Skip Navigation

European Journal of Heart Failure 2003 5(1):27-31; doi:10.1016/S1388-9842(02)00165-4
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Sirera, R.
Right arrow Articles by Rivera, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sirera, R.
Right arrow Articles by Rivera, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2002 European Society of Cardiology

Quantification of proinflammatory cytokines in the urine of congestive heart failure patients. Its relationship with plasma levels

Rafael Sireraa, Antonio Salvadorb, Ildefonso Roldánb, Raquel Talensa, Andrés González-Molinaa and Miguel Riveraa,*

a Centro de Investigación Hospital La Fe, Av. de Campanar, 21, 46009 Valencia, Spain
b Servicio de Cardiologí Hospital Peset, Valencia, Spain

* Corresponding author. Tel.: +34-9638-627-97; fax: +34-9638-687-18 E-mail address: rivera_jmi{at}gva.es


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Aims: Proinflammatory cytokines are important mediators for the development of heart failure and increased plasma levels of these cytokines have been reported in patients with this condition. The purpose of the study was to investigate whether urine, a non-invasively obtained biological sample, was an appropriate medium in which to measure the concentration of tumor necrosis factor-alpha (TNF-{alpha}) and interleukin-6 (IL-6) in patients in the advanced stages of the disease.

Methods and results: Thirty consecutive patients who had severe congestive heart failure (NYHA classes III and IV) and 30 matched healthy control subjects were enrolled. Plasma and the first urine of the day were collected and TNF-{alpha} and IL-6 were quantitatively analyzed by enzyme-linked immunosorbent assays. For every subject there were no differences in the amount of cytokine determined in plasma and urine. Both urine and plasma levels of IL-6 and TNF-{alpha} were greater in heart failure patients than in controls.

Conclusion: Our results show that plasmatic and urinary levels of proinflammatory cytokines did not differ significantly. Thus, urine may be a good milieu in which to study these cytokines and may have diagnostic, prognostic and therapeutic implications.

Key Words: Congestive heart failure • Urine • Cytokines • Interleukin-6 • Tumor necrosis factor-alpha

Received March 5, 2002; Revised June 5, 2002; Accepted July 22, 2002


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The mechanisms that explain the progressive dysfunction of the failing myocardium remain largely unknown. An imbalance of immune mediators together with an inappropriate enhancement of proinflammatory cytokines or even reduction of anti-proinflammatory mediators have been recently recognized to play an important role in the pathophysiology of cardiac diseases, not only in acute disorders but also in chronic conditions such as congestive heart failure (CHF) [1,2]. From in vitro and in vivo studies, the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-{alpha}) have been proposed as important mediators in the development of heart failure [3,4] but their precise pathophysiological role is still unclear.

Cytokines are extremely pleiotropic proteins, synthesized and secreted after stimulation of different cell types, that exert their biological function through the interaction with specific cell membrane receptors in an autocrine or paracrine fashion. Activated cells present in cardiac tissue [5] such as macrophages, endothelial cells and myocytes are producers of TNF-{alpha}, and TNF-{alpha} receptors of type 1 and type 2 have been identified in cardiac myocytes [6]. In addition, TNF-{alpha} mediates a negative inotropic effect on the myocardium by several mechanisms [7] and apoptosis in cardiac myocytes [8] and also contributes to progressive left ventricular remodeling [9]. Recently, its implication in the increased oxidative stress [10] observed in patients with dilated cardiomyopathy or mild cardiac insufficiency has been demonstrated.

IL-6 is expressed in cells present in the cardiac parenchyma [11] such as activated macrophages and endothelial cells. An interesting point is that the site of production of both proinflammatory cytokines, IL-6 and TNF-{alpha}, in CHF patients does not seem to be restricted to cardiac tissue [12]. Despite the known pathophysiological actions of TNF-{alpha} and IL-6 in experimental models, its clinical importance in CHF is still unclear, though these cytokines have developed a potential significance. In fact, blood levels of these cytokines have been found to be moderately elevated in CHF patients [13]. Recent studies reveal that several treatments for cardiac diseases including angiotensin-converting enzyme inhibitors [14] and other experimental treatments, such as positive inotropic substances [15] or anti-proinflammatory cytokines [16], were downregulators of these mediators. Other drugs that act directly against the cytokines, as a TNF-{alpha} receptor analogue, are being tested in clinical trials [17].

Nothing is known about cytokine levels in the urine of patients with CHF despite the interest in their role as potential mediators of disease progression and even though the urine levels of cytokines have demonstrated a high diagnostic specificity in other pathologies [18] and the sample is non-invasively obtained. Therefore, the aim of this study was to determine the urinary excretion of IL-6 and TNF-{alpha} in patients with severe CHF and healthy subjects and to compare them with the plasma levels, in order to explore if the observed plasma alteration of proinflammatory cytokines is also evident in urine.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patients
Sixty subjects were included in this study. Thirty of them were consecutive patients who had severe heart failure established on the basis of medical history, electrocardiogram, chest X-ray and echo-Doppler study [19]. The cause of heart failure was dilated cardiomyopathy (n=11), ischemic cardiomyopathy (n=9), hypertension (n=5) and valvular heart disease (n=5). The patients were functionally defined according to New York Heart Association in classes III (15 subjects) and IV (15 subjects) and were receiving standard medical treatment following the guidelines of the American Heart Association [20] and the European Society of Cardiology [21]. Gender distribution was 20 males and 10 females and the mean age 67±11 years. Subjects with acute coronary syndromes and those with acute and chronic liver disease, rheumatoid arthritis, chronic infections, pulmonary diseases, malignancy or urinary tract pathologies were excluded. All patients were on stable medical therapy for at least 1 month before enrollment in the study and none were receiving anti-inflammatory drugs. The other 30 subjects were healthy controls matched in age and gender and without any history of cardiac disease, hypertension, diabetes mellitus or hyperlipidemia. The Investigation Review Boards of our Institutions approved the study protocol and all participants gave written consent before entering the study.

2.2. Blood and urine samples
As there is a high correlation between 24-h cytokine concentrations and spot samples [22], patients and controls were asked to collect the first urine of the day. The same morning, in a fasted state, peripheral blood was collected by venipuncture into pyrogen-free vacuum tubes containing EDTA as anticoagulant. Urine and anticoagulated blood tubes were immediately centrifuged (15 min at 400xg) and the sediment-free urine and plasma samples were obtained. Both plasma and urine samples were aliquoted and stored at –80 °C until further analysis and thereafter were only thawed once.

2.3. Cytokine measurement
Plasma and urine concentrations of IL-6 and TNF-{alpha} were measured in duplicate by specific commercial sandwich enzyme-linked immunosorbent assays (ELISA) following manufacturer's recommendations (Quantikine, R&D systems Inc, Minneapolis, MN). The tests were quantified at 450 nm in a dual wavelength microplate reader (Sunrise, TECAN, Austria) with the help of Magellan software (version 2.5 TECAN, Austria). The ELISAs were suitable for the quantitative determinations of cytokines in both plasma and urine and analyte concentrations found in samples were in the range of linearity of each assay. Results were expressed as pg/ml.

2.4. Statistical analysis
SPSS 7.0 package (SPSS Inc, Illinois, USA) was used for statistical analysis. Student's t-test assuming equal variances was used to assess differences in continuous variables for unpaired data (patients and controls) and the paired data (plasma vs. urine determinations). Data is presented as the mean±S.E.M. (standard error of the mean). A P value lower than 0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Plasma and urine concentrations of IL-6
As can be seen in Fig. 1, in CHF patients the mean±S.E.M. plasma concentration of IL-6 was 28.6±5.4 pg/ml while in the control group it was 8.2±2.1. This difference was statistically significant (P<0.001). In the same figure the mean urine values of IL-6 in the CHF group and in the control group, being 24.0±3.8 and 8.1±0.8, respectively, are shown. In urine, the elevation observed in CHF patients, when compared with healthy controls, was also significant (P<0.001).


Figure 1
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Mean±S.E.M. plasma and urine concentrations of IL-6 in CHF patients (n=30) and healthy controls (n=30). **P<0.001.

 
When we compared for every subject and in every group the plasma and the urine concentrations of IL-6, we did not find any statistically significant differences.

3.2. Plasma and urine concentrations of TNF-{alpha}
Fig. 2 shows the mean±S.E.M. values of TNF-{alpha} in plasma and urine samples for both groups. In CHF patients we found that plasma and urine concentrations of TNF-{alpha} were 5.1±1.5 and 3.9±1.3, respectively. In the control group plasma and urine levels of TNF-{alpha} were 2.0±0.5 and 1.8±0.3, respectively. When we compared with normal subjects we found that the plasma TNF-{alpha} elevation observed in CHF patients was significant (P<0.05) while the urine elevation seen did not reach statistical significance.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Mean±S.E.M. plasma and urine concentrations of TNF-{alpha} in CHF patients (n=30) and healthy controls (n=30). *P<0.05.

 
For every subject and in every group plasma and urine concentrations of this cytokine did not differ significantly.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
CHF is characterized by complicated cardiorenal, hemodynamic and neurohormonal alterations [23]. In an attempt to delineate a biochemical mechanism for the cachexia that occurs in patients with advanced heart failure, it was discovered that patients with advanced heart failure expressed elevated levels of TNF-{alpha} in their peripheral circulation [24]. In the ensuing years since this observation, there has been increasing evidence that TNF-{alpha} might also directly contribute to the progression of heart failure [25,26] by virtue of the direct toxic effects that this molecule exerts on both the heart [27] and the circulation [28].

Recent studies suggest that IL-6 may be physiopathologically linked to impaired function in advanced heart failure by influencing critical determinants of cardiac performance systemically and by direct effects on the heart [29]. One of the signals that up-regulates IL-6 synthesis is TNF-{alpha}, which has been implicated in the development of ventricular hypertrophy and depression of myocardial contractility through stimulation of its receptor which is expressed in cardiac myocytes [30].

Our study's objective was to analyze the urinary excretion and the plasma levels of proinflammatory cytokines in patients with severe CHF, in order to confirm if the previously described enhanced plasma concentrations of IL-6 and TNF-{alpha} were also detectable in urine. As cytokines are glycoproteins of relatively low molecular mass, rarely more than 20 kDa, and cationic molecules smaller than this molecular mass are free filtered through glomeruli as easily as water [31], we have speculated that proinflammatory cytokines could be present in the urine of patients without urinary tract pathologies. In the present study, we have demonstrated for the first time that proinflammatory cytokines could be measured by commercial immunoassay in the urine of CHF patients, and that their concentrations are significantly greater than those obtained from healthy controls.

Our results therefore confirm the findings from several other reports demonstrating elevated levels of proinflammatory cytokines in the plasma of CHF patients [32,33]. We have shown a significant increase in the plasma levels of IL-6 and TNF-{alpha} in patients with heart failure in NYHA classes III and IV. The same findings can be observed in the urine of these patients when compared with healthy matched controls, but only IL-6 elevation in the urine of CHF patients attains statistical significance. Several reasons may explain why TNF-{alpha} levels in the urine of heart failure patients were elevated, like IL-6, but were not statistically significant. It is possible that high levels of interfering factors could cause unusual results in some cases. In fact, the presence of soluble receptors for these cytokines, usually present in biological samples, could interfere with the measurement of their ligands in the samples. Another drawback of this study was that an important number of determinations in urine were slightly above the minimum detectable concentration of TNF-{alpha}, around the limits of the test's precision, and this could affect the statistical significance.

A limitation of the study concerning the determinations of proinflammatory cytokines in urine is that these molecules are mainly produced and consumed locally in an autocrine or paracrine manner. If, as some authors have proposed, the failing myocardium is the principal synthesizer and receptor of proinflammatory cytokines [11,19,26], then the levels may not be as elevated in peripheral blood as in cardiac tissue and therefore the leakage into the urine could be so difficult to measure, that very sensitive analytical procedures would be needed. In this regard, it is worth mentioning that there are some contradictory data demonstrating that the elevation of cytokine concentration in the myocardial tissue is a late event, and it is preceded by elevated plasma levels of proinflammatory cytokines [34].

A common limitation with these kind of studies is that patients, as expected, are receiving conventional therapy for their disease, and it is known that several kinds of drugs can reduce the blood levels of these proinflammatory cytokines. In fact, angiotensin-converting enzyme inhibitors and glycosides have been shown to decrease plasma levels of both IL-6 and TNF-{alpha} because some of them are inhibitors of proinflammatory cytokine synthesis. However, this study confirms that a high degree of immune activation persists in heart failure patients even during standard therapy, and can be detected in plasma and urine. On the other hand, it is known that certain substances can diminish plasma levels of important pathophysiological mediators, such as myocardial steroids [35], while expression at the heart can still be elevated. This suggests that the relationship between tissue, plasma and urinary levels of proinflammatory cytokines, as well as the relationship between cytokine reduction in plasma and the efficacy of heart failure drugs requires further investigation.

In conclusion, this study expands on previous publications that have described elevated levels of IL-6 and TNF-{alpha} in heart failure, by demonstrating that urinary levels of proinflammatory cytokines increase in patients in relation to heart failure symptomatology, and that these levels are similar to those obtained in plasma. These findings are consistent with the point of view that overexpression of proinflammatory cytokines in patients with CHF and deteriorated functional status may be one of several different maladaptive biochemical mechanisms that change the patient status from asymptomatic ventricular dysfunction to symptomatic heart failure [23]. If confirmed, urine may be a good medium to study the cytokine imbalance observed in CHF patients. These new data may have diagnostic implications and can provide the basis for the development of new suitable methods with prognostic value that would need to be clarified in specifically designed studies and might also enable us to evaluate the efficacy of therapeutic interventions.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Mann D.L., Young J.B. Basic mechanisms in congestive heart failure: recognizing the role of proinflammatory cytokines. Chest (1994) 105:897–904.[Free Full Text]
  2. Matsumori A., Yamada T., Suzuki H., Matoba Y., Sasayama S. Increased levels of cytokines in patients with myocarditis and cardiomyopathy. Br. Heart J. (1994) 72:561–566.[Abstract/Free Full Text]
  3. Finkel M.S., Oddis C.V., Jacob T.D., Watkins S.C., Hattler B.G., Simmons R.L. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science (1992) 257:387–389.[Abstract/Free Full Text]
  4. Torre-Amione G., Kapadia S., Benedict C., Oral H., Young J.B., Mann D.L. Proinflammatory cytokine levels in patients with depressed left ventricular ejection fraction: a report from the studies of left ventricular dysfunction (SOLVD). J. Am. Coll. Cardiol. (1996) 27:1201–1206.[Abstract]
  5. Kapadia S., Lee J.R., Torre-Amione G., Birdsall H.H., Ma T.S., Mann D.L. Tumor necrosis factor gene and protein expression in adult feline myocardium after endotoxin administration. J. Clin. Invest. (1995) 96:1042–1052.[Web of Science][Medline]
  6. Torre-Amione G., Kapadia S., Lee J., Bies R.D., Levobitz R., Mann D.L. Expression and functional significance of tumor necrosis factor receptor in human myocardium. Circulation (1995) 92:1487–1493.[Abstract/Free Full Text]
  7. Gulick T.S., Chung M.K., Pieper S.J., Lange L.G., Schreiner G.F. Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte β-adrenergic responsiveness. Proc. Natl. Acad. Sci. USA (1989) 86:6753–6757.[Abstract/Free Full Text]
  8. Krown K.A., Page M.T., Nguyen C., et al. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. J. Clin. Invest. (1996) 98:2854–2865.[Web of Science][Medline]
  9. Boluyt M.O., O'Neill L., Meredith A.L., et al. Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure: marked upregulation of genes encoding extracellular matrix components. Circ. Res. (1994) 75:23–32.[Abstract/Free Full Text]
  10. Tsutamoto T., Wada A., Matsumoto T., et al. Relationship between tumor necrosis factor-alpha production and oxidative stress in the failing hearts of patients with dilated cardiomyopathy. J. Am. Coll. Cardiol. (2001) 37:2086–2092.[Abstract/Free Full Text]
  11. Neumann F.J., Ott I., Gawaz M., et al. Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. Circulation (1995) 92:748–755.[Abstract/Free Full Text]
  12. Petretta M., Condorelli G.L., Spinelli L., et al. Circulating levels of cytokines and their site of production in patients with mild to severe chronic heart failure. Am. Heart. J. (2000) 140:E28.[CrossRef][Medline]
  13. Meldrum D.R. Tumor necrosis factor in the heart. Am. J. Physiol. (1998) 274:577–595.
  14. Gullestad L., Aukrust P., Ueland T., et al. Effect of high-versus low-dose angiotensin converting enzyme inhibition on cytokine levels in chronic heart failure. J. Am. Coll. Cardiol. (1999) 34:2061–2067.[Abstract/Free Full Text]
  15. Matsumori A., Shioi T., Yamada T., Matsui S., Sasayama S. Vesnarione, a new inotropic agent, inhibits cytokine production by stimulated human blood from patients with heart failure. Circulation (1994) 89:955–958.[Abstract/Free Full Text]
  16. Li D., Zhao L., Liu M., et al. Kinetics of tumor necrosis factor alpha in plasma and the cardioprotective effect of a monoclonal antibody to tumor necrosis alpha in acute myocardial infarction. Am. Heart. J. (1999) 137:1145–1152.[CrossRef][Web of Science][Medline]
  17. Louis A., Cleland J.G., Crabbe S., et al. Clinical trials update: CAPRICORN, COPERNICUS, MIRACLE, STAF, RITZ-2, RECOVER and RENAISSANCE and cachexia and cholesterol in heart failure. Highlights of the Scientific Sessions of the American College of Cardiology, 2001. Eur. J. Heart Fail. (2001) 3:381–387.[Abstract/Free Full Text]
  18. Gormley S.M., McBride W.T., Armstrong M.A., et al. Plasma and urinary cytokine homeostasis and renal dysfunction during cardiac surgery. Anesthesiology (2000) 93:1210–1216.[Web of Science][Medline]
  19. European Society of Cardiology. Remme W.J., Swedberg K. Comprehensive guidelines for the diagnosis and treatment of chronic heart failure. Task force for the diagnosis and treatment of chronic heart failure of the European Society of Cardiology. Eur. J. Heart Fail. (2002) 4:11–22.[Free Full Text]
  20. Cohn J.N. The management of chronic heart failure. N. Engl. J. Med. (1996) 335:490–498.[Free Full Text]
  21. Remme W.J. Towards the better treatment of heart failure. Eur. Heart J. (1998) (Suppl_L):L36–42.
  22. Rivera S., Sattler F.R., Boyd H., Auffenberg T., Nakao S., Moldawer L.L. Urinary cytokines for assessing inflammation in HIV-wasting. Cytokine (2001) 13:305–313.[CrossRef][Web of Science][Medline]
  23. Cohn J.N., Levine T.B., Olivari M.T., et al. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N. Engl. J. Med. (1984) 311:819–823.[Abstract]
  24. Levine B., Kalman J., Mayer L., Fillit H.M., Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N. Engl. J. Med. (1990) 323:236–241.[Abstract]
  25. Hegewish S., Weh H.-J., Hossfeld D.K. TNF-induced cardiomiopathy. Lancet (1990) 2:294–295.[Web of Science]
  26. Maury C.P.J., Teppo A.-M. Circulating tumor necrosis factor-{alpha} (cachectin) in myocardial infarction. J. Intern. Med. (1989) 225:333–336.[Web of Science][Medline]
  27. Yokoyama T., Vaca L., Rossen R.D., Durante W., Hazarika P., Mann D.L. Cellular basis for the negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian heart. J. Clin. Invest. (1993) 92:2303–2312.[Web of Science][Medline]
  28. Munger M.A., Johnson B., Amber I.J., Callahan K.S., Gilbert E.M. Circulating concentrations of proinflammatory cytokines in mild or moderate heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am. J. Cardiol. (1996) 77:423–427.[CrossRef][Web of Science][Medline]
  29. Plenz G., Song Z.F., Tjan T.D.T., et al. Activation of the cardiac interleukin-6 system in advanced heart failure. Eur. J. Heart Fail. (2001) 3:415–421.[Abstract/Free Full Text]
  30. Kunisada K., Hirota H., Fujio Y., et al. Activation of JAK-STAT and MAP kinases by leukemia inhibitory factor through gp130 in cardiac myocytes. Circulation (1996) 94:2626–2632.[Abstract/Free Full Text]
  31. Dworkin L.D., Brenner B.M. Biophysical basis of glomerular filtration. In: The kidney: physiology and pathophysiology—Seldin D.W., Giebish G., eds. (1992) 2nd ed. New York: Raven Press.
  32. Tsutamoto T., Hisanaga T., Wada A., et al. Interleukin-6 spillover in the peripheral circulation increases with the severity of heart failure, and the high plasma level of interleukin-6 is an important prognostic predictor in patients with congestive heart failure. J. Am. Coll. Cardiol. (1998) 31:391–398.[Abstract/Free Full Text]
  33. Dutka D.P., Elborn J.S., Delamere F., Shale D.J., Morris G.K. Tumour necrosis factor alpha in severe congestive cardiac failure. Br. Heart J. (1993) 70:141–143.[Abstract/Free Full Text]
  34. Kubota T., Miyagishima M., Alvarez R.J., et al. Expression of proinflammatory cytokines in the failing human heart: comparison of recent-onset and end-stage congestive heart failure. J. Heart Lung Transplant (2000) 19:819–824.[CrossRef][Web of Science][Medline]
  35. Takeda Y., Yoneda T., Demura M., Miyamori I., Mabuchi H. Sodium-induced cardiac aldosterone synthesis causes cardiac hypertrophy. Endocrinology (2000) 141:1901–1904.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Eur J Heart FailHome page
R. Cortes, M. Portoles, A. Salvador, V. Bertomeu, F. G. de Burgos, L. Martinez-Dolz, E. R. Lleti, V. Climent, A. Jordan, R. Paya, et al.
Diagnostic and prognostic value of urine NT-proBNP levels in heart failure patients
Eur J Heart Fail, October 1, 2006; 8(6): 621 - 627.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Sirera, R.
Right arrow Articles by Rivera, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sirera, R.
Right arrow Articles by Rivera, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?