Julio Nuñez

MD Marifina Chilet

MD Juan Sanchis

Vicent Bodi

Eduardo Nuñez

MD Gema Miñana

MD Nuria Tormo

MD Maria A Clari

MD Mauricio Pellicer

MD Francisco J Chorro

M.D. Julio Núñez

MD Vicent Bodí

MD Eduardo Núñez

MD Angel Llàcer

MD David Navarro

Prevalence and Prognostic Implications of Active Cytomegalovirus Infection in Patients with Acute Heart Failure

Keywords: Acute Heart Failure. Cytomegalovirus Infection. Inflammation. Mortality. Risk stratrification systolic blood pressure, DBP, diastolic blood pressure, LVEF, left ventricular ejection fraction, HR, heart rate, NT-proBNP, N-terminal probrain natriuretic peptide, IgM, immunoglobulin M, M, male, F, female, HF, heart failure, CHD, coronary heart disease, CABG, Coronary artery bypass graft surgery, HMC, hypertensive myocardiopaty

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INTRODUCTION

Despite recent therapeutic innovations, advanced heart failure (HF) remains a serious medical condition with morbidity and mortality rates comparable or even higher than that for most malignancies [START_REF] Stewart | More 'malignant' than cancer? Five-year survival following a first admission for heart failure[END_REF]. Several studies support the involvement of inflammatory pathways in triggering the initiation and favoring the progression of HF [START_REF] Von Haehling | Inflammatory biomarkers in heart failure revisited: much more than innocent bystanders[END_REF][START_REF] Yndestad | Systemic inflammation in heart failure--the whys and wherefores[END_REF][START_REF] Torre-Amione | Immune activation in chronic heart failure[END_REF]. In this context, proinflammatory cytokines, such as tumor necrosis factor-(TNF-) and interleukin-6 (IL-6) appear to have detrimental effects on cardiac function [START_REF] Von Haehling | Inflammatory biomarkers in heart failure revisited: much more than innocent bystanders[END_REF][START_REF] Yndestad | Systemic inflammation in heart failure--the whys and wherefores[END_REF][START_REF] Torre-Amione | Immune activation in chronic heart failure[END_REF]; Nevertheless, the prognostic implication of the inflammatory responses in HF remains unclear. Recently, some authors have highlighted the role of hyperinflammatory states in promoting cytomegalovirus (CMV) reactivation [START_REF] Cook | Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice[END_REF]. CMV infection causes significant morbidity and mortality in immunocompromised patients, not only as a result of virus cytopathogenicity ("direct effects"), but also by virtue of its immunomodulatory properties ("indirect effects") [START_REF] Crough | Immunobiology of human cytomegalovirus infection: from bench to bedside[END_REF]. The latter effects may account for the reported association between CMV infection and chronic allograft rejection in solid organ transplant recipients, particularly transplant-related vasculopathy, whose histopathology remarkably resembles that of atherosclerosis [START_REF] Bruggeman | Does cytomegalovirus play a role in atherosclerosis?[END_REF]. In line with this, a role for CMV in the pathogenesis of cardiovascular diseases has been postulated [START_REF] Bruggeman | Does cytomegalovirus play a role in atherosclerosis?[END_REF][START_REF] Zhu | Host response to cytomegalovirus infection as a determinant of susceptibility to coronary artery disease: sex-based differences in inflammation and type of immune response[END_REF].

Data obtained in the murine cytomegalovirus model indicate that a proinflammatory state is a critical factor in promoting CMV reactivation [START_REF] Cook | Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice[END_REF]. In this mechanistic model, TNF-, a cytokine known to directly stimulate immediate early (IE) CMV gene expression in vitro [START_REF] Prösch | Stimulation of the human cytomegalovirus IE enhancer/promoter in HL-60 cells by TNF-alpha is mediated via induction of NF-KappaB[END_REF], may play a pivotal role [START_REF] Döcke | ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the diagnosis and treatment of acute and chronic heart failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM)[END_REF]. In fact, intraperitoneal administration of well-known inflammatory mediators in HF, such as lipopolysaccharide (LPS) and TNF-, were capable of reactivating CMV from latency in lungs of previously healthy mice [START_REF] Cook | Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice[END_REF]. We thus hypothesized that active CMV infection was likely to occur in acute heart failure (AHF) patients and that it may have prognostic implications. In the current study, we sought to determine in a cohort of patients with AHF: 1) the prevalence of CMV infection; 2) whether active CMV infection was associated with an increased risk for the composite endpoint of short-term all-cause mortality or readmission for AHF and; 3) whether those patients with an active CMV infection displayed higher rates of hospitalized-day per weeks of follow-up. A c c e p t e d M a n u s c r i p t

METHODS

Study group

We prospectively studied a cohort of 138 patients consecutively admitted to the cardiology department of Hospital Clínico Universitario de Valencia from 1 st November 2008 to 19 th May 2009 with the diagnosis of AHF. Diagnosis of AHF was defined as the rapid onset of symptoms and signs secondary to abnormal cardiac function and the presence of objective evidence of structural or functional abnormality of heart at rest (cardiomegaly, third heart sound, cardiac murmur, abnormality of the echocardiogram or raised natriuretic peptide), following current guidelines [11] and established by a trained cardiologist. Exclusion criteria were known or suspected infections, any systemic inflammatory disease different from heart failure, immunodeficiency, end-stage renal disease undergoing dialysis, cancer, blood transfusion within 30 days, and the need for mechanical ventilation before blood sampling. One hundred thirty eight patients were initially selected, but 6 patients were later excluded because of death before blood sampling (n=2), a final diagnosis of acute coronary syndrome (n=2), pneumonia (n=1) and cancer (n=1), leaving the study sample in 132. Demographic information, medical history, vital signs, 12-leads electrocardiogram, laboratory data and drug utilization were routinely determined in emergency department and throughout the hospital course following pre-established registry questionnaires. Left ventricular ejection fraction (LVEF) was assessed with two-dimensional echocardiography in all patients during index hospitalization (Agilent Sonos 5500-Phillips).

All patients received intravenous treatment with furosemide at least during the first 48 hours of admission. Treatment with angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), betablockers, aldosterone antagonist, anticoagulants and other therapeutic strategies was individualized following established guidelines [11].

Patients' follow-up was censored if death occurred or by having undergone cardiac transplantation or cardiac valve replacement surgery. The composite of all-cause mortality and readmission for AHF was selected as the main clinical endpoint. As secondary endpoint, the cumulative incidence rate of hospitalized-day per week of follow-up was compared between those with and without CMV DNAemia. This study was approved by an institutional review committee and patients gave written informed consent.

CMV Assays

Following an overnight fasting, venous blood samples were drawn from the antecubital vein between 8:30 h and 9:30 h with patients in resting supine position for at least 15 min prior to sampling. IgG and IgM antibodies against CMV were detected by means of a commercial immunoassay (Architect CMV IgG assay, Abbott Diagnostics, Des Plaines, IL, USA). CMV DNA load was measured by real-time PCR with the Abbott CMV PCR kit (produced by Qiagen GmbH, Hilden, Germany, for Abbott Diagnostics, Des Plaines, IL, USA), performed using the m2000RT system (Abbott Molecular) and following the manufacturer's instructions, as previously described [START_REF] Gimeno | Quantification of DNA in plasma by an automated real-time PCR assay (CMV PCR Kit, Abbott) for surveillance of active[END_REF]. DNA extractions (from 500 µl of plasma) were performed using the Abbott mSample preparation system DNA kit on the m2000 SP instrument (Abbott Molecular). The reported limit of detection of the assay (95% C.I.) is 25 CMV DNA copies/mL [START_REF] Gimeno | Quantification of DNA in plasma by an automated real-time PCR assay (CMV PCR Kit, Abbott) for surveillance of active[END_REF]. The assay is, however, able to reliably quantify lower CMV DNA loads (Navarro D, unpublished observation). Specimens with CMV DNA loads <25 copies/mL were reassayed using a different

Plasma cytokines analysis

Plasma levels of TNF-, IL-6, interleukin-1beta (IL-1 ) and interleukin-10 (IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (eBioscience human IL-1 , IL-6, TNF-and IL-10 ELISA Ready-SET-Go!). The sensitivity claimed by the manufacturer for the different cytokine assays were: 3.0 pg/mL for TNF-, 4.0 pg/mL for IL-1 , 2 pg/mL for IL-6, and 2.0 pg/mL for IL-10. High-sensitivity ELISAs were used for cytokines measurements in samples giving values below the detection limit in the respective low-sensitivity assay. The concordance between the values obtained in the low-sensitive assay (for those above the detection limit) and those in the high-sensitive assay approached 100%.

Statistical analysis

Continuous variables were expressed as median (interquartile range). Discrete variables were presented as percentages. Baseline characteristics were compared among those patients with and without CMV DNAemia. Using standard cutpoints CMV DNAemia was also categorized by the presence of >25 copies/mL, 25 copies/mL or negative (undetectable CMV DNA) results. The composite of mortality/AHF rehospitalization rates was depicted among the CMV DNAemia status using the Kaplan-Meier method, and their differences tested by the Peto-Peto Prentice test. The independent association between CMV DNAemia categories and the main outcome was assessed with Cox regression analysis. Candidate covariates for the initial multivariable model included all variables listed in table 1, including cytokines. Then, a reduced and parsimonious model was derived by using the Royston and Altman model-selection algorithm [13]. The final Cox model included the following covariates: previous admission for AHF, systolic blood pressure at admission (mmHg), Charlson comorbidity index, serum sodium (mEq/L), serum NTpro brain natriuretic peptide (BNP) (pg/ml) and serum antigen carbohydrate 125 (CA125). The proportionality assumption for the hazard function over time was tested by means of the Schoenfeld residuals. The model's discriminative was assessed by the Harrell´s C-statistic while its calibration tested by the Gronnesby and Borgan test [14]. For the secondary analysis, negative binomial regression was used to determine the incidence rate ratio (IRR) of patient's hospitalized-day per week of follow-up according to CMV DNAemia status. Independent predictors included in this model were: age, smoking status, last NYHA class measured under stable conditions, previous admission for AHF, valvular heart disease, anemia, lymphocyte relative count, CA125, uric acid, TNF-, left ventricular ejection fraction and treatment with furosemide, espironolactone, statins and treatment with inotropic drug support.

A 2-sided p-value of <0.05 was considered to be statistically significant for all analyses. All analyses were performed using STATA 11 (StataCorp. 2009. Stata Statistical Software: Release 11. College Station, TX: StataCorp LP).

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RESULTS

Prevalence of active CMV infection

One hundred and twenty nine patients (97.7%) were CMV IgG-seropositive. A single analysis for the presence of CMV DNA in plasma was performed (median of 3 days -IQR 1-5 days-following admission). CMV DNA was detected in plasma from 11 (8.3%) patients, 4 of whom had CMV DNAemia levels of >25 copies/mL (median 43 copies/mL; range, 31 to 72 copies/mL). Lower CMV DNAemia levels (median 10 copies/mL; range, 6 to 12 copies/mL) were consistently detected in the remaining 7 patients. Six patients (4.6%) tested positive for CMV IgMs, 2 of whom tested positive for CMV DNAemia. Time to sampling after admission did not differ significantly (p=0.656) between patients with or without CMV DNAemia.

Characteristics of patients across CMV status

The mean age of patients in the sample was 76±13 years; 64 patients (48.5 %) were female, 70 (53%) had been previously admitted for AHF and 78 (59.1%) exhibited left ventricular ejection fraction 45%. Table 1 shows the clinical characteristics of the study population according to the CMV DNAemia status. Patients with active CMV infection displayed more frequently LVEF<45%, had lower median systolic and diastolic blood pressures, and exhibited a trend for a higher median NT-proBNP serum values. Interestingly, no differences were found in age, gender, cardiovascular risk factors, ethiology, presentation, comorbidity and treatment between groups.

Inflammatory status and active CMV infection

Plasma levels of several proinflammatory and immunoregulatory cytokines were measured in patients with (at the time of CMV DNA detection) and without active CMV infection. Patients exhibiting CMV DNAemia showed lower median IL-10 values and higher median TNF-/IL-10 and TNF-/IL-6 serum values (Table 1). These differences were particularly evident when patients were stratified on the basis of their CMV DNAemia load (none, and >25 copies/mL) (Figure 1).

CMV infection and death/readmission for AHF

During a median follow-up of 120 days (IQR: 60-240), 23 (17.4%) deaths, 34 (24.2%) readmissions for AHF and 45 (34.1%) deaths/readmission for AHF were identified. The cumulative rates for death, readmissions for AHF and the composite endpoint were higher for patients with CMV DNAemia (45.5% vs. 14.9%, 63.6% vs. 20.7% and 81.8% vs. 29.8%, respectively; p<0.001 for all comparisons) than for those not experiencing it, with tangible differences observed since the first days of follow-up for death and the composite endpoint of allcause mortality/readmission for AHF (Figure 2a and 2b, respectively). Detailed medical history and evolution of 11 patients who exhibited CMV DNAemia are shown in Table 2.

Under a multivariate context, after adjusting for established prognostic factors and potential confounders, including previous admission for AHF, systolic blood pressure, Charlson comorbidity index, serum sodium, serum NT-proBNP and tumor marker antigen carbohydrate 125 (CA125), the occurrence of CMV DNAemia remained strongly associated with the endpoint (HR=4.39, 95% CI: 2.02-9.52; p<0.001) (Table 3). In a sensitivity analysis including in the final multivariate model TNF-, IL-6 and IL-10 values, independently of their p-value, CMV DNAemia remains associated with the main endpoint (HR=4.84, 95% CI: 2.10-11.15; p<0.001). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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In addition, a risk gradient was found according to the magnitude of the viral load. Thus, compared to patients with undetectable CMV DNAemia, patients with >25 copies/ml showed a prohibitive adjusted increased risk of death or readmission (HR=13.6, CI 95%: 3.98-46.58; p<0.001) whereas patients with CMV DNAemia of 25 copies/mL showed and intermediate increased risk (HR=2.93, CI 95%: 1.11-7.71; p<0.030) (Table 3). It is noteworthy to point out that all patients who displayed CMV DNAemia of >25 copies/mL died within the first 100 days following index admission (one of them died during index admission and in the rest, death was preceded by a readmission for AHF) (Table 2).

Likewise, patients with detectable CMV-specific IgMs displayed an increased risk of the composite endpoint (Figure 3). This excess of risk attributable to the presence of CMV-specific IgMs remained strongly significant in multivariate analysis, including CMV DNAemia as a variable (HR: 6.91, CI 95%: 1.95-24.5; p=0.003). Since presence of DNAemia and CMV-specific IgMs yielded similar prognostic and biological meanings, collapsing them into one category, led us to identify a subgroup of subjects (n=15) with a very-high risk of death/rehospitalization for AHF (HR: 4.70, CI 95%: 2.29-9.61; p<0.001).

Harrell´s C-statistics of the multivariate models that included CMV DNAemia either as binary or three-level variable showed higher discriminative ability (0.787 and 0.789) than the model without it (0.764). The Gronnesby and Borgan test of goodness-of-fit showed a good model's calibration (p = 0.579).

CMV and cumulative incidence hospitalized-day rates

The presence of CMV DNAemia identified a subset of patients with a higher: a) median (IQR) length of stay during the index hospitalization [23 days (11) vs. 11 days (10), p<0.001], b) cumulative patient's hospitalized-day [42 days (24) vs. 15 days (18), p=0.001] and patient's rates of hospitalized-day/per week of follow-up (1.07 vs. 0.78 days/week of follow-up, p=0.031). Using negative binomial regression, the IRR calculated for the presence of CMV DNAemia was 1.99, CI 95% CI: 1.04-3.82; p=0.037, association that remained significant and in the same direction after multivariate adjustment (IRR: 2.40, 95% CI: 1.44-4.02; p=0.001).

DISCUSSION

To the best our knowledge, this is the first study reporting that active CMV infection occurs in patients with AHF, and that in this clinical setting, appears to exert detrimental effect on prognosis.

It is now widely accepted that an activation of the immune system occurs in patients with chronic heart failure, leading to an inflammatory state that appears to be determinant in disease progression [START_REF] Von Haehling | Inflammatory biomarkers in heart failure revisited: much more than innocent bystanders[END_REF][START_REF] Yndestad | Systemic inflammation in heart failure--the whys and wherefores[END_REF][START_REF] Torre-Amione | Immune activation in chronic heart failure[END_REF]15]. In this sense, serum levels of TNF-, IL-6 and soluble TNF-receptors I and II have been shown to correlate with disease stage and progression, and ultimately with a poorer prognosis in chronic and in AHF [2-4, 16, 17]. TNF-has been shown to trigger CMV reactivation from latency at the cellular level [START_REF] Cook | Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice[END_REF]. In this context, active CMV infection has been shown to occur frequently in critically ill patients without canonical immunosuppression [18-20], especially in those with severe sepsis, a biological condition leading to CMV reactivation in the murine CMV model [START_REF] Cook | Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice[END_REF]. In this setting, CMV may perpetuate and aggravate the inflammatory state by virtue of its immunomodulatory properties [21,22]. This may account for the reported association between CMV infection and prolonged hospitalization, longer duration of mechanical ventilation and higher rates of nosocomial infections and overall mortality in this clinical setting [18][19][20].

On the basis of the above assumptions, we reasoned that active CMV infection was likely to occur in patients with HF, especially during an acute episode. Indeed, we found that CMV DNAemia occurred at a low level (<100 copies/mL) in 8.7% of cases. The above findings are reminiscent to those reported in critically ill patients without canonical immunosuppression experiencing an active CMV infection, in which a rapid expansion of CMV-specific functional T cells in response to CMV replication keeps systemic CMV load at a low level until complete resolution of the episode [23]. In addition, in our cohort, the occurrence of CMV DNAemia was independently associated with an increased risk of morbidity and mortality. This association was particularly evident when patients were stratified according to their level of CMV DNAemia, those displaying CMV DNAemia levels >25 copies/mL being at the highest risk. In fact, the 4 patients with in the upper CMV DNAemia category died during the first 100 days following the index admission. In line with our findings, the magnitude of the CMV DNA load has been recently reported to be independently and quantitatively associated with prolonged hospitalization and death after adjusting for parameters indicative of severity such as APACHE II score and other potential confounders in a cohort of critically ill patients attended at different intensive care units [19].

Further, the presence of CMV-specific IgMs, a well-known marker of CMV reactivation in immunocompetent individuals harboring latent CMV [24], was independently associated to an increased risk of death/readmission for AHF. The possibility exists nevertheless that IgMs detected were not truly CMV specific [24].

Whether CMV is a direct cause of morbidity and mortality in patients with AHF or it is simply a surrogate marker for disease severity could not be elucidated in the current study. CMV may reactivate in AHF patients in the setting of a pro-inflammatory state. As an immunomodulatory agent, CMV may enhance inflammation which would result in further myocardial injury [START_REF] Torre-Amione | Immune activation in chronic heart failure[END_REF]15,[25][26][27] or may even increase the risk of secondary infections [22], a well recognized precipitating factor for HF descompensation [11]. The fact that patients with active CMV infection exhibited an enhanced immunoinflammatory state (higher plasma TNF - A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

9

/IL-10 and IL-6/IL-10 ratios) when compared to those without active infection provides support to the plausibility of this hypothesis.

Our results, however, must be interpreted with caution: First, this is a preliminary observational study that includes a limited number of patients with AHF; second, despite the strength of the association, the low number of patients with active CMV infection precludes the inference of more precise estimates; third, as no sequential measurements were performed, peak CMV DNAemia values for some patients may have been missed, and finally, some patients might have been miscategorized due to the intrinsic interassay coefficient of variation of the qPCR used in this study (estimated to be within 20-30% for low CMV DNA loads).

CONCLUSIONS

This preliminary study indicates that active CMV infection may occur in immunocompetent patients admitted for AHF, albeit at a low frequency and that the presence of CMV DNAemia was independently associated with a higher risk for the composite endpoint of death or readmission for AHF. Further studies are warranted in order to: 1) confirm the presence of active CMV infection in AHF, and 2) to determine whether active CMV infection plays a direct role in the progression of the disease or if it is merely an epiphenomenon linked to the severity of the inflammatory state. Should the data presented herein be confirmed, then patients displaying CMV DNAemia may constitute a subgroup of patients with AHF amenable to be managed aggressively or with specific therapeutic modalities such as immunomodulation, antiinflammatory therapies or even antiviral agents. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

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Clinical

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Clinical

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  aliquot and were regarded as positive if they yielded a reproducible CMV DNA load value. Measurements were performed by a technician completely blinded to patient diagnosis and evolution.

  Clinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162

Clinical

  Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162 A c c e p t e d M a n u s c r i p t

Clinical

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12 cytomegalovirus

 12 infection and guidance of pre-emptive therapy for allogeneic hematopoietic stem cell transplant recipients. J Clin Microbiol 46, 3311-8 13 Royston, P. and Sauerbrei, W. (2008) Multivariable Model-building: A Pragmatic Approach to Regression Analysis Based on Fractional Polynomials for Modelling Continuous Variables. Wiley-Blackwell, Chichester, UK 14 May, S. and Hosmer, D.W. (2004) Hosmer and Lemeshow type goodness-of-fit statistics for the Cox proportional hazards model. In Advances in Survival Analysis: Handbook of Statistics (Balakrishnana, N. and Rao, C.R., eds), Vol 23. pp. 383-394. Amsterdam. Elsevier, North-Holland. 15 Fildes, J.E., Shaw, S.M., Yonan, N. and Williams, S.G. (2009) The immune system and chronic heart failure: is the heart in control? J Am Coll Cardiol 53, 1013-20 16 Miettinen, K.H., Lassus, J., Harjola, V.P., Siirilä-Waris, K., Melin, J., Punnonen, K.R., Nieminen, M.S., Laakso, M. and Peuhkurinen, K.J. (2008) Prognostic role of pro-and antiinflammatory cytokines and their polymorphisms in acute decompensated heart failure. Eur J Heart Fail 10, 396-403 17 Chen, D., Assad-Kottner, C., Orrego, C. and Torre-Amione, G. (2008) Cytokines and acute heart failure. Crit Care Med 36, S9-16 18 Kalil, A.C. and Florescu, D.F. (2009) Prevalence and mortality associated with cytomegalovirus infection in nonimmunosuppresed patients in the intensive care unit. Crit Care Med 37, 2350-8 19 Limaye, A.P., Kirby, K.A., Rubenfeld, G.D., Leisenring, W.M., Bulger, E.M., Neff, M.J., Gibran, N.S., Huang, M.L., Santo Hayes, T.K., Corey, L. and Boeckh, M. (2008) Cytomegalovirus reactivation in critically ill immunocompetent patients. JAMA 300, 413-22 20 Osawa, R. and Singh, N. (2009) Cytomegalovirus infection in critically ill patients: a systematic review. Crit Care 13, R68 21 Söderberg-Nauclér, C. (2006) Does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer? J Intern Med 259, 219-46 22 Varani, S., Frascaroli, G., Landini, M.P. and Söderberg-Nauclér, C. (2009) Human cytomegalovirus targets different subsets of antigen-presenting cells with pathological consequences for host immunity: implications for immunosuppression, chronic inflammation and autoimmunity. Rev Med Virol 19, 131-45 23 von Muller, L., Klemm, A., Durmus, N., Weiss, M., Suger-Wiedeck, H., Schneider, M., Hampl, W. and Mertens, T. (2007) Cellular immunity and active cytomegalovirus infection in patients with septic shock. J Infect Dis 196, 288-95 24 Vornhagen, R., Plachter, B., Hinderer, W., The, T.H., Van Zanten, J., Matter, L., Schmidt, C.A., Sonneborn, H.H. and Jahn, G. (1994) Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens. J Clin Microbiol 32, 981-86 25 Lin, L., Kim, S.C., Wang, Y., Gupta, S., Davis, B., Simon, S.I., Torre-Amione, G. and Knowlton, A.A. (2007) HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis. HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis. Am J Physiol Heart Circ Physiol 293, H2238-47 26 Kim, S.C., Stice, J.P., Chen, L., Jung, J.S., Gupta, S., Wang, Y., Baumgarten, G., Trial, J. and Knowlton, A.A. (2009) Extracellular heat shock protein 60, cardiac myocytes, and apoptosis. Circ Res 105, 1186-95 27 Bason, C., Corrocher, R., Lunardi, C., Puccetti, P., Olivieri, O., Girelli, D., Navone, R., Beri, R., Millo, E., Margonato, A., Martinelli, N. and Puccetti, A. (2003) Interaction of Clinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  antibodies against cytomegalovirus with heat-shock protein 60 in pathogenesis of atherosclerosis. Lancet 362, 1971-7 Clinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162

Figure 1

 1 Figure 1 Serum levels of cytokines stratified by CMV DNAemia IL-1 : interleukin 1 beta; IL-6: interleukin 6; IL-10: interleukin 10; TNF-: tumor necrosis factor alpha; TNF-/IL-10: tumor necrosis factor alpha/interleukin 10 ratio; IL-6/IL-10 ratio: interleukin 6/interleukin 10 ratio

Table 1

 1 Baseline characteristics stratified by CMV statusLicenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited Clinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162

		Clinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162
		CMV	No CMV
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162	DNAemia n=11 (8.3%) Demographic and medical history DNAemia n=121 (91.7%) 77 (54-79) 76 (67-81) Gender (male), n (%) Age, years 8 (72.7) 60 (49.6) Previous admission for AHF, n (%) 7 (63.6) 63 (52.1) Hypertension, n (%) 8 (72.7) 98 (81) Dyslipidemia, n (%) 6 (54.5) 63 (52.1) Current smoker, n (%) 1 (9.1) 9 (7.4) Diabetes Mellitus, n (%) 8 (72.7) 57 (47.1) Ischemic heart disease, n (%) 7 (63.6) 45 (37.2) Valvular heart disease, n (%) 2 (18.2) 40 (33.1) De novo HF 4 (36.4) 51 (42.1) ADHF, n (%) 9 (81.8) 88 (72.7) Acute pulmonary edema, n (%) 1 (9.1) 24 (19.8) NYHA class III/IV, n (%) § 1 (9.1) 15 (12.4) COPD, n (%) 1 (9.1) 16 (13.2) Stroke, n (%) 0 14 (11.6) PAD, n (%) 1 (9.1) 5 (4.1) Radiological pleural effusion, n (%) 6 (54.5) 53 (43.8) Peripheral edema, n (%) 7 (63.6) 77 (63.6) A c c e p t e d M a n u s c r i p t p-value 0.705 0.141 0.462 0.509 0.875 0.843 0.104 0.086 0.310 0.709 0.513 0.384 0.748 0.695 0.233 0.450 0.493 1.000

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Table 2

 2 Characteristics and prognosis of patients with CMV DNAemiaClinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162

	Clinical features

Table 3

 3 Hazard ratios for the composite endpoint (all-cause mortality or readmission for acute heart failure) attributable to the presence of CMV infection *Final multivariate Cox model adjusted by previous admission for AHF, systolic blood pressure on admission, Charlson comorbidity index, serum sodium, serum NTproBNP, and serum CA125. CMV: cytomegalovirus; AHF: acute heart failure; NT-proBNP: NT-pro brain natriuretic peptide; CA125: antigen carbohydrate 125 Clinical Science Immediate Publication. Published on 24 Jun 2010 as manuscript CS20100162 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100162

		HR (CI 95%)	p-value
	Unadjusted
	Detectable CMV DNAemia	4.40 (2.11-9.19)	<0.001
	No CMV DNAemia	1	-
	CMV DNAemia 20 copies/ml	2.9 (1.12-7.30)	0.028
	CMV DNAemia > 20 copies/ml	16.3 (5.26-50.28)	<0.001
	Adjusted*
	Detectable CMV DNAemia	4.39 (2.02-9.52)	<0.001
	No CMV DNAemia	1	-
	CMV DNAemia 20 copies/ml	2.93 (1.11-7.71)	0.030
	CMV DNAemia > 20 copies/ml	13.6 (3.98-46.6)	<0.001

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgments

This study was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, RED HERACLES RD06/0009/1001 (Madrid, Spain). Julio Núñez had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.


INTRODUCTION

The European Water Framework Directive requires a monitoring of pesticide contamination in surface water. In France, 15 million analyses have been collected since 1997. These data are hard to interpret, partly because the surveillance is carried out under multiple monitoring networks. Each network has its own purpose (general knowledge of water quality, control of drinking water standards, or specific knowledge of pesticide contamination) and area of concern, leading to different monitoring strategies in terms of sampling date, location and rate.

A study has been carried out to find suitable methods to assess the current situation and trends of the contamination and to highlight the link between pesticide pressure and water contamination.

METHODS OF ASSESSING CONTAMINATION

Geographic grouping

Analyses from a single sampling site are not often sufficient to assess a trend in contamination. Moreover, samplings date and analyzed pesticides vary from a site to another. To get round these difficulties, we tested a method of interpretation by gathering data on a geographic basis. We assumed that pesticide transfers, and as a consequence temporal patterns of concentration in water courses, are similar within regions that have homogeneous topography, climate, soil type, bedrock and water flow. We used the hydro-ecoregions (HER), that were defined to implement the European Water Framework Directive, which requires a typology of aquatic ecosystems based on geographical features in order to define reference conditions (Figure 1).

Descriptors

Each analysis is described by its sampling location, date and the substance analysed. The result is either the molecule concentration, the limit of detection (LOD) if the molecule was not detected, or the limit of quantification (LOQ) if traces of the molecule were detected but not quantified. A relevant descriptor is the 90 th percentile of the results (LOD are replaced by 0 and LOQs by LOQ/2) twice per month (the first 15 days and the rest of the month, thus years can be compared with each other or accumulated) (see Figure 2).

We also used the percentage of concentrations above 0.1 µg/L against all the analyses. These two descriptors are statistically correlated.

Link with application periods, marketing authorisations and land use

Seasonal variations appear to be similar from one year to another. For example, isoproturon, a herbicide mostly used for cereals, shows two periods of contamination per year (Figure 2): the first one at the end of the winter and the second one in autumn.

They fit with the application periods of this pesticide: at the vegetation regrowth and at the winter cereals' sowing. A Principal Component Analysis (PCA) was conducted on the percentages of the concentrations above 0.1 µg/L per substance and HER. It showed that substances applied mostly on vineyards, such as norflurazon, procymidone and dimethomorph, are mostly detected in HER 15 (Plain of the Saône), more specifically in the Beaujolais area. Similarly, substances used on beets (metamitron and chloridazon) or potatoes (prosulfocarb) are detected in the north: HER 20 (sand-clay deposits) and 9 (limestone tables). Substances used on corn and/or sunflower (acetochlor, dimethenamid, metolachlor and alachlor) are detected in HER 14 (Hillsides of Aquitaine). Contamination of water, as estimated with this method, is therefore consistent with land use, but there are regions particularly contaminated, as for example the plain of the Saône. Is it because larger amounts of products are applied there or because their environment is more vulnerable?

This way of describing contamination is interesting because it shows patterns which are not always visible at the scale of a stand alone sampling site, and which are consistent with application periods, marketing authorisations and land use. It also highlight regions with comparatively high contamination. We then used pesticide sold quantities data to pursue the study of the link between pesticide pressure and water contamination.

LINK BETWEEN PESTICIDE PRESSURE AND WATER CONTAMINATION

Database of pesticide sales

Since 2008, pesticide distributors have had to pay a tax on non-point pollution. Thus sellers must declare the sold quantities of each product for agricultural or nonagricultural uses. At request of the ministry in charge of the environment, Onema (the French national agency for water and aquatic environments) built a tool to access these data for each plant protection product or for each substance.

This leads to a valuable database to assess the quantities of each pesticide applied.

However are uncertainties about where the products are used remain. Indeed, some companies have point of sales in much of France, or sell products on the Internet, but declare at their head office the overall quantity sold by the firm.

Selling periods versus contamination periods

In 2009, sales were declared from January to June and from July to December. This provides information about selling periods. Figure 3 shows a relation between the ratio of contamination from April to September on contamination from October to March and the ratio between quantities sold from January to June and quantities sold along the year. That is, the substances mostly sold during the first part of year are mostly detected in surface water in the middle of the year, and vice versa.

Sold quantities versus contamination levels

For this study, we did not group the data within HER but within units based both on HER and land use (31 units). We allocated the quantities sold by a firm to the unit where the city of the firm is located.

First, we studied the relation between sold quantities and contamination levels for some pesticides, for example glyphosate (cf Figure 4). Contamination in a geographic unit is related to the amount of glyphosate sold, but 2 regions have a comparatively high contamination: Paris area and Flanders. This may be due to a higher vulnerability of surface water in these regions (in particular, large surfaces are waterproof in Paris area) or to the uncertainties on the application locations. Then, we studied pesticide transfer for each geographic unit, for example the Cretaceous chalk of Paris Basin. We can see on Figure 5 that, similarly, there is a relation between sold quantities and contamination, but that some substances are comparatively more detected in surface water in this region: aminotriazole, diuron, nicosulfuron and metaldehyde. We can assumed that these substances have a higher transfer ability or are applied in conditions that favour transfers towards surface water.

Conclusion

Methods of interpretation of pesticide contamination monitoring data by grouping data on a geographic basis are interesting to describe contamination and link it to pesticide applications (dates, regions and quantities). Comparison with quantities sold highlights substances and regions with comparatively high contamination, and further studies are necessary to interpret these results.

  Figure 1: Map of French HER

Figure

  Figure 2: 90 th percentile of analysis results per 15 days, for isoproturon, in the armorican HER (12) This method also highlights trends between years. For example, Atrazine was detected above all between May and September. Its contamination level decreased steadily until 2003, and sharply between 2003 and 2004. This may be due to the reduction in the applied amounts, as local ban on atrazine use have been implemented since 1997, complete ban on sale at the end of 2002 and complete ban on use at the end of 2003.

Figure 3 :

 3 Figure 3: Period of contamination according to period of sales per pesticide and per unit in 2009

Figure 4 :

 4 Figure 4: Percentage of concentrations of glyphosate above 0.1 µg/L according to sold quantities, per geographic unit and per year (2008 or 2009)

  Figure 5

2,4-MCPA, 2009 Diuron, 2009 Nicosulfuron, 2009 2,4-MCPA, 2008 Nicosulfuron, 2008 Isoproturon, 2009 Chlortoluron, 2009 Diuron, 2008 Isoproturon, 2008 Chlortoluron, 2008 Aminotriazole, 2009 Metaldehyde, 2008 Aminotriazole, 2008 Metaldehyde, 2009 Glyphosate, 2008 Glyphosate, 2009


Fabien Souris

Jules Grucker

Jacques Dupont-Roc

Philippe Jacquier

Observation of metastable hcp solid helium

Keywords: 67, 80, -s -Quantum crystals 64, 60, My -Metastable phases 67, 25, dt -Sound in 4 He

published or not. The documents may come

Crystalline solids can be brought into metastable state with respect to fusion if surface melting is avoided. Experimentally this is achieved by heating small metal or dielectric crystals embedded in stronger matrices beyond the bulk melting temperature [1,2]. Metals are the most frequently studied materials. Fast heating of bulk solids by subpicosecond laser pulses has also provided evidence of such metastable states [3,4]. Numerous computer simulations have been used to unveil mechanisms limiting the metastability domain [2,[5][6][7][8].

Solid helium offers novel opportunities. At low temperatures (0 < T < 1.4 K), hcp solid helium-4 only exists for pressures higher than P m (T ) > ∼ 25 bar. The melting line is nearly horizontal in the T -P plane, in contrast with other simple solids where it is nearly vertical. In this unique situation, pressure is the efficient control parameter to explore the metastability region below the melting line. In addition, solid helium offers the peculiarity that quantum zero point motion plays a more important role than thermal motion. For melting, it does not obey the empirical Lindemann criterion [9] 1 . Metastable expanded solid helium-4 has also been considered in the past in view of achieving the Andreev-Lifshitz-Thouless scenario for supersolidity [11,12] by lowering the vacancy energy [13]. Although this scenario is now considered less relevant [14,15], (a) E-mail: grucker@lkb.ens.fr 1 For hcp solid helium √ < u2 >/a ∼ 0.26 at melting instead of 0.1. See [10] the interstitial and vacancy energies in a stretched lattice has been computed [14], and a limit of the metastable region has been proposed to lie 35 bar below the melting line [16]. None of these prospects ever came to an experimental realization 2 and a presumably large domain for the solid metastable phase remains unexplored.

In this letter, we provide full experimental evidence that metastable solid helium-4 can be obtained well below P m (T ) by a focused pressure wave inside bulk solid. This technique was used to study the liquid phase of helium in a metastable state with respect to its vapor or to the solid phase [18,19]. For the hcp lattice of solid helium-4, the sound velocity is anisotropic. A pressure wave front originating from a point source is not a sphere, but a prolate surface along the c-axis [20]. Hence hemispherical transducers do not produce focused sound waves in that case. Decompression of hcp solid helium with an hemispherical transducer has actually been tried with only a modest success [21]. Here we use an appropriate shaping of the ultrasound emitter (piezo-electric transducer PZT) to compensate for the sound velocity anisotropy 3 . The den-sity modulation, below and above the coexistence density, is accurately measured using an interferometric imaging technique. The experimental cell containing solid helium is cooled by a helium-4 cryostat with four optical ports. The temperature can be regulated from 1.1 K to 2.1 K. Internal pressure is measured by a capacitive gauge [22] calibrated along the liquid-solid coexistence line against an external pressure gauge 4 . The symmetry axis of the PZT is vertical and is taken as z-axis (Fig. 1). The orientation of solid helium should be such that its symmetry axis (perpendicular to the c-facet) coincide with the z-axis. Helium density modulations are monitored via the refractive index using a Jamin interferometer (Fig. 1). One of its arms passes through the acoustic focal region and is taken as the y-axis, while the other one crosses the cell in an unperturbed zone. The focal region is imaged onto a CCD camera using an appropriate lens. The imaged surface is about 4 × 2.4 mm 2 . The measurement technique has been described elsewhere [23] and may be summarized as follows: optical phase shifts ϕ(x, z) induced by density modulations are extracted from several images taken with different phases of the reference beam. This process is then reproduced for different values of the delay t between the sound pulse emission and the imaging laser pulse to construct a phase map movie. Because of the rotational invariance of the sound field around the z-axis, the variation of the refractive index δn depends only on the coordinate z and on the distance r to the z-axis. The optical phase map is then the integral along the y-axis of δn(r, z), i.e. its Abel transform. An inverse Abel transform [24] of ϕ(x, z) provides the radial profile δn(r, z). Density maps can be computed from δρ(r, z) = ρ 0 δn(r, z)/(n 0 -1) where ρ 0 and n 0 are the unperturbed density and refractive index of the solid. This method of determining sound fields has been quantitatively tested in water by comparison with an optical fiber hydrophone [23]. For an easier appreciation of the modulation achieved, conversion to pressure is [25]. Solid dots are experimental data provided by the inner pressure gauge. In this example, δP0 = 0.54 bar.

CCD

To obtain an oriented and homogeneously pressurized crystal at (T 0 , P 0 ), a crystal seed with its c-facet horizontal is first grown at a temperature below the roughening transition of the c-facet (1.3 K) [28]. The horizontality of the facet is controlled optically in both x and y directions with 1 • accuracy. Filling the cell with the crystal and pressurizing it from P m (T 0 ) to P 0 at constant temperature T 0 would be harmful for crystal quality. Indeed, because helium is a very compressible solid, material would have to be brought into the cell including into the PZT cup. This would result in plastic deformation of the crystal, possibly breaking it into several differently oriented pieces. Pressure gradients could also be created. Instead we follow the procedure shown in Fig. 2. The cell with the seed in equilibrium with the liquid is warmed up to some temperature T C chosen so that P m (T C ) > P 0 > P m (T 0 ). This warming along the melting line is done slowly enough to keep a quasi-static equilibrium between the pressure in the filling line and the melting pressure at the cell temperature. The crystal is then grown at T C until the cell is filled and the filling capillary is blocked by the solid phase. Eventually, the whole cell is slowly cooled at constant volume down to (T 0 , P 0 ) in a little more than two hours. This slow isochoric cooling is expected to avoid crystal breaking, as well as temperature and pressure gradients. For an easier appreciation of the wave field around the focus, the map is supplemented on the left of the axis by a mere symmetry.

The experiment then runs as follows. A four-oscillation resonant RF pulse (frequency 1.25 MHz, amplitude V tunable from 10 to about 250 V) is applied to the PZT and generates a convergent pressure wave train in the crystal. Phase maps are recorded for delays t ranging from 10 µs to 20 µs with 0.05 µs time steps. Radial refractive index maps δn(r, z, t) are obtained from them. Such a map is shown in Fig. 3. From those maps, one can find the acoustic focus z f as the location where δn(0, z, t) has its maximum amplitude. In Fig. 4, the refractive index variation at acoustic focus δn(0, z f , t) is plotted versus time. The pressure scale of the right axis is computed using equation (1). The pressure reaches its minimum at t min = 15.53 µs and its variation from the static pressure P 0 is δP min = -2.80 bar ±0.12 bar. The error bar is computed from the r.m.s. noise on δn and corresponds to one standard deviation. The initial offset pressure being δP 0 = 0.87 bar, one concludes that the solid is decompressed under its melting pressure by ∆P = δP min + δP 0 = -1.93 ± 0.12 bar. In another run with δP 0 = 0.23 bar, the value ∆P = -2.2 ± 0.05 bar was obtained.

Does helium stay in the solid phase? If a liquid bubble had nucleated at the focus, it would have expanded for about a quarter of the sound period at a velocity close to the sound velocity. Thus it would have reached a diameter of the order of 100 µm. Taking into account the refractive index difference between liquid and solid phases (about 0.0035), the liquid bubble would have created a phase shift as large as 4 radians on a size larger than the 20 µm image resolution (to be compared to maximum phase shifts of about 1 radian induced by the pressure wave). More- For the last point on each run, a defect appears at focus (see inset), which is also responsible for the larger noise. The crystal stability limit in ∆P is between the two dash-dotted lines.

Is the crystal modified by the pressure wave? Recent experiments [29,30] show that creation and motion of dislocations are involved in the response of solid helium-4 even to very low shear strain (ǫ ≈ 10 -8 ). The temperature used here (1.1 K) is significantly higher than the temperature where the dislocations become free to move. Hence, it is likely that with the strain produced in this experiment (typically 2×10 -4 at the PZT's surface at 200 V, 4×10 -3 at focus) the solid becomes full of dislocations. These dislocations can organize themselves into grain boundaries, leading to a subdivision of the crystal into slightly disoriented crystallites [START_REF] Friedel | Dislocations[END_REF]. We looked for such a crystal modification in two ways. First, we have performed a set of experiments where the driving voltage (V ) is gradually increased from a reference voltage V ref = 60 V up to 220 V. In between each different driving voltage acquisition, a test acquisition at the reference voltage was taken. We then compared the successive test phase maps to the reference map and found them quite similar. For instance, the minimum depressions ∆P for the reference voltage are within 20% as shown in Fig. 5 (open circles). For comparison, a misaligned crystal with its c-axis at 20 • from the PZT axis or a solid made of a few crystals give very different density maps. As a second test, after sonication at 100 V, the crystal was melted carefully at its coexistence pressure, reversing the procedure used for its growth. The liquid/solid interface reveals the existence of grain boundaries around the focal region. The conclusion is that the local disorientation of the crystal is not sufficient to modify significantly the pressure wave propagation.

In the same series of measurements, we looked for the maximum achievable depression. It was found that the crystals break when V exceeds a threshold lying between 185 V and 220 V. An extended defect with a different density appears at the focal point (see inset of Fig. 5). The corresponding value ∆P is between -1.93 and -2.40 bar. This threshold zone is determined as follows. For a given run at a given temperature, its upper limit is the lowest pressure achieved without breaking the crystal. To estimate its lower limit, we extrapolate ∆P from the last two points where the crystal remains stable up to the voltage where the crystal is broken. For the two runs of Fig. 5, the given threshold zone is the union of the corresponding threshold zones.

Once defects are created in the crystal, they disappear in a few seconds after the sound pulse. However, subsequent sound focusing is degraded, indicating a substantial crystal reorganization in the central region. Hence to carry out a new experiment, the crystal has to be melted again and a new one regrown using the procedure shown in Fig. 2. Clearly the limit encountered here is not the limit of the solid phase discussed in ref. [16] which is expected at much lower depression (-35 bar). In order to get some hints about its origin, 24 different runs were performed varying the temperature, the initial pressure offset δP 0 , the repetition rate of sound pulses. Each run was made with a new crystal. It appears that ∆P somewhat depends on the crystal, although they are grown in similar conditions. Actually, ∆P may change by a factor 2 from one sample to another. It was found that ∆P is sensitive to the emission rate of sound pulses received by the crystal. A smaller rate tends to promote larger ∆P . Surprisingly, we did not find a strong correlation between δP 0 and ∆P . Finally, ∆P does not depend much on temperature from 1.1 K to 1.4 K as shown in Fig. 6. It represents the lowest thresholds achieved for five temperatures in this temperature range. From this graph, it seems unlikely that the instability is related to the hcp/bcc coexistence line or its extrapolation in the metastable region. Note that these data were taken at slower sound pulse rates than the data of Fig. 5. This may explain the lower minimum pressures achieved.

A further indication appears in Fig. 5. The values of ∆P do not scale linearly with V above V = 60 V. This reveals some non linear process in the production or the propagation of the pressure wave, possibly dislocation creation or motion. The initial dislocation density may most probably vary from one crystal to another; their proliferation might depend on the number of strong sound pulses sent through the sample. If confirmed, this would appear as a technical limitation of the ultrasound technique to achieve larger depressions using a bowl shaped PZT. One can nevertheless try to double ∆P by using two such PZTs, assembled face to face, to make a nearly complete wave surface.

To summarize, we have demonstrated the production of metastable solid helium between 1.1 K and 1.4 K using pressure as a control parameter. For amplitudes up to -2.0 bar below the melting line, the ultrasound technique used does not seem to alter drastically the crystal. An unexpected instability of the solid phase was found. Nevertheless, a metastable region for hcp solid helium-4 is now accessible where crystal properties can be investigated. Among these, bulk compressibility can be addressed through Brillouin scattering. Vacancy energy will be more difficult to measure, but no less interesting.

Fig. 1 :

 1 Fig.1:A convergent pressure wave is produced in hcp solid helium at 1.1 K by the excitation of a well shaped piezotransducer (PZT). The density modulations near focus are monitored using a Jamin interferometer. The lens L images the focal region onto the CCD camera

Fig. 2 :

 2 Fig. 2: Experimental path in helium-4 phase diagram to obtain a crystal at T0 and P0. A → B: liquid pressurization at constant T . B: nucleation of an oriented crystal seed. B → C: warming of the crystal seed in equilibrium with the liquid up to TC ∼ 1.4 K. C: growth of the crystal until the whole cell is filled. C → D(T0, P0): isochoric cooling. Coexistence curves are taken from Grilly[25]. Solid dots are experimental data provided by the inner pressure gauge. In this example, δP0 = 0.54 bar.

Fig. 3 :

 3 Fig. 3: Refractive index map δn(r, z) of a convergent pressure wave in hcp helium single crystal. PZT voltage is V = 185 V and propagation time from the PZT surface is t = 15.53 µs.For an easier appreciation of the wave field around the focus, the map is supplemented on the left of the axis by a mere symmetry.

Fig. 4 :Fig. 5 :

 45 Fig. 4: Time evolution of refractive index and pressure variations at focus for V = 185 V. Maximum transient depression (δPmin = -2.8 bar ±0.2 bar) is obtained at tmin = 15.53 µs and is clearly below the static melting line indicated by the dotted line at -δP0 = -0.87 bar.

Fig. 6 :

 6 Fig.6: Threshold zones (crystal stability limit, see text) at different temperatures between 1.1 K and 1.4 K

Attempts to obtain metastable solid helium at much higher pressures and temperatures using temperature as the control parameter are reported in[17]

3 A cylinder of bulk ceramic PZT401 from Morgan Electroceramics was grinded on a CNC lathe prior to metallization and poling. The inner surface matches half the wave surface of the quasilongitudinal sound mode. The outer surface is parallel to the inner one providing a constant thickness of 2 mm. The equatorial part is then cut away over 1 mm in order to make the focal region visible.

Keller PAA-35/30bar/80797 made using:δP (r, z) = χ δn(r, z) n 0 -1(1)where χ = 269 bar[25] is the bulk modulus

and n 0 = 1.036[27]. This formula is the linear approximation of the equation of state, valid up to δP ≃ 5 bar with an error less than a few percent.When producing solid under depression, nucleation of the liquid phase on the PZT's surface must be avoided. Let δP P ZT (t) be the acoustic pressure at the PZT surface, T 0 the temperature of the cell and P m (T 0 ) the static melting pressure. We note P 0 the uniform pressure in the cell and δP 0 = P 0 -P m (T 0 ) its initial offset from the melting line (Fig.2). P 0 is chosen so that the condition δP P ZT (t) + δP 0 > 0 is always fulfilled, even at the troughs of the pressure wave.

The use of formula(1) is justified because solid helium compressibility is nearly isotropic (c 11 + c 12 = c 33 + c 13 ). See[26]

We thank S. Balibar and F. Caupin for their help and the technical staff of the Laboratoire Kastler Brossel p-4 Observation of metastable hcp solid helium. and of the Département de Physique de l' École Normale Supérieure for a strong support. This research has been supported by ANR grant-05-BLAN-0084-01.