs. 2135 +/-107.4pg/ml; p < 0.001) (Figure 2a). No significant differences were found in cytokine release following treatment with the TLR1/2 agonist Pam3Csk (Figure 2b). TNFα release was found to be significantly elevated in IBS compared to HC blood (1462 +/-127.9 vs. 934.6 +/-94.93 pg/ml; p < 0.001) following treatment with the TLR2 agonist HKLM (Figure 2c). IL-8 release was found to be significantly elevated in IBS compared to HC blood (2582 +/-122.7 vs. 2015 +/-168.3 pg/ml; p < 0.05) following treatment with the TLR3 agonist Poly I:C (Figure 2d). IL-1β was found to be significantly elevated in IBS compared to HC (1715 +/-100.8 vs. 1157 +/-95.4 pg/ml; p < 0.001) following treatment with the TLR4 agonist LPS as well as TNFα levels (2427 +/-175.5 vs. 1723 +/-118.5 pg/ml; p < 0.001) (Figure 2e). IL-1β release was found to be significantly elevated in IBS compared to HC (947. 3a). No significant differences were found following treatment with the TLR6/1 agonist FSL1 (Figure 3b). IL-8 release was found to be significantly elevated in IBS compared to HC blood (3408 +/-112.2 vs. 2128 +/-147.9 pg/ml; p < 0.001) following treatment with the TLR7 agonist Imiquimod (Figure 3c). IL-1β release was found to be significantly elevated following treatment with the TLR8 agonist ssRNA40 (1404 +/-118.5 vs. 913.8 +/-88.15 pg/ml; p < 0.001), IL6 release was found to be significantly elevated (2037 +/-144.3 vs. 1326 +/-137.6 pg/ml; p < 0.01); in addition IL-8 release was found to be significantly elevated (3438 +/-126.9 vs. 2477 +/-146.8 pg/ml; p < 0.001); finally, TNFα release was found to be significantly elevated in IBS compared to HC blood following treatment with the ssRNA40 (1944 +/-161.2 vs. 1076 +/-110.3 pg/ml; p < 0.001) (Figure 3d). No significant changes in cytokine levels were detected following treatment with the TLR9 agonist ODN2006 (Figure 3e). A separate analysis was carried out of male and female participants. There was no significant difference between these groups with the exception of LPS (TLR4) induced cytokine release, where there was significantly more IL1β, IL6 and TNFα released in female IBS compared to male IBS patients (Data not shown). release. IL-8 release was greater following activation with agonists for TLR3, 7 and 8. All of these receptors recognise viral nucleic acids. Finally, increased TNFα release was related to increased TLR2, 4, 5 and 8 activities. Enhanced TLR8-mediated cytokine release was the common factor in relation to each of these cytokines. At present, viral nucleic acids are the only known ligand for TLR8 17 , although a yet undiscovered endogenous or exogenous ligand(s) may also activate this receptor. In addition, there were some differences between male and female patients for LPS stimulation, indicating changes in TLR4 activity may be linked not only to the condition but also sex, and further studies are needed to investigate this matter.

IBS patients have been shown to have a different microbiota to that of healthy controls [START_REF] Kassinen | The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects[END_REF] . The gut microbiota are known to have an influence on a number of physiological parameters including the immune system and may prime immune cells that enter the peripheral circulation [START_REF] Rhee | Principles and clinical implications of the brain-gut-enteric microbiota axis[END_REF] . This may impact on the TLR expression profile of immune cells and result in altered TLR activity. Indeed, our group has recently reported increased expression of TLR4 in mucosal biopsies from IBS patients [START_REF] Brint | Differential Expression of Toll-Like Receptors in patients with Irritable Bowel Syndrome[END_REF] . Secondly, IBS patients appear to be particularly susceptible to stress [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF] and this seems to have effect on gut motility [START_REF] Tache | Stress and the gastrointestinal tract III. Stress-related alterations of gut motor function: role of brain corticotropin-releasing factor receptors[END_REF][START_REF] Tache | CRF1 receptor signaling pathways are involved in stress-related alterations of colonic function and viscerosensitivity: implications for irritable bowel syndrome[END_REF] . It is apparent from both animal models and clinical studies that chronic stress may affect the immune response [START_REF] Glaser | Stress-induced immune dysfunction: implications for health[END_REF] .

Repeated social defeat stress in mice enhanced the bactericidal activity of splenic macrophages, and led to an increase in the expression of TLR2 and TLR4 on these cells [START_REF] Bailey | Repeated social defeat increases the bactericidal activity of splenic macrophages through a Toll-like receptor-dependent pathway[END_REF] . Social defeat can also activate splenic dendritic cells and enhance TLR activity on these cells as assessed by cytokine output in response to ligand stimulation [START_REF] Powell | Repeated social defeat activates dendritic cells and enhances Toll-like receptor dependent cytokine secretion[END_REF] . Furthermore, chronic restraint stress resulted in immune suppression which was mediated by TLR4 [START_REF] Zhang | Toll-like receptor 4 mediates chronic restraint stress-induced immune suppression[END_REF][START_REF] Zhang | Chronic restraint stress promotes immune suppression through toll-like receptor 4-mediated phosphoinositide 3kinase signaling[END_REF] . Elevations in the expression of TLR mRNA was observed in the colonic mucosa of two different rat models of stress-induced visceral hypersensitivity when compared to normosensitive rats [START_REF] Mckernan | Toll-like receptor mRNA expression is selectively increased in the colonic mucosa of two animal models relevant to irritable bowel syndrome[END_REF] . These rats develop visceral hypersensitivity, changes in immune response and differences in the gut microbial populations [START_REF] O'mahony | Early life stress alters behavior, immunity, and microbiota in rats: implications for irritable bowel syndrome and psychiatric illnesses[END_REF] . Thirdly, cytokines and glucocorticoids may synergise to alter TLR expression [START_REF] Hermoso | Glucocorticoids and tumor necrosis factor alpha cooperatively regulate toll-like receptor 2 gene expression[END_REF][START_REF] Sakai | Glucocorticoids synergize with IL-1beta to induce TLR2 expression via MAP Kinase Phosphatase-1-dependent dual Inhibition of MAPK JNK and p38 in epithelial cells[END_REF] . Plasma cytokines and cortisol have been shown to be elevated in this cohort of patients and this potentially may explain why there are increases in TLR activity in these patients.

A limitation of the current study is that the TLR receptor activity in the periphery may not resemble that of the gut mucosa. This study has provided novel insights into relationships between TLR activation, plasma cytokine levels and clinical phenotype among a well characterised group of IBS patients. We have described for the very first time, distinctive patterns of TLR activity in the periphery. Based on observed relationships between certain phenotypic features and demonstrated associations between stress and TLR expression and activity in animal models, it is plausible to suggest that there maybe a link between stress, TLR activation and cytokine profiles in IBS patients.

Table 1:

Shown is a comparison of demographic data collected on the study's cohort of healthy controls and IBS patients. Data was collected from medical and family history questionnaires while additional information was taken from specific questionnaires regarding IBS severity (IBS-SS).

Data is expressed as mean +/-SEM. Student's t-test was used to determine significant differences (p-values shown).

Table 2:

Shown is a table of cytokine and cortisol levels in the plasma of healthy controls and IBS patients.

Data is expresses as mean +/-SEM. Student's t-test was used to determine significant differences.

  reviewer comments below which we feel are very important.

  Irritable bowel syndrome (IBS) is a stress-related disorder with disturbed brain-gut communication, gastrointestinal homeostasis and, based on recent evidence, low grade inflammation and an altered microbiota. The immune system is a critical regulator of the braingut axis. Toll-like receptors (TLRs) are pattern recognition molecules regulating innate immunity.

  blood (15 ml) was added to an equal volume of Histopaque 1077 (Sigma, St.Louis, MO, USA) in a sterile 50 ml tube and centrifuged at 400xg for 30 mins at room temperature. Plasma on the upper layer was transferred to a separate tube and stored at -80ºC for future use. Collected whole blood (2 ml) was diluted 1:10 in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Dublin, Ireland). Blood was aliquoted into 24-well plates and cultured in 37ºC incubator with 5% CO 2 . Each blood sample was cultured in DMEM cell culture medium supplemented with 10% Fetal Calf Serum (Sigma, Dublin, Ireland) with or without the following Toll-like receptor ligands from Human TLR agonist kit (InvivoGen, San Diego, CA, USA) for 24 hours: TLR1/2 -Palmitoyl-3-cysteine-serine-lysine 4 (Pam3Cys); TLR2 -Heat-killed Listeria Monocytogenes (HKLM); TLR3 -Polyriboinosinic polyribocytidylic acid (Poly I:C); TLR4lipopolysaccharide (LPS); TLR5 -Salmonella typhimurium Flagellin; TLR6/2: FSL-1; TLR7 -Imiquimod; TLR8 -ssRNA40. Agonists were reconstituted in endotoxin-free water (supplied)

  the Cortisol Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI, USA) with a detection limit of 26.99 pg/ml.

  viral nucleic acid. Increased TLR8 activity was associated with IL-6

  Figure Legends:

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

  8 +/-91.27 vs. 570.8 +/-65.95 pg/ml; p < 0.001) as well as TNFα (1354 +/-128.8 vs. 921.7 +/-

	85.61 pg/ml; p < 0.01) following treatment with the TLR5 agonist Flagellin (Figure
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Table 1 :

 1

		Healthy Controls	IBS	p value
	Age	36.24 +/-1.835	40.9 +/-2.034	0.09
	Sex	10 M, 20 F	5 M, 25 F
	Height	1.71 +/-0.017	1.67 +/-0.017	0.0876
	Weight	68.4 +/-2.558	69.74 +/-2.673	0.7192
	BMI	23.19 +/-0. 6084	24.89 +/-0.7459	0.0827
	Family History IBS 2	13
	IBS Subtype	N/A	11A, 10C, 9D
	IBS-SS	4.83 +/-4.83	258.8 +/-13.58	p < 0.0001

Table 2 :

 2

		Control	IBS	p-value
	Cortisol	78220 ± 7590	107000 ± 12340*	0.0488
	Cytokine
	IL-1β	0.7751 ± 0.09349 0.9111 ± 0.1270	0.3922
	IL-6	1.581 ± 0.1462	2.875 ± 0.3350*** 0.0008
	IL-8	7.474 ± 0.3745	9.397 ± 0.7667 *	0.0282
	TNFα	7.487 ± 0.4606	7.500 ± 0.2472	0.9808
	IL-2	0.7668 ± 0.09624 0.9327 ± 0.2535	0.5430
	IL-12	8.227 ± 2.990	13.84 ± 4.923	0.3342
	IFNγ	4.381 ± 0.7624	5.484 ± 0.9830	0.3789
	IL-4	2.114 ± 0.6566	1.120 ± 0.2286	0.1585
	IL-5	3.708 ± 0.7518	5.121 ± 1.387	0.3745
	IL-10	16.62 ± 2.327	20.50 ± 3.762	0.3840
	IL-13	14.32 ± 4.894	9.615 ± 3.226	0.4257

Alimentary Pharmacology & Therapeutic

The Alimentary Pharmabiotic Centre is a Centre for Science and Technology (CSET) funded by Science Foundation Ireland (SFI), through the Irish Government's National Development Plan. The authors and their work were supported by SFI and GlaxoSmithKline through grant 07/CE/B1368.

Discussion:

The exact pathophysiology of IBS is still unknown. It has been suggested to have an immune component [START_REF] Ohman | Pathogenesis of IBS: role of inflammation, immunity and neuroimmune interactions[END_REF] , with low level immune activation reported [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Liebregts | Immune activation in patients with irritable bowel syndrome[END_REF][START_REF] Kindt | Immune dysfunction in patients with functional gastrointestinal disorders[END_REF][START_REF] Ohman | T-cell activation in patients with irritable bowel syndrome[END_REF][START_REF] Ohman | B-cell activation in patients with irritable bowel syndrome (IBS)[END_REF][START_REF] Chadwick | Activation of the mucosal immune system in irritable bowel syndrome[END_REF][START_REF] Scully | Plasma Cytokine Profiles in Females With Irritable Bowel Syndrome and Extra-Intestinal Co-Morbidity[END_REF] . These studies have shown increases in proinflammatory cytokines, [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Liebregts | Immune activation in patients with irritable bowel syndrome[END_REF][START_REF] Kindt | Immune dysfunction in patients with functional gastrointestinal disorders[END_REF][START_REF] Scully | Plasma Cytokine Profiles in Females With Irritable Bowel Syndrome and Extra-Intestinal Co-Morbidity[END_REF] as well as alterations in certain immune cell populations in IBS patients [START_REF] Kindt | Immune dysfunction in patients with functional gastrointestinal disorders[END_REF][START_REF] Ohman | T-cell activation in patients with irritable bowel syndrome[END_REF][START_REF] Ohman | B-cell activation in patients with irritable bowel syndrome (IBS)[END_REF][START_REF] Chadwick | Activation of the mucosal immune system in irritable bowel syndrome[END_REF] . Given the key role of toll-like receptors in the activation of cytokine release, we hypothesized that TLR activity may be altered in IBS patients. IBS patients were shown to have elevated plasma cortisol levels, in agreement with previous reports from this laboratory [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF] . Cortisol is a product of the hypothalamic-pituitary-adrenal (HPA) axis and an important hormonal indicator of stress. The HPA axis has been demonstrated to be dysregulated in IBS patients by a number of groups including our own [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Chang | Dysregulation of the hypothalamicpituitary-adrenal (HPA) axis in irritable bowel syndrome[END_REF][START_REF] Videlock | Childhood trauma is associated with hypothalamic-pituitary-adrenal axis responsiveness in irritable bowel syndrome[END_REF] . Stress is known to disrupt the function of the immune system, including cytokine levels [START_REF] Glaser | Stress-induced immune dysfunction: implications for health[END_REF] . Of cytokines measured, only IL-6 and IL-8 were significantly elevated in IBS patients, in agreement with previous studies [START_REF] Dinan | Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker?[END_REF][START_REF] Scully | Plasma Cytokine Profiles in Females With Irritable Bowel Syndrome and Extra-Intestinal Co-Morbidity[END_REF] .

The effects of elevated cytokine levels on the pathophysiology of IBS remain to be defined. Cytokines play a role in central sensitization. IL-1β, IL-6 and TNFα have been shown to affect transmission in the spinal cord resulting in hyperalgesia [START_REF] Kawasaki | Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord[END_REF] . This finding is relevant to IBS, in which both elevations in cytokines and hypersensitivity to pain have been demonstrated [START_REF] Arebi | Review article: the psychoneuroimmunology of irritable bowel syndrome--an exploration of interactions between psychological, neurological and immunological observations[END_REF] .

Furthermore, cytokines are known to have an effect on the HPA-axis and stress response [START_REF] John | Cytokines: regulation of the hypothalamopituitary-adrenocortical axis[END_REF] . IL-1β, IL-6 and TNFα have all been reported to stimulate secretion of corticotrophin-releasing hormone (CRH) in rat as well as in humans [START_REF] Chrousos | The hypothalamic-pituitary-adrenal axis and immunemediated inflammation[END_REF][START_REF] Dantzer | From inflammation to sickness and depression: when the immune system subjugates the brain[END_REF] . Cytokines can cross the blood brain barrier and stimulate cells at the barrier to release more cytokines within the brain [START_REF] Dantzer | From inflammation to sickness and depression: when the immune system subjugates the brain[END_REF] . However, it is not clear whether the elevations in cytokine levels observed in IBS patients are responsible for the alteration in HPA-axis activity or vice versa.

Whole blood stimulations revealed a distinct pattern of peripheral TLR activity in IBS patients. Accordingly, we demonstrated an association between increased TLR4, 5 and 8 activities and IL1β production; these receptors recognise bacterial lipopolysaccharide (LPS),


Armen Sargsyan

Claude Leroy

Yevgenya Pashayan-Leroy

Rafayel Mirzoyan

Aram Papoyan
email: papoyan@ipr.sci.am

David Sarkisyan

High contrast D 1 line electromagnetically induced transparency in nanometric-thin rubidium vapor cell

Electromagnetically induced transparency (EIT) on atomic D 1 line of rubidium is studied using a nanometricthin cell with atomic vapor column length in the range of L = 400 -800 nm. It is shown that the reduction of the cell thickness by 4 orders as compared with an ordinary cm-size cell still allows to form an EIT resonance for L = λ (λ = 794 nm) with the contrast of up to 40 %. Remarkable distinctions of EIT formation in nanometric-thin and ordinary cells are demonstrated. Despite the Dicke effect of strong spectral narrowing and increase of the absorption for L = λ/2, EIT resonance is observed both in the absorption and the fluorescence spectra for relatively low intensity of the coupling laser. Well resolved splitting of the EIT resonance in moderate magnetic field for L = λ can be used for magnetometry with nanometric spatial resolution. The presented theoretical model well describes the observed results.

Introduction

Continuous interest to coherent population trapping (CPT) and the related electromagnetically induced transparency phenomena is stipulated by a number of important applications in a variety of fields such as laser cooling, information storage, magnetometry, spectroscopy, atomic frequency references etc. [1,[START_REF] Arimondo | Progress in Optics[END_REF][START_REF] Wynands | [END_REF]4,5,6]. EIT resonance can occur in a Λ-system with two long-lived states and one excited state coupled by two laser fields and displays a strong reduction in absorption where a maximum is expected in the absence of the coupling laser field (EIT can occur also in ladder Ξ-and V -systems). For many applications, it is important to reduce the dimensions of a cell containing atomic metal vapor where the EIT resonance is formed, at the same time keeping good resonance parameters. The linewidth of the EIT resonance in a Λ-system for the case of low laser intensity is given by γ EIT = 2Γ 21 + Ω 2 /γ N [START_REF] Wynands | [END_REF], where Γ 21 is the coherence dephasing rate, with Ω being the Rabi frequency and γ N the natural linewidth. For the case when nanometric-thin cells (NTC) are employed [7,8,9,10], the value of Γ 21 is highly affected by atom-wall (cell window) collisions; it takes place, though less pronounced, also for sub-millimeter thin cells [11,12]. It is known that a unique collision with a dielectric surface of an uncoated vapor cell is sufficient to thermalize the ground hyperfine levels, with depolarization probability 0.5 -1 (see [13] and references therein). In order to preserve the atomic coherence in wall collisions and to detect ultra-narrow linewidths with the help of coherent processes like EIT, either coated walls or admixture of a buffer gas were used [11]. As the size of a vapor cell is reduced, the lifetime of the ground-state coherence becomes shorter because of collisions of atoms with cell windows: Γ 21 = (2πt) -1 , where t = L/u (L is the distance between windows and u is the most probable thermal velocity). Also the contrast of EIT resonance depends strongly on Γ 21 . Hence, one could expect that the EIT effect would completely wash out in the case of L < 1 µm (2Γ 21 > 100 MHz). Nevertheless, it was demonstrated that the EIT resonance can be observed in thin cells with thickness as small as ∼ 1 µm [9,10]. The explanation for this non-intuitive behavior is as follows: when the coupling laser is resonant with an atomic transition, only the atoms flying nearly parallel to cell windows and hence not experiencing wall collisions do contribute to the formation of the EIT resonance.

In [14] it was shown that for an ordinary cm-size cell, the excitation of the D 1 line results in greater EIT contrast as compared to that for the D 2 line. One of the goals of the present study was to verify whether this statement holds also for the case of NTC by comparing the measured EIT contrast with that observed earlier for the D 2 line [9,10]. A high contrast is needed, in particular, to detect the splitting of the EIT resonance in an external magnetic field for the thickness as small as L = λ = 794 nm with a better spectral resolution than that presented in [15]. This may allow one to develop an optical magnetometer with nanometric-range spatial resolution. Switching from D 2 line to D 1 line is also expected to be favorable for forming the EIT resonance both in the absorption and the fluorescence spectra when the cell thickness is λ/2 (397 nm).

Experiment

Nanometric-thin cell

The design of a NTC is similar to that of extremely thin cell described earlier [7]. The modification implemented in the present work is as follows. The rectangular 20 x 30 mm, 2.5 mm-thick window wafers polished to < 5 nm surface roughness are fabricated from commercial optical grade sapphire (Al 2 O 3 ), which is chemically resistant to hot vapors of alkali metals. The wafers are cut across the c -axis to minimize the birefringence. In order to exploit variable vapor column thickness in the range of 30 -2000 nm, the cell is vertically wedged by placing a 2 µm-thick platinum spacer strip between the windows at the bottom side prior to gluing. The NTC is filled with a natural rubidium (72.2 % 85 Rb and 27.8 % 87 Rb). The photograph of the NTC cell is presented in Fig. 1. Since the gap between the windows (i.e. the thickness of Rb atomic vapor column) L is of the order of visible light wavelength, an interference pattern is clearly seen visualizing a smooth thickness variation from 30 nm to 2000 nm. A thermocouple TC is attached to the sapphire side arm (SA) at the boundary of metallic Rb to measure the temperature, which determines the vapor pressure. The SA temperature in present experiment was 120 -140 • C, while the windows temperature was kept some 20 • C higher to prevent condensation. This temperature regime corresponds to atomic number density N = 1.6 × 10 13 -4.8 × 10 13 cm -3 . The NTC operated with a special oven with four optical outlets: a pair of in line ports for laser beam transmission and two orthogonal ports to collect the side fluorescence. This geometry allows simultaneous detection of transmission and fluorescence spectra. The oven and NTC assembly was rigidly attached to a translation stage for smooth vertical movement to adjust the needed vapor column thickness without variation of thermal conditions. For more details see [8,16]. The experimental arrangement is sketched in Fig. 2. The beams of two separate single-frequency extended cavity diode lasers (ECDL) with λ ≈ 794 nm and ∼ 1 MHz linewidth are well superimposed and directed onto the NTC at near-normal incidence by the first Glan prism (G 1 ). The coupling and probe beams have an elliptical shape of 3 × 2 mm and have linear and perpendicular polarizations. Faraday isolators (1 ) are used to prevent an extraneous feedback from reflected beams. Nanometric-thin cell (4 ) is placed inside the three pairs of mutually perpendicular Helmholtz coils (2 ) providing the possibility to cancel laboratory magnetic field as well as to apply homogeneous magnetic field. The transmission and fluorescence radiations are recorded by the photodiodes (3 ) followed by operation amplifiers; the signal of the photodiodes is recorded by a two-channel digital storage oscilloscope Tektronix TDS 3032B. The radiation power of the coupling and probe lasers was varied throughout the measurement in the range of 1 -30 mW and 0.01 -3 mW, respectively using neutral density filters F. An improved Dichroic-Atomic-Vapor-Laser-Locking technique realized with the help of a separate NTC with L = λ/2, a permanent ring magnet (7 ), λ/4 plate, Glan prism (G 3 ), and an error signal unit for electronic subtraction of two photodiodes signals is used for the coupling laser frequency stabilization (see [16]). The frequency reference spectrum was formed with the help of an auxiliary NTC (5 ) with L = λ [16]. A second Glan prism (G 2 ) was used for separating the coupling and probe beams, so that only the probe beam transmission could be monitored. In Fig. 3 is shown the transmission spectrum of the probe laser (the upper curve) for the thickness L = λ (λ = 794 nm) when the 20 mW coupling laser radiation is resonant with F = 3 → F ′ = 3 transition, while the 0.04 mW probe laser radiation frequency is scanned across the F = 2 → F ′ = 2, 3 transitions (see Fig. 8 in case of zero coupling laser detuning ∆ = 0). The full width at half maximum (FWHM) of the EIT resonance is 25 MHz, while the contrast defined as the ratio of the EIT amplitude to the height of the shoulders is 34 %. For a higher coupling laser radiation power, 28 mW, the the linewidth broadens to ∼ 35 MHz, while the contrast reaches nearly 40 %. Note that the best contrast for D 2 line obtained under similar experimental conditions was 5 -6 % [9,10]. The grey line shows the transmission spectrum when the coupling laser is off. The side-arm temperature of NTC is 140 • C (Rb atomic vapor density N = 4.8 × 10 13 cm -3 ). As is seen, the velocity selective optical pumping (VSOP) peak demonstrates an increase of the absorption, since the coupling laser transfers a number of atoms from F = 3 to F = 2, thus increasing the probe absorption for F = 2 → F ′ = 2, 3 transitions (also the EIT resonance is superimposed on the VSOP at F = 2 → F ′ = 3 transition). The theoretical spectrum as obtained from the numerical calculations (see Section 3) is shown in Fig. 7 (the second curve from the bottom, case of ∆ = 0) for the same parameters as in Fig. 3 except the probe laser power taken to be 1 mW.

Experimental setup

EIT in nanometric-thin cell: the coupling laser is resonant with atomic transition

Fig. 4 presents the EIT resonance linewidth as a function of the coupling laser power for ∆ = 0. The intersection of the curve with the vertical axis gives the residual coherence dephasing rate of γ ′ 21 = 6 MHz, which is caused by several reasons, including atom-atom, atomwall collisions in NTC, time-of-flight broadening, as well as due to the fact that the lasers are not coherently coupled [START_REF] Wynands | [END_REF].

Earlier, it was demonstrated that there is a dramatic difference for the absorption (and also fluorescence) spectrum when the thickness of NTC is reduced from L = λ to L = λ/2, which is caused by the revival of Dicke-type coherent narrowing effect. Particularly, the linewidth of the sub-Doppler absorption spectrum for L = λ/2 is ∼ 3 times narrower than that for L = λ, while showing the same peak absorption value [18,19,20]. Moreover, as it was recently demonstrated, for L = λ/2 it is impossible to form any type of sub-Doppler dip (VSOP resonance) neither in transmission nor in fluorescence spectra, even for the laser intensity as high as several tens of W/cm 2 , as opposed to L = λ case where only a few mW/cm 2 probe laser intensity is sufficient for a dip formation. For this reason, the L = λ/2 case is of a particular interest for EIT resonance formation.

The striking point is that it is possible to form an EIT resonance (i.e. a peak of reduced absorption) for the thickness L = λ/2 already at a coupling laser intensity as low as ∼ 0.1 W/cm 2 (see Fig. 5(a)). The grey curve shows the 120 MHz-wide sub-Doppler transmission spectrum for L = λ/2 when the coupling laser is off. The EIT for the thickness L = λ/2 was theoretically addressed in [10].

The EIT resonance demonstrating reduction of the fluorescence (dip of reduced fluorescence) for L = λ/2 is shown in Fig. 6(a). The grey curve shows the sub-Doppler fluorescence spectrum of 70 -80 MHz linewidth [8] when the coupling laser is off. The theoretical spectra for the parameters matching exactly the corresponding experimental conditions of Figs. 5(a As it is seen, there is a good agreement between the experiment and the theory. Thus, despite the Dicke effect of strong spectrum narrowing and increase of the absorption for L = λ/2, the increase of coupling laser intensity to ∼ 100 mW/cm 2 makes it possible to form an EIT resonance both in the absorption and the fluorescence spectra.

EIT in nanometric-thin cell: dependence on coupling laser detuning

One of the drastic differences in behavior of EIT realized in NTC and in an ordinary 1 -10 cm long cell is exhibited by the EIT linewidth and amplitude dependence on the coupling laser detuning ∆ (see Fig. 8). The EIT linewidth and amplitude (contrast) as a function of blue detuning ∆ of the coupling laser for L = λ can be evaluated from the experimental and theoretical transmission spectra shown in Fig. 7. The side-arm temperature of NTC is 140 • C, P c ≈ 20 mW. In order to get more prominent EIT resonance for the large values of detuning, the probe laser radiation power was increased to 1 mW. As it is seen, the increase of detuning ∆ from 0 to 40 and 85 MHz results in rapid broadening of EIT resonance linewidth from 25 MHz to 50 and 80 MHz FWHM, correspondingly, meanwhile the EIT amplitude rapidly decreases. This behavior has the following physical explanation. For the case of an exact atomic resonance (∆ = 0), mainly the atoms flying parallel to NTC windows contribute to formation of the EIT resonance. The interaction time for these atoms is locity projection v z = 2π∆/k contribute to the formation of EIT, and for these atoms the flight time between the cell windows shortens with the detuning increase: τ L = L/v z = kL/(2π∆). This causes frequent quenching collisions of atoms with the cell windows leading to the increase of γ ′ 21 and consequently, to strong linewidth broadening and contrast reduction of the EIT resonance. For this case γ EIT can be described by a simple expression good agreement with the experimental values. We should note that measurement of EIT resonance linewidth γ EIT at large coupling laser detuning could serve as a convenient tool to study atom-window collisions and properties of window material. This behavior completely differs from that observable in ordinary 1 -10 cm long cell where formation of EIT resonance on the wings of the Doppler profile of the absorption line can lead to its narrowing [21] as opposed to the case of NTC.

τ D = D/v,
γ EIT (∆) = γ EIT ∆=0 + 2γ ′ 21 (∆), (1)
It is well known that addition of a buffer gas into 1 -10 cm long ordinary cell improves EIT resonance parameters [START_REF] Wynands | [END_REF]4]. The preliminary results have shown that admixture of 20 Torr of N 2 buffer gas to Rb vapor in NTC leads to decrease of EIT broadening rate with the increase of ∆. This is explained by substantial shortening of the mean free path of Rb atoms due to Rb-N 2 collisions (∼ 300 nm), which becomes less than the NTC thickness (L = 794 nm). However, at ∆ = 0 there appears undesirable supplementary broadening: Rb atoms flying parallel to the NTC windows experience velocity changing collisions with N 2 , change their direction and collide with the windows, which leads to the increase of Γ 21 .

Splitting of EIT resonance in magnetic field, L = λ

In [9] it was shown that using NTC with Rb vapor of L = 2λ thickness (λ = 780 nm), one can measure a magnetic field by splitting EIT resonances formed at D 2 line excitation. However, due to a small contrast of the EIT resonance it was impossible to detect the splitting at smaller L values. Switching to D 1 excitation allowed us to improve the EIT contrast and thus to detect the splitting for L = λ = 794 nm as is shown in Fig. 9. The measurement shown in the upper curve was done with application of B = 14 G longitudinal magnetic field (B k), for laser radiation powers P c = 5 mW and P p = 0.05 mW. Three well resolved EIT resonances are clearly seen in the upper curve; the lower curve presents the EIT resonance when B = 0. In [9] it was shown that in this case three Λ-systems are formed, and the frequency separation between these EIT components is 1.4 MHz/G × 14 G = 19.6 MHz. The inset presents the results of fitting by Gaussian function resulting in the linewidth of 10 MHz FWHM for an individual EIT component. Note that the exact splitting value for strong B fields is given by the Rabi-Breit formula [START_REF] Wynands | [END_REF].

So-called "λ-Zeeman technique" (LZT) to investigate atomic transitions in magnetic field was presented in [15]. Rb atoms are confined in a NTC with L = λ. Narrow VSOP resonances of 30 -35 MHz linewidth formed in a single beam in transmission spectrum are split into several components in a magnetic field; their frequency positions and transition probabilities depend on the B field. As one can see in Fig. 9, the splitting of EIT resonance provides a spectral resolution, which is better than that for LZT by a factor of 3 [15]. Moreover, among the inherent advantages of NTC is the possibility to apply very high magnetic field using widely available strong permanent ring magnets (our Helmholtz coils produce B < 200 G). In spite of a strong inhomogeneity of the B field (it can reach ∼ 150 G/mm in axial direction), the variation of B inside the atomic vapor column does not exceed ∼ 0.1 G (several orders less than the applied B value) because of sub-micrometer thickness of the NTC. Our preliminary results show that it is realistic and straightforward to determine a spatiallylocalized value of a B field in the range of 1 -1000 G with nearly the same precision throughout the whole range, by measuring the frequency interval between the EIT components. This is especially valuable for mapping of strongly non-homogeneous magnetic field.

Theoretical model

To calculate the observed EIT resonances we consider four-level Λ-type atomic systems consisting of two ground hyperfine levels (F g = 2, 3) and two excited levels (F e = 2, 3) in the D 1 line of 85 Rb (see Fig. 8) confined in a cell of thickness L. The probe field E p (with center angular 10). For simplicity, we restrict ourselves to one dimensional situation where the two driving linearly polarized laser radiations are in the ±z-direction. The probe and coupling lasers are detuned from the corresponding atomic resonances by

∆ p1,p2 = ω p -ω 3,4;1 -k p v z , ∆ c1,c2 = ω c -ω 3,4;2 -k c v z , (2)
where the subscripts p(c) mark the detunings for the probe (coupling) lasers, ω ij is the frequency difference between levels i and j, and k p,c v z are the Doppler shifts for atoms corresponding to the velocity component v z along the propagation vectors k p,c . The total Hamiltonian of the system is given by Ĥ = Ĥ0 + ĤI , where Ĥ0 describes a free atom and ĤI describes the interaction between the atom and the electric fields. With electricdipole and rotating-wave approximations, the interaction Hamiltonian ĤI of the system is given by

ĤI = -h Ω p1 |3 1| + Ω p2 |4 1| +Ω c1 |3 2| + Ω c2 |4 2| + H.c., (3)
where Ω p1,p2 = µ 1;3,4 E p /2h and Ω c1,c2 = µ 2;3,4 E c /2h are the Rabi frequencies of the probe and coupling fields, respectively, with µ ij being the electric-dipole matrix element associated with the transition from j to i, and H.c. represents Hermitian conjugate. To describe the dynam- 10 Λ-type four-level atomic system. ics of the system driven by this Hamiltonian we adopt here the theoretical model presented in Ref. [10]. Interested readers may refer to this work and to references therein for the mathematical details of the calculations. We summarize here the main features of the approach used. The basic assumptions made in the model are as follows: the atomic number density is assumed to be low enough so that the effect of collisions between the atoms can be ignored; the atoms experience inelastic collisions with the cell walls; the incident beam diameters largely exceed the cell thickness. The internal atomic dynamics is described by a semiclassical density matrix ρ = ρ ij |i j|, the time evolution of which is given by the Liouville equation of motion :

c E p E 2 p ∆ 4 2 1 3 1 c ∆ 2 c ∆ 1 p ∆ Fig.
ρ = -i/h[ Ĥ, ρ] + Γ ρ, ( 4
)
where Γ is the relaxation operator :

Γ ij = 1 2 (γ i + γ j ) + γ ij δ ij .
Here γ i , γ j are the natural decay rates of states |i and |j , and γ ij is the pure dephasing rate.

To obtain the probe transmission spectra, we provide an exact numerical solution of the set of optical Bloch equations deduced from Eq. ( 4) and coupled to Maxwell equations [START_REF] Shore | The Theory of Coherent Atomic Excitation[END_REF]. In accordance with the assumptions made, we neglect the collisional broadening of optical transitions compared to the spontaneous decay rate, the relaxation of atoms traveling out of the diameter of the laser beam. The dephasing rate of the ground-state coherence, Γ 21 , is determined in the model by the finite time of flight of atoms between the cell windows. This parameter, usually introduced into theoretical calculations as a phenomenological constant to fit the experimental data, is taken into account in the model exactly by solving the temporal density-matrix equations with proper boundary conditions for each atom separately.

A number of relevant parameters, both inherent to the atomic system and the experimental setup, are included into the model such as Doppler broadening, phase fluctuations in the laser fields [START_REF] Agarwal | [END_REF]24], non-correlation of the laser fields [START_REF] Wynands | [END_REF]. The light reflections on the locally highly-parallel cell walls of a nano-cell behaving as a Fabry-Pérot cavity [25] is taken into account. For the Rabi frequency the estimates are obtained from Ω/2π = aγ(I/8) 1/2 , where I is the laser intensity in mW/cm 2 , γ is the decay rate of the excited state, and a is a fit parameter (for our case a ≈ 0.2) [27]. A general agreement between the results of our calculations and that of the experiment is seen.

Discussion

Miniaturization of EIT setup without loss of resonance characteristics is among the challenges for technological implementation of the electromagnetically induced transparency. The results obtained in the present study are not intuitive, and it is worth to compare them with the relevant results presented in literature, and analysing the distinctions. In [26] a similar setup was employed for the EIT studies: two separate lasers of 1 MHz linewidth and approximately the same power were used to form the EIT resonance on D 1 line of Rb. The vapor was confined in a 3 cm-long glass cell. The following key parameters have been achieved: ∼ 60 % for the EIT resonance contrast, and ∼ 20 MHz for the spectral width. The corresponding values obtained in the present work with the use of L = 794 nm NTC are ∼ 40 % and ∼ 35 MHz, respectively. Thus, decreasing the cell length by a factor of 3.8 × 10 4 does not lead to a dramatic deterioration of the key parameters of EIT.

Sub-millimeter-thick cells have been used in [11,12] for EIT formation. In recent works [28,29] EIT resonance is formed in Rb vapor generated in hollow-core photonic band-gap (HCPBG) of inner diameter 6 µm and L = 25 cm. Large L together with high laser intensity achieved in HCPBG leads to the decrease of threshold laser power for EIT formation. However, the width of EIT resonance reaches ∼ 100 MHz, which exceeds by an order of magnitude the width of EIT resonance in NTC. Moreover, the realization of HCPBG method is rather complicated technically.

There are yet other papers where suggestions to improve the EIT characteristics in thin cells are addressed. In [30] formation of narrow EIT resonance in nano-cells is proposed by using supplementary anti-relaxation coating for windows, aiming at decrease of coherence dephasing rate. However, the realisation of such a coating is not yet feasible. Formation of regions of few tens of micrometer in quartz substrate-window is realized using chemical etching with subsequent filling these regions with Rb vapor [17], meanwhile the realization of EIT in such a structure has not been yet discussed. Based on the above analysis, we believe the most convenient and technically justified option for obtaining narrow-linewidth and highcontrast EIT resonances is the NTC presented in this work (note that the NTC has been successfully used also for EIT registration in a ladder Ξ-system [31]).

As it follows from the obtained results, the decrease of L is compensated by an increase of Rb atomic density, so that the product N × L practically does not change. An important particularity of EIT is the weak influence of 5P 1/2 level broadening on the EIT parameters (in contrast, 5P 1/2 level broadening dramatically affects the amplitude of the VSOP resonance). The latter can allow one to form EIT resonance for L < 100 nm by increasing the density N and improving the laser parameters (for example, by the technique of phase-locking the coupling and probe lasers [32]), realization of lock-in detection, etc. Such a small thickness of the NTC is of interest for the study of van der Waals interaction of atoms with dielectric windows [33].

Conclusion

The peculiarities of the EIT phenomenon are studied in transmission and fluorescence spectra under D 1 line excitation of rubidium atoms with the help of nanometricthin vapor cell with a possibility to exploit vapor column thickness L in the range of 400 -800 nm. The striking point of the present work is that the reduction of L by more than 4 orders as compared with an ordinary cmsize cell does not worsen the high contrast of EIT peak (up to 40 %). This relatively weak thickness dependence is explained by involvement of atoms with particular velocity components in EIT resonance formation: when the coupling laser is resonant with the atomic transition only the atoms moving parallelly to the NTC windows contribute to the EIT formation, and the quenching collisions with the windows are not essential. The situation is dramatically different when the coupling laser is detuned from the atomic transition. In this case the atoms having velocity component towards the windows are responsible for the EIT, they experience collisions with the windows resulting in broadening and contrast worsening of the resonance. It is justified that with the help of the NTC with L = λ and for a large frequency detuning ∆ of the coupling laser from atomic resonance it is possible to directly reveal and evaluate the strong influence of atomwalls collisions on the EIT linewidth. By changing the value of ∆ one can smoothly control the EIT linewidth and contrast; measurement of the EIT linewidth makes it is possible to determine the atom-wall collision rate. The theoretical model well describes the observed results. Formation of the EIT resonance in a Λ-system for even smaller thickness (below 100 nm) can offer a possibility for quantitative study of atom-surface van der Waals interaction.

For the first time, it is demonstrated that despite the Dicke-effect of strong spectrum narrowing and increase of the absorption when thickness is L = λ/2 (397 nm), the relatively low intensity of the coupling laser is sufficient to form the EIT resonance both in the absorption and the fluorescence spectra. This is the minimal L for the coherent process reported so far.

It is shown that the EIT resonance in a NTC with L = λ in an external magnetic field to well-resolved components that may allow one to develop magnetometer with nanometric-range spatial resolution. This can be of importance for measuring and mapping the strongly non-homogeneous magnetic field.

Acknowledgement

Fig. 1

 1 Fig. 1 Photograph of the NTC. The gap thickness L varies from 30 to 2000 nm across the window as visualized by an interference pattern. Regions of L = 30 nm and 2000 nm are marked. SA: sapphire side-arm; TC: thermocouple.

Fig. 2

 2 Fig. 2 Sketch of the experimental setup. 1 -Faraday isolators; G1,2,3-Glan prisms; 2 -Helmholtz coils; 3 -photodiodes; 4 -main NTC; 5 -auxiliary NTC of L = λ; 6 -auxiliary NTC of L = λ/2; 7 -permanent ring magnet (for details, see the text); BD-beam damp.

Fig. 3

 3 Fig.3EIT spectrum for 85 Rb D1 line (upper curve). L = λ = 794 nm. Coupling laser is resonant with F = 3 → F ′ = 3 transition, probe laser is scanned across F = 2 → F ′ = 2, 3 transitions. Grey line: the spectrum without the coupling laser (vertically shifted for convenience).

Fig. 4

 4 Fig. 4 Dependence of 85 Rb D1 line EIT resonance linewidth on coupling laser power for ∆ = 0. L = λ = 794 nm.

  ) and 6(a) with Ω c = 1.5γ N , γ ′ 21 = 6 MHz are shown in Fig. 5(b) (transmission spectrum, Ω p = 0.06γ N ,) and Fig. 6(b) (fluorescence spectrum, Ω p = 0.5γ N ).

Fig. 5

 5 Fig. 5 Probe laser transmission spectrum for 85 Rb D1 line. L = λ/2 = 397 nm. Coupling laser (Pc = 27 mW) is resonant with F = 3 → F ′ = 3 transition, probe laser (Pp = 0.04 mW) is scanned across F = 2 → F ′ = 2, 3. Grey line: spectrum when coupling laser is off. Graph a: experiment; graph b: numerical calculations for the experimental parameters, Ωc = 1.5γN , Ωp = 0.06γN , γ ′ 21 = 6 MHz.

Fig. 6

 6 Fig. 6 Transmission spectrum of the probe laser for 85 Rb D1 line, L = λ/2 = 397 nm. Coupling laser (Pc = 27 mW) is resonant with F = 3 → F ′ = 3 transition, probe laser (Pp = 3 mW) is scanned across F = 2 → F ′ = 2, 3. Grey line: the coupling laser is off. Graph a: experiment; graph b: numerical calculations for the experimental parameters, Ωc = 1.5γN , Ωp = 0.5γN , γ ′ 21 = 6 MHz.

Fig. 7

 7 Fig.7EIT spectra for L = λ = 794 nm when the coupling laser is detuned by ∆ from F = 3 → F ′ = 3 transition. The spectra are shifted vertically for convenience. The lower grey curve is the transmission spectrum when the coupling laser is off. Graph a: experiment; graph b: numerical calculations for the experimental conditions, Ωc = 1.3γN , Ωp = 0.3γN , γ ′ 21 = 6 MHz. Additional dephasing rate γ ′ 21 (∆) is responsible for the EIT behavior seen for ∆ = 0 (see the text).

Fig. 8 Fig. 9

 89 Fig. 8 Relevant energy levels of 85 Rb D1 line involved in EIT process. The coupling laser detuning ∆ varies from F = 3 → F ′ = 3 transition in the range of 0 -100 MHz.

The authors are grateful to A. Sarkisyan for his valuable participation in fabrication of the NTC and to Charles Adams for useful discussions. Research conducted in the scope of the International Associated Laboratory IR-MAS. Armenian team thanks for the ANSEF Opt-2428 support. R. M. acknowledges the support from the NF-SAT, NAS RA and CRDF (grant no. ECSP-10-10 GRSP).


Marco Autili
email: marco.autili@univaq.it

Davide Di Ruscio
email: davide.diruscio@univaq.it

Paola Inverardi
email: paola.inverardi@univaq.it

James Lockerbie
email: james.lockerbie.1@city.ac.uk

Massimo Tivoli
email: massimo.tivoli@univaq.it

A Development Process for Requirements Based Service Choreography

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

I. INTRODUCTION

The near future in software production envisions an ubiquitous world of available services that can be easily discovered and coordinated to fit users' needs. The Internet of Services paradigm [START_REF]CORDIS ICT Programme. Service and Software Architectures, Infrastractures and Engineering[END_REF] emerges from the convergence of the Future Internet (FI) and the service-oriented software paradigm. Services play a central role in this vision as effective means to achieve interoperability between heterogeneous parties of a business process and independence from the underlying infrastructure. At the same time they offer an open platform to build new value added service-based systems as a choreography of available services discovered within the FI.

Choreography is put forward as a generic abstraction of any possible collaboration among multiple services, and integrates previously established views on service composition, among which service orchestration. In principle, any possible view on collaboration and previously established view on composition among multiple services (e.g., service orchestration) can be abstracted as a choreography. Three often overlapping viewpoints and related terminologies can be distinguished [START_REF] Barros | Standards for web service choreography and orchestration: Status and perspectives[END_REF], [START_REF] Dijkman | Service-oriented design: A multiviewpoint approach[END_REF]: [START_REF] Alexander | Writing Better Requirements[END_REF] Choreography captures collaborative processes involving multiple services and their interactions from a global perspective; (2) Behavioral interface captures the behavior of a single service that participates in the choreography (i.e., the signature and the interaction protocol with the environment that the service supports); and (3) Orchestration deals with the description of the interactions in which a set of services can be engaged, as well as the internal steps between these interactions.

In the FI, choreographies must be reliable and meet users' requirements. Additionally, they should be adaptive to context changes concerning both user-centric and resourcecentric data, and quality of service (QoS). New method-ologies are needed to address the specification, adaptation, synthesis, scalability, validation, simulation, dynamic execution, and re-configuration aspects of a choreography. The whole choreography development process is affected by the high dynamicity and evolution degree of a highly-scalable choreography. Thus, certain phases of the development process together with their proper artefacts, need to become pervasive, accompanying the choreography implementation at run-time.

This paper describes our experience in this direction and presents our approach to choreography development within the CHOReOS project (www.choreos.eu). The core objective of CHOReOS is to leverage, as far as possible, relevant SOA standards, while making choreography development a fully dynamic, scalable, and domain-expert-centric process so as to face the challenges posed by the Ultra Large Scale (ULS) FI.

Towards the definition of the choreography dynamic development process, CHOReOS focuses on the following (macro-)activities: (i) a domain-expert requirements specification framework for adaptable QoS-aware highly-scalable choreographies; (ii) methods and mechanisms for large scale service base management; (iii) choreography synthesis; (iv) service-oriented middleware for the FI to support choreography execution; and (v) analysis of choreography scalability, governance and V&V.

Although we provide an overview of the entire development process model (Section III), in this work we focus on the first and third activity only (Sections IV and V). Related state of the art is discussed in Section II. Conclusions and future work are given in Section VI.

II. RELATED WORK

An example of a dynamic development process model has been proposed within the FP6 IST EU PLASTIC project (www.ist-plastic.org/). The PLASTIC development process relies on Model Driven Development (MDD) [START_REF] Selic | The pragmatics of model-driven development[END_REF] solutions to build adaptable context-aware service-oriented applications. The CHOReOS dynamic development process (and its related tools), is inspired by the PLASTIC development process model and will be based on the models@runtime approach [START_REF] Blair | Models@run.time[END_REF]. We will follow a conceptual-model-centric strategy, as in PLASTIC, and we will exploit model transformation technologies in order to make models available, and synchronized, at each phase of the process life-cycle from design-to run-time, as in the models@runtime approach. Existing research on models@runtime mainly focuses on models representing software structures and procedural representations of them such as architectural models. As pointed out in [START_REF] Blair | Models@run.time[END_REF], the models@runtime vision should go further by raising the level of run-time model abstraction to that of requirements. For instance, a research challenge concerns the development of methods and tools for observing the systems requirements and goals during execution. To this aim, the domain-expert specification framework proposed by CHOReOS (see Section IV) represents a step forward.

In general, requirements engineering research has not recognized the importance of open systems and serviceoriented computing, considering it to be downstream from requirements activities, although some work has been undertaken (e.g., [START_REF] Fickas | Requirements monitoring in dynamic environments[END_REF]). However, most of this work has not directly addressed recognized challenges in service-oriented computing. Some of the most relevant work in serviceoriented computing has been in QoS ontologies and measurement [START_REF] Sawyer | Faceted service specification[END_REF], however this work focuses more on servicelevel qualities and SLA, but not the originating requirements and their expression. In [START_REF] Rao | A survey of automated web service composition methods[END_REF], the authors propose a general framework for automatic Web service composition. Waldinger [START_REF] Waldinger | Web agents cooperating deductively[END_REF] elaborates an idea for service synthesis by theorem proving. Most end-users, however, will lack the education and/or training to be able to express requirements using even semi-formal notations supported by UML or first-order languages. Most functional requirements will be expressed in, at best, structured natural language. Zachos et al. [START_REF] Zachos | Ontology-aided translation in the comparison of candidate service quality[END_REF], for example, present techniques to match semistructured natural language QoS requirements to service qualities during service selection using ontologies.

Current approaches for synthesizing service choreographies, involve having a central unit responsible for selecting and composing the system [START_REF] Brogi | Automated generation of bpel adapters[END_REF], [START_REF] Melliti | Distributed behavioural adaptation for the automatic composition of semantic services[END_REF], [START_REF] Salaün | Generation of service wrapper protocols from choreography specifications[END_REF] (just to cite some). The centralization is a clear drawback when considering the expected Ultra Large Scale of the Future Internet. Dealing with the synthesis phase of decentralized choreographers is among the main objectives of the CHOReOS dynamic development process. We define choreography patterns by considering as baseline our previous work described in [START_REF] Autili | Synthesis of decentralized and concurrent adaptors for correctly assembling distributed component-based systems[END_REF], so that the choreography may be distributed in a set of wrappers. We will introduce model transformation techniques [START_REF] Mei | A software architecture centric self-adaptation approach for internetware[END_REF] to refine the domain-expert choreography requirements specification into a peer-style specification of the choreography. Thus, the choreography synthesis process shall result in producing the decentralized model of an abstract coordination delegate that, accounting for service functional and non-functional abstractions, enforces the composition of the discovered services to adhere to the specified choreography.

III. CHOREOS DYNAMIC DEVELOPMENT MODEL

The evolutionary nature of choreographies in the Future Internet makes unfeasible a standard development process and all the artefacts and models might be exploited also by the deployment-and run-time activities hence leading to a dynamic development process model. Therefore, central to the development process is the notion of models and models@runtime techniques. The CHOReOS Development Process Model, decomposes into:

• First, CHOReOS intends to support the systematic development of choreographies from their design to their actual enactment, i.e., execution. CHOReOS uses two orthogonal model-transformation approaches: (i) topdown and (ii) cross-cutting. The former will serve to refine the natural language-based domain-expert requirements specification into analysis-technique-specific models derived from a peer-style specification. The latter will serve to integrate the different modeling/reasoning technologies by passing from a technique-specific model to a different technique-specific model. • A distributed (large scale) service base will further manage information about available services offered by service providers. The service base organizes available services into functional and non-functional views. Each view is characterized by corresponding abstractions (functional/non-functional) and a set of available services that are represented by each abstraction. • The approach envisioned for the choreography synthesis then starts from (i) the peer-style specification of the choreography and from (ii) the set of services discovered from the large scale service base. Input (i) is based on the functional and non-functional abstractions, and it comes from the refinement of the domain expert specification obtained by means of the top-down transformational process. Input (ii) comes from the exploitation of the service base management mechanisms. That is, the synthesis process assumes that the services into the registry/base have been discovered so that they satisfy the local (to the service) functional and non-functional requirements that have been specified for the choreography and, hence, can be considered as potential candidates to participate in the global choreography process. The choreography synthesis produces the abstract coordination delegates that, on top of the CHOReOS middleware, support the enactment of choreographies in a distributed way. • Finally, the process accounts for complex analysis and validation steps at run-time.

IV. REQUIREMENTS SPECIFICATION As neither the role of domain expert or service consumer can assume requirements knowledge, a core challenge to address is how quality requirements on future services can be expressed as simply as possible. The most straightforward form of expression of requirements from these two roles is natural language. However, some degree of structure to free-text expression is needed to capture the service quality needs of the stakeholders effectively. To do this we use qualifiers [START_REF] Alexander | Writing Better Requirements[END_REF], which transform the functional root of a requirement into a non-functional requirement (NFR). Rather than asking stakeholders to express requirements using typical NFR types our approach prompts them to express qualities on the service through 5 simple questions: "How quick?", "How secure?", "How precise?", "How robust?", and "How simple to use?". These questions were selected based on a model of qualities and measures developed for the Cloud Commons Consortium (www.cloudcommons.com) and revised for the CHOReOS project. Figure 1 presents a simple representation of the model, and shows how the qualifier questions map onto service-based application qualities and QoS on services aggregated in these applications. It would not be realistic for domain experts and service consumers to express requirements on qualities such as scalability, flexibility and serviceability as these areas are the concern of service developers and providers. However, through associations in the model these can be implied, for example, high performance may require a particular level scalability and flexibility of the services. Our approach also uses the concept of customer satisfaction from the VOLERE specification [START_REF] Robertson | Mastering the Requirements Process[END_REF] to measure how happy the user will be if the requirement is implemented and how unhappy they will be if it is not. All user responses on quality and satisfaction are prioritized relative to each other using a sliding scale recorded notionally from 1 to 5. Prioritization of requirements is particularly useful when considering scalability and the potentially hundreds of requirements for domain experts to manage.

The specification process (see Figure 2) begins with the service consumer who expresses requirements on their service needs. The domain expert then receives consumer requirements through the evolving requirements specification and can also act as a surrogate for individual service consumers by expressing their own domain specific requirements. The domain expert can constantly review the latest requirements/queries to extract the overall requirements on the services. Domain expert analysis helps to identify emerging patterns or similarities based on functionality, quality and user type or preference. The resulting set of prioritized The service choreography patterns encapsulate different complex choreography decisions made in the presence of user requirements, by linking codified domain knowledge of user tasks to classes of service solutions. The user requirements are matched to user task models that, in turn, are used to reformulate service queries with terms describing classes of services in the task model. Each pattern considers the qualities of the services to be invoked, the qualities that different choreographies offer and how, when combined, these service and choreography qualities deliver serviceoriented applications with different qualities. Service behavioral models are automatically synthesized by the Synthesis of Behavior Protocol activity starting from the descriptions of services that have been discovered within the Service Base. The synthesis process is able to understand how to actually coordinate the discovered services to suitably realize the specified choreography. For instance, it might be the case that the discovered services, although potentially suitable in isolation, when interacting together can lead to, e.g., concurrency and interaction mismatches such as deadlocks, or safety and timeliness violations. Thus, although a given set of services, if coordinated in the right way, can be used to achieve the specified choreography, they can completely miss the choreography's goal if coordinated in a different way. Further applying Model-to-Model Transformation, the synthesis method will produce abstract coordination delegate models. Such models, accounting for service functional and non-functional abstractions, force the collaboration of the discovered services to guarantee the specified choreography. To actually realize the choreography, the abstract coordination delegate models are then concretized into actual software artifacts by means of Model-to-Code Transformations. The generated coordination delegates are deployed on the CHOReOS choreography engine and, by means of the CHOReOS service-oriented middleware, they suitably access and coordinate the discovered services.

In the CHOReOS deliverable D3.1 [START_REF]Deliverable D3.1: Initial Architectural Style for CHOReOS Choreographies[END_REF], we discuss an example concerning the development of a choreography-based travel agency system that can be realized by choreographing four services: a Booking Agency service, two Flight Booking services, and a Hotel Booking service. The example shows how coordination delegates correctly and distributively interact, at run-time, with each other, hence ensuring the behavior globally specified by the Choreography Model. For space reasons, it is not possible to describe this example here and, hence, for it we entirely refer to [START_REF]Deliverable D3.1: Initial Architectural Style for CHOReOS Choreographies[END_REF].

VI. CONCLUSIONS AND FUTURE WORK

In this paper we have briefly described our initial experience of choreography development within the CHOReOS project (www.choreos.eu). The core objective of CHOReOS is to leverage relevant SOA standards, while making choreography development a fully dynamic, scalable, and domainexpert-centric process so as to face the challenges posed by the FI. After providing an overview of the entire development process model, we have focused on requirements specification and choreography synthesis.

As future work we plan to instantiate the process model and apply it to the case studies we have promised to target in CHOReOS.

Figure 1 .

 1 Figure 1. Simplified overview of the CHOReOS quality model

Figure 2 .

 2 Figure 2. Goal and requirements specification quality-based requirements is then associated with choreography strategies, which are expressed in the form of patterns.The service choreography patterns encapsulate different complex choreography decisions made in the presence of user requirements, by linking codified domain knowledge of user tasks to classes of service solutions. The user requirements are matched to user task models that, in turn, are used to reformulate service queries with terms describing classes of services in the task model. Each pattern considers the qualities of the services to be invoked, the qualities that different choreographies offer and how, when combined, these service and choreography qualities deliver serviceoriented applications with different qualities.

  V. CHOREOGRAPHY SYNTHESISA choreography can be seen as a collaborative process among interacting participants. The message exchange globally defined by the choreography has to be projected (in a peer-style fashion) among different participants according to the "local" roles they play to fulfill the global choreography. Choreography synthesis concerns the realization of distributed coordination delegates that, supported by the CHOReOS middleware, cooperatively work to support the enactment and execution of the choreography. By relying on the CHOReOS service base management mechanisms, Model-to-Model Transformation techniques[START_REF] Huang | Runtime recovery and manipulation of software architecture of component-based systems[END_REF] are used to refine the Choreography Model together with the service Behavioral Models into a Peer-style Specification (see Figure3).

Figure 3 .

 3 Figure 3. Choreography synthesis

This work is supported by the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement number 257178 (project CHOReOS -Large Scale Choreographies for the Future Internetwww.choreos.eu).


Hau Duc

Sylvain Nguyen

Guillou

Dan Kim

Damien Nguyen
email: dan.nguyen@saint-venant-lab.fr

Julien Pham Van Bang
email: damien.pham-van-bang@saint-venant-lab.fr

Chauchat

Pham Van Bang

Julien Chauchat
email: julien.chauchat@hmg.inpg.fr

Simulation for the convective descent phase of dredged-sediment releases in the seawater by a two-fluid model

Keywords: Dredged sediment release, sediment transport, two-phase flow modelling, fine sand I

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

INTRODUCTION

The process of dredged-sediment disposal generally follows three different steps [START_REF] Johnson | Development and verification of numerical models for predicting the initial fate of dredged material disposed in open water[END_REF]: (i) convective descent during which the sediment falls under the influence of gravity; (ii) dynamic collapse, occurring when the descending cloud or jet impacts the bottom (iii) passive transportdispersion, commencing when the sediment transport and spreading are determined more by ambient currents and turbulence than by the dynamics of the disposal operation.

Previous numerical studies have shown the limits of the "passive scalar" approach in simulating dredged sediment disposal [START_REF] Garapon | -3D numerical modelling of sediment disposal[END_REF]. This is due to the very high concentration of the sediment cloud. In recent work, Freson et al. [START_REF] Farout-Freson | Modèle numérique de clapagephase de chute[END_REF] has used a bi-specie model, which is based on a degeneration of the two-phase model, and considers the sediment-water mixture. Interesting results were obtained for the falling phase whereas the bottom transport phase remains still problematic.

The purpose of this work is to study the convective descend step using a two-phase model [START_REF] Nguyen | A two-phase numerical model for suspended-sediment transport in estuaries[END_REF]. This model has been applied [START_REF] Guillou | Simulation of the dredged sediment's release with a two-phase flow model[END_REF] to the experimental configuration of Villaret et al. [START_REF] Villaret | Etude expérimentale de la dispersion des rejets par clapage[END_REF]. The process of dredged-sediment disposal is studied with different values of the current, the sediment diameter, and the initial sediment concentration.

II GOVERNING EQUATIONS

The two-phase flow model is based on the Eulerian-Eulerian (or two-fluid) description. The model is fully described in [START_REF] Nguyen | A two-phase numerical model for suspended-sediment transport in estuaries[END_REF]. Herein we briefly recall the basic equations with special closures for the sediment dumping case. The governing equations (1) are written for each phase. The subscript k could be "f" for fluid and "s" for solid phase:

( ) ( ) ( )        + + + + - ⋅ ∇ =       ⊗ ⋅ ∇ + ∂       ∂ =       ⋅ ∇ + ∂ ∂ → → → → → → → → k k k k k k k k k k k k k k k k k M ρ g α I p α ρ u u ρ α t u α u α t α 1 1 0 Re τ τ (1)
where k Villaret et al. [START_REF] Villaret | Etude expérimentale de la dispersion des rejets par clapage[END_REF] realised a laboratory experiment of sand release in a straight channel of 72 m long, of 1.5 m wide and of 1.5 m height. A specifically designed recipient (maximum capacity of 60 litters) is placed at 15 cm below the free-water surface. The ambient current is maintained with the help of a hydraulic pump. The sand-water mixture is initially filled-up in the recipient at a given concentration. Then the bottom of recipient is suddenly opened (Figure 1b) and the measurement (photo-camera and concentration) starts simultaneously. Table 1 gives an index of the different testing conditions.

Table 1. Testing conditions and nomenclature: W inj is the injection velocity from the recipient, D p the sediment particle diameter (90 µm), ρ the dry density of the solid (2650 kg/m 3 ), Cm is the concentration of the mixture (450 g/l), Vr the volume of dumped material (60 l), Uc the ambient velocity [START_REF] Villaret | Etude expérimentale de la dispersion des rejets par clapage[END_REF].

Tests

IV TWO-PHASE MODELING & RESULTS

The 2-D X/Z computation domain extends over 14m in the x-direction (centred at the release location) and over 1m on the vertical one (z-ascendant coordinates). A regular mesh of 61x1401 nodes as well as a time step of ∆t=0,001 s are used. The numerical parameters are set in Table 1. The lateral ends are considered as open boundaries. At the injection location (inlet diameter equal to 10 cm), the fluid and sediment fluxes are imposed following a Poiseuille profile with a given maximum velocity (W inj ). Figure 2a presents the time evolution of the maximum radius R of the sediment cloud obtained by the two-phase model and the single-phase one, compared with the experiment [START_REF] Villaret | Etude expérimentale de la dispersion des rejets par clapage[END_REF] and the analytical solution proposed by Krisnappan [START_REF] Krishnappan | Dispersion of granular material dumped in deep water[END_REF]. Clearly, the two-phase model results are fairly in agreement with the experiment and the analytical solution, whereas the single-phase model results more and more differ the experimental values during the second half period of convective descent phase. Indeed Figure 2a shows that over this period, the single-phase model gives a radius, which is nearly constant (≈18cm). This can explain by the fact that the development of the sediment cloud in a single-phase model is only engendered by the density gradient, and no solid-fluid interaction is taken in consideration. Yet this interaction that express by the drag force is dominant in such a flow. The longitudinal profiles of vertical velocity plotted in Figure 2b show that the vertical velocity of the solid phase is always larger than the fluid phase one. Figure 3 presents the iso-value contours of sediment concentration as well as streamlines at t=1.5 s obtained from the two-phase model (Figure 3a) and the single-phase one (Figure 3b) for test e11. Obviously the two-phase model reproduces two counterrotating vortices, which allow a full development of the sediment cloud. As the cloud growths, the surrounding water is entering inside the cloud while the iso-value contour is expanding. This illustrates that the horizontal velocity component of fluid-and solid-phase are unequal in the direction and magnitude. No counter-rotating vortices are observed in the single-phase model results. This is why the sediment cloud obtained by the single-phase model is not developed as physically expected. The experimental observations are therefore qualitatively well captured by the two-phase model. Figure 4 presents the evolution of the sediment cloud as well as solid velocity fields in still water (Figure 4a) and in a water flow of velocity, Uc=25 cm/s (Figure 4b). Note that the conservation of mass in the computation domain is good enough as far as the relative error is around 0.05%.

V CONCLUSIONS

This study presents numerical simulations of dredged sediment disposal with and without ambient currents by using a two-phase flow model. This modelling well suit to such cases with fluid and solid phase velocities (all the components) differ each other in amplitude and in direction. The numerical results obtained by the model are fairly in agreement with experimental data during the convective descent phase. The formation and propagation of density current are also realistic with experiments. For the parameters remaining inaccessible from the experiment such as the concentration field, the formation of counter-rotating vortices, the influence of ambient current or particle diameter, the numerical model provides an estimate or a representation that is in qualitative accordance with other published data. The actual study deals with non-cohesive sediments. For such a case, no consolidation effect is encountered. To deal with cohesive sediment, the consolidation routine should be activated but more important is the need to account for the flocculation/defloculation processes that should play a fundamental role in the dynamic of the system.

ρFigure 1 :

 1 Figure 1: Definition sketch: a) location of optical sensors (OP) for turbidity measurements; b) muddumping[START_REF] Villaret | Etude expérimentale de la dispersion des rejets par clapage[END_REF]

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: Test 11: a) numerical and experimental radius of the sediment cloud; b) vertical velocity profile at z=-0.83 m

Corresponding author


J Chauchat

S Guillou

K D Nguyen

may come     Numerical simulation of the water-sediment mixed flow in a periodic channel by a two-phase model

Dresdner

  Wasserbauliche Mitteilungen Heft 32 Numerical simulation of the water-sediment mixed flow in a periodic open channel by a two-phase model

J. Chauchat, S. Guillou and K. D. Nguyen

Wasserbaukolloquium 2006: Strömungssimulation im Wasserbau

Dresdner Wasserbauliche Mitteilungen Heft 32