HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Amicosante, M. Articles by Saltini, C. Search for Related Content PubMed PubMed Citation Articles by Amicosante, M. Articles by Saltini, C.

HLA-DP-unrestricted TNF- release in beryllium-stimulated peripheral blood mononuclear cells

¹ Laboratory of Clinical Pathology, and ² Division of Respiratory Diseases of the University of "Tor Vergata", National Institute for Infectious Diseases (L. Spallanzani, IRCCS), Rome, ³ Division of Occupational Medicine, Policlinico, Modena, and ⁴ Division of Pulmonary Medicine, Policlinico, Modena, Italy. ⁵ Pulmonary and Intensive Care Medicine, Cleveland Clinic Foundation, Cleveland, OH, USA

CORRESPONDENCE: M. Amicosante, Laboratory of Clinical Pathology, INMI L. Spallanzani-IRCCS, Via Portuense 292, I-00149, Rome, Italy. Fax: 39 065582237. E-mail: amicosante@inmi.it

Keywords: berylliosis, cytokines, human leukocyte antigen-DPGlu69, interferon-, T-cell proliferation, tumour necrosis factor-

Received: November 16, 2001
Accepted June 12, 2002

This study was supported by grants from US Dept of Energy (DoE) grant DE-FG02-93ER61714 and grant 99060855594 from MIUR, Italy.

	Abstract

TOP Abstract Methods Results Discussion Acknowledgements References

 
Berylliosis is a granulomatous disorder of the lung caused by inhalation of beryllium (Be) and dominated by the accumulation of CD4+ T-helper (Th)1 memory T-cells proliferating in response to Be in the lower respiratory tract. Two gene markers have been associated with susceptibility to berylliosis: 1) the human leucocyte antigen (HLA)-DP gene whose allelic variants, carrying glutamate in position 69 of the ß-chain (HLA-DPGlu69), can bind Be directly and present it to interferon (IFN)- releasing Th1 T-cell clones from patients with berylliosis; and 2) the cytokine gene tumour necrosis factor (TNF)- which has been shown to increase berylliosis risk independent of HLA-DPGlu69.

In order to determine whether TNF- release was triggered by Th1 T-cell activation by Be stimulation in the context of HLA-DPGlu69 molecules, the proliferation of BeSO₄-stimulated blood mononuclear cells and the release of IFN-, TNF-, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 by BeSO₄-stimulated blood mononuclear cells was quantified in 11 individuals with berylliosis using an anti-HLA-DP antibody as a probe for HLA-DP restricted T-cell activation.

While proliferation and IFN- release were completely abrogated by HLA-DP inhibition (inhibition with anti-HLA-DP monoclonal antibody (mAb): 88±16 and 77±16%, respectively; anti-HLA-DR: 29±38 and 14±10%, respectively), the release of TNF- was not (inhibition with anti-HLA-DP mAb: 8.9±7.8%). No other cytokine was detected at significant levels. Moreover, Be was able to induce TNF- production in healthy control subjects not exposed to Be in the absence of T-cell proliferation and IFN- production.

In conclusion, these data suggest that the tumour necrosis factor- response of mononuclear cells is independent of the activation of beryllium-specific human leucocyte anitgen-DP restricted T-cells, which is consistent with the finding that the tumour necrosis factorA2 and the human leucocyte anitgen-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk.

Berylliosis is a chronic granulomatous disorder of the lung caused by inhalation of beryllium (Be) dusts affecting 1–16% of Be-exposed individuals 1, 2. The immunopathology of this disorder is dominated by the accumulation of CD4+ CD45RO+ T-helper (Th)-1 memory T-cells proliferating in response to Be in the lower respiratory tract 3–5. Previously, the current authors have shown that allelic variants of the human leucocyte antigen (HLA)-DP molecule carrying a glutamate residue in position 69 of the HLA-DP ß-chain (HLA-DPGlu69) 6–8 play a central role in disease pathogenesis by directly binding Be in the absence of antigen processing 9 and by restricting the response to Be of Th1 T-cell clones derived from patients with berylliosis 10. In addition, the current authors have shown that the tumour necrosis factor (TNF)A2 allele of the cytokine gene TNF-, which is expressed at exaggerated levels by lung and blood mononuclear cells from berylliosis patients in response to Be 11, 12, is also associated with susceptibility to berylliosis and positively interacts with the HLA-DPGlu69 marker to increase berylliosis risk 13.

Since the Th1 cytokines interferon (IFN)- and TNF-, both hyperexpressed by berylliosis patient mononuclear cells in response to Be 12, play a central role in granulomatous reactions 14, one may question whether the expression of TNF- is the consequence of Be-specific CD4 T-cell activation or is a related but independent event, as the genetic association finding may suggest.

To answer this question, blood mononuclear cells were obtained from individuals with berylliosis and both T-cells and mononuclear phagocytes were stimulated with Be using an anti-HLA-DP monoclonal antibody (mAb) to separate the response to the metal of Be-specific HLA-DPGlu69-restricted Th1 CD4+ T-cells from that of the mononuclear phagocytes.

Interestingly, consistent with the finding that the TNFA2 and HLA-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk, the data indicate that the TNF- response of the mononuclear phagocytes is independent of the activation of Be-specific HLA-DP restricted T-cells.

	Methods

TOP Abstract Methods Results Discussion Acknowledgements References

 
Study population
Eleven individuals with berylliosis were enrolled in the study after giving informed consent: 10 males and one female, all Caucasians, mean age 45±7 yrs and average time of employment 15±6 yrs. The study protocol was approved by the Cleveland Clinic IRB (Cleveland, OH, USA). As a control, five healthy non-Be-exposed individuals were enrolled: four males and one female, all Caucasians and mean age 32±3 yrs.

Lymphocyte proliferation
Peripheral blood mononuclear cells (PBMCs) obtained from patients with berylliosis were isolated from heparinised whole blood by density centrifugation on a Ficoll Hypaque gradient. PBMCs (2x10⁵ cells·well^–1) were then cultured in 96-well flat-bottomed microtitre plates in Roswell Park Memorial Institute (RPMI) 1640 tissue culture medium supplemented with 2 mM L-glutamine, 10% foetal bovine serum, 100 U·mL^–1 penicillin, and 100 µg·mL^–1 streptomycin in the presence of beryllium sulfate (BeSO₄[4H₂O]) at 10, 50 and 100 µM (all reagents form Sigma Co., St. Louis, MO, USA). Phytohaemoagglutinin (PHA; 5 µg·mL^–1; Sigma) and Candida albicans (10 µg·mL^–1) were used as positive controls. T-lymphocyte proliferation was measured by thrytiated deossiribonucleotide thymidine ([³H]TdR) incorporation. After 5 days of culture, cells were pulsed with 1 µCi of [³H]TdR (Amersham International, Amersham, UK) and harvested onto glass-fibre filters 18 h later. Proliferation was measured as [³H]-TdR incorporation by liquid scintillation spectroscopy. The results are expressed as the mean of triplicate cultures. The stimulation index was calculated as the ratio of mean counts per minute (cpm), in antigen-stimulated wells, to the mean cpm in control wells.

In order to evaluate possible contamination with the bacterial endotoxin product of the BeSO₄ solution used as antigen, the BeSO₄ batch solution used throughout the study was tested with the Limulus Amebocyte Lysate test (CAPE CODE Inc., Woods Hole, MA, USA). The stock solution of BeSO₄ (0.2 M) showed undetectable levels of endotoxin contamination (<0.03 U·mL^–1 of Escherichia coli endotoxin, similar to endotoxin-free water). Similarly, undetectable endotoxin levels were revealed for BeSO₄ at the working concentration in RPMI complete medium.

Measurement of cytokines in supernatants
The levels of IFN-, TNF-, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 release in the culture supernatants of Be-stimulated PBMCs were evaluated with commercially available solid-phase, two-site enzyme-linked immunosorbent assays (CytElisa; CytImmune Science, San Diego, CA, USA). Cell supernatants were collected after 3 days (RANTES, GM-CSF, IL-4 and IL-12) and 5 days (IFN-, TNF-, IL-6, IL-8 and IL-10) of PBMC culture before [³H]TdR incorporation, and frozen at –80°C until use. Cytokine concentrations were evaluated in triplicate and the results expressed as the mean of triplicate cultures.

Monoclonal antibody inhibition of lymphocyte activation
Protein-A sepharose purified mAb directed against HLA-DR (L243) 10, HLA-DP (B7/21) 10, HLA-DQ (L2) 10, HLA-class I (W6/32) 10 and the 19 kDa Mycobacterium tuberculosis protein (HYT6) 15 were used at increasing concentrations (10, 20 and 50 µg·mL^–1) to inhibit antigen presentation, lymphocyte proliferation and cytokine production as previously described 5.

HLA typing
HLA-DP and TNFA1/A2 typing was carried out as previously described 13.

Statistical analysis
All the data were expressed as mean±sd. Comparisons between groups were made with t-tests. Comparisons between frequencies were made using the Chi-squared test with the Yates and Bonferroni corrections where necessary.

	Results

TOP Abstract Methods Results Discussion Acknowledgements References

 
HLA-DP restriction of beryllium-stimulated lymphocyte proliferation
All PBMCs obtained from berylliosis patients (n=11), carrying glutamate-69-positive HLA-DP alleles, showed positive responses to PHA and the recall antigen C. albicans (table 1). Of these, nine had a proliferative response to BeSO₄ that was >3 times the mean SI of unstimulated cells (table 1). In all cells the anti-HLA-DP specific mAb induced a marked inhibition of Be-stimulated proliferation (88±16%) that was significantly greater than that obtained with mAbs against HLA-DR (29±38%), HLA-DQ (2±2%), class-I major histocompatability complex (MHC; 1±2%), and the M. tuberculosis 19 kDa-specific protein (0±1%; p<0.05 for all comparisons with anti-HLA-DP; fig. 1).

View larger version (8K): [in this window] [in a new window]   Fig. 1.— Inhibition of beryllium-induced a) proliferation, b) interferon- and c) tumour necrosis factor- release by monoclonal antibodies directed against human leucocyte antigen (HLA)-DR, HLA-DP, HLA-DQ, HLA-class I and the 19 kDa Mycobacterium tuberculosis protein in peripheral blood mononuclear cells from berylliosis patients. DR: anti-HLA-DR; DP: anti-HLA-DP; DQ: anti-HLA-DQ; CI: anti-HLA-class I; Mtb: anti-19 kDa M. tuberculosis.

HLA-DP restriction of interferon- beryllium-stimulated release by mononuclear cells
PBMCs from all 10 patients tested released IFN- in response to PHA and C. albicans (table 1). Be-stimulated IFN- release was significantly higher than control in nine of 10 patients (table 1). The release of this cytokine was strongly blocked by the anti-HLA-DP mAb (77±16%) but not by the other mAbs used (anti-HLA-DR, 14±10%; anti-HLA-DQ, 1±2%; anti-class-I, 1±1%; anti-M. tuberculosis 19 kDa-specific protein, 0±1%; p<0.05 for all comparisons with anti-HLA-DP; fig. 1).

Finally, none of the five healthy non-Be-exposed controls released IFN- in response to BeSO₄ (data not shown).

Lack of HLA-DP restriction of tumour necrosis factor- beryllium-stimulated release by mononuclear cells
PBMCs from all of the nine berylliosis patients tested released TNF- in response to PHA and C. albicans (750.1±426.6). PBMCs from seven of the subjects evaluated also released TNF- in response to Be stimulation at levels that were significantly higher than controls (table 1). However, in contrast to IFN-, the release of TNF- was not blocked by the anti-HLA-DP specific mAb (fig. 1). Similarly to berylliosis patients, PBMCs obtained from four of five non-Be-exposed healthy subjects released TNF- in response to BeSO₄ (195.3±76.2 pg·mL^–1, p=0.02 with respect to Be-unstimulated PBMCs (91.9±15.8); p=0.09 with respect to PBMCs from berylliosis patients stimulated with BeSO₄), while anti-HLA class II monoclonal antibodies had no effect on the cytokine release (data not shown).

No significant levels of the cytokines IL-4, IL-6, IL-8, IL-10, IL-12, GM-CSF and RANTES were found in Be-stimulated culture supernatants of PBMCs from any of the patients tested, although all cytokines were released in response to PHA stimulation (table 1). Finally, when TNF- release levels were analysed in relation to the carriage of the TNFA2 allele (TNFA2/TNFA1 or TNFA2/TNFA2) and those who carried only the TNFA1 allele, no differences were found either in the response to Be (TNFA2-negative, n=5, 281±90 pg·mL^–1; TNFA2-positive, n=4, 312±108 pg·mL^–1; p>0.05) or to PHA (TNFA2-negative, 1,232±778 pg·mL^–1; TNFA2-positive, 1,385±761 pg·mL^–1; p>0.05) and C. albicans (TNFA2-negative, 741±469 pg·mL^–1; TNFA2-positive, 633±546 pg·mL^–1; p>0.05).

	Discussion

TOP Abstract Methods Results Discussion Acknowledgements References

 
Studies previously carried out by this and other groups 6–8 have suggested that the HLA-DP-ß chain allelic variant coding for a glutamate residue in position 69 is the immune response gene associated with Be binding and the induction of the Th1 lymphocyte response that characterises berylliosis 9–11, 16, 17. In addition, consistent with the observation that TNF- is released at exaggerated rates in the lungs of individuals with berylliosis 18, the current authors have shown that the TNF- gene allelic variant TNFA2, implicated in the regulation of TNF- release 19, is also associated with susceptibility to berylliosis, positively interacting with the HLA-DPGlu69 marker in the determination of berylliosis risk 13.

In granulomatous disorders, such as tuberculosis, leprosy and sarcoidosis, IFN--producing Th1 T-cells dominate the disease immune response 20–23. The observation that proliferation and IFN- release by fresh blood mononuclear cells in response to Be are completely abrogated by the anti-HLA-DP specific mAb suggests that in berylliosis-affected individuals the Th1 response to Be is systemic. The fact that HLA-DP glutamate 69-positive molecules are capable of directly binding Be 9, may suggest that Be presentation in the presence of this molecule may itself drive the Th1 response, as has been described in animal models where the Th1 or Th2 polarisation of the immune response is dictated by a specific MHC/peptide combination 24.

TNF-, together with IFN-y, is the dominant cytokine produced in the reaction to Be by mononuclear cells from berylliosis patients. Since TNF- is primarily released by mononuclear phagocytes 25, it is reasonable to question whether cytokine production is driven by, or independent of, Th1 T-cell activation and IFN- release. The antibody blocking experiments presented here clearly show that Be-stimulated blood mononuclear phagocytes were able to release TNF- independently of HLA-DP restricted IFN- release. Moreover, the ability of PBMCs from non-Be-exposed healthy subjects to release TNF- in response to Be strongly support this notion.

Finally, the current authors were unable to demonstrate a correlation between TNF- levels in Be-stimulated cultures and the TNFA1/TNFA2 genotypes. However, although it has been proposed that TNF- release is under the control of a polymorphic sequence with two known alleles (TNFA1 and TNFA2), whereby TNFA2 is associated with higher PHA/phorbol myristate acetate-stimulated cytokine release 19, not all studies have confirmed this finding 26, 27.

The cell function data in this study further support the concept that the tumour necrosis factor- and human leucocyte antigen-DP genes are immunogenetic independent factors of berylliosis and that they may interact in the development of the granulomatous reaction induced by beryllium in the lung.

	Acknowledgements

TOP Abstract Methods Results Discussion Acknowledgements References

 
The authors would like to thank G. Lombardi (Dept of Immunology, Imperial College of Science, Technology and Medicine, London, UK) for helpful advice; V. Vezzani, (Gaiato Modena, Italy) for assistance at the beginning of the study and K.M. O'Donnell for editing the manuscript.

	References

TOP Abstract Methods Results Discussion Acknowledgements References

 

1. Saltini C, Markham TN, Jones Williams W. Berylliosis. The Granulomatous DisordersLondon, Cambridge University Press, 1999; pp. 336–351.
2. Kreiss K, Wasserman S, Mroz MM, Newman LS. Beryllium disease screening in the ceramics industry. Blood lymphocyte test performance and exposure-disease relations. Occup Med 1993;35:267–274.
3. Epstein PE, Dauber JH, Rossman MD, Daniele RP. Bronchoalveolar lavage in a patient with chronic berylliosis: evidence for hypersensitivity pneumonitis. Ann Intern Med 1982;97:213–216.
4. Rossman MD, Kern JA, Elais JA, et al. Proliferative response of bronchoalveolar lymphocytes to beryllium. Ann Intern Med 1988;108:687–693.
5. Saltini C, Winestock K, Kirby M, Pinkston P, Crystal RG. Maintenance of alveolitis in patients with chronic beryllium disease by beryllium-specific helper T cells. N Engl J Med 1989;320:1103–1109.[Abstract]
6. Richeldi L, Sorrentino R, Saltini C. HLA-DPB1 glutamate 69: a genetic marker of beryllium disease. Science 1993;262:242–244.[Abstract/Free Full Text]
7. Wang Z, White PS, Petrovic M, et al. Differential susceptibilities to chronic beryllium disease contributed by different Glu69 HLA-DPB1 and -DPA1 alleles. J Immunol 1999;163:1647–1653.[Abstract/Free Full Text]
8. Richeldi L, Kreiss K, Mroz MM, Zhen B, Tartoni P, Saltini C. Interaction of genetic and exposure factors in the prevalence of berylliosis. Am J Ind Med 1997;32:337–340.[CrossRef][ISI][Medline] [Order article via Infotrieve]
9. Amicosante M, Sanarico N, Berretta F, et al. Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate ß69. Hum Immunol 2001;62:686–693.[CrossRef][ISI][Medline] [Order article via Infotrieve]
10. Lombardi G, Germain C, Uren J, et al. DP allele-specific T cell responses to beryllium account for DP-associated susceptibility to chronic beryllium disease. J Immunol 2001;166:3549–3555.[Abstract/Free Full Text]
11. Tinkle SS, Schwitters PW, Newman LS. Cytokine production by bronchoalveolar lavage cells in chronic beryllium disease. Environ Health Perspect 1996;104:Suppl. 5, 969–971.
12. Tinkle SS, Kittle LA, Schwitters BA, Addison PW Jr, Newman LS. Beryllium stimulates release of T helper 1 cytokines IL-2 and IFN- from BAL cells in chronic beryllium disease. J Immunol 1997;158:518–526.[Abstract]
13. Saltini C, Richeldi L, Losi M, et al. Major histocompatibility locus (MHC) genetic markers of beryllium sensitization and disease. Eur Respir J 2001;18:677–684.[Abstract/Free Full Text]
14. Romagnani S. Biology of human TH1 and TH2 cells. J Clin Immunol 1995;15:121–129.[CrossRef][ISI][Medline] [Order article via Infotrieve]
15. Khanolkar-Young S, Kolk AH, Andersen AB, et al. Results of the third immunology of leprosy/immunology of tuberculosis antimycobacterial monoclonal antibody workshop. Infect Immun 1992;60:3925–3927.[Abstract/Free Full Text]
16. Fontenot AP, Torres M, Marshall WH, Newman LS, Kotzin BL. Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease. Proc Natl Acad Sci USA 2000;97:12717–12722.[Abstract/Free Full Text]
17. Fontenot AP, Falta MT, Freed BM, Newman LS, Kotzin BL. Identification of pathogenic T cells in patients with beryllium-induced lung disease. J Immunol 1999;163:1019–1026.[Abstract/Free Full Text]
18. Bost TW, Riches DW, Schumacher B, et al. Alveolar macrophages from patients with beryllium disease and sarcoidosis express increased levels of mRNA for tumor necrosis factor- and interleukin-6 but not interleukin-1ß. Am J Respir Cell Mol Biol 1994;10:506–513.[Abstract]
19. Higuchi T, Seki N, Kamizono S, et al. Polymorphism of the 5'-flanking region of the human tumor necrosis factor (TNF)-alpha gene in Japanese. Tissue Antigens 1998;51:605–612.[ISI][Medline] [Order article via Infotrieve]
20. Zhang M, Lin Y, Iyer DV, Gong J, Abrams JS, Barnes PF. T-cell cytokine responses in human infection with Mycobacterium tuberculosis. Infect Immun 1995;63:3231–3234.[Abstract]
21. Modlin RL, Melamcon-Kaplan J, Young SMM, et al. Learning from lesions: patterns of tissue inflammation in leprosy. Proc Natl Acad Sci USA 1988;85:1213–1217.[Abstract/Free Full Text]
22. Bergeron A, Bonay M, Kambouchner M, et al. Cytokine patterns in tuberculous and sarcoid granulomas: correlations with histopathologic features of the granulomatous response. J Immunol 1997;159:3034–3043.[Abstract]
23. Manca F, Rossi G, Valle MT, et al. Limited clonal heterogeneity of antigen-specific T cells localizing in the pleural space during mycobacterial infection. Infect Immun 1991;59:503–513.[Abstract/Free Full Text]
24. Murray JS. How the MHC selects Th1/Th2 immunity. Immunol Today 1998;19:157–163.[CrossRef][ISI][Medline] [Order article via Infotrieve]
25. Beutler B, Cerami A. The biology of cachectin/TNF- primary mediator of the host response. Ann Rev Immunol 1989;7:625–635.[ISI][Medline] [Order article via Infotrieve]
26. Uglialoro AM, Turbay D, Pesavento OA, et al. Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-a gene promoter. Tissue Antigens 1998;52:359–367.[ISI][Medline] [Order article via Infotrieve]
27. Skoog T, van't Hooft FM, Kallin B, et al. A common functional polymorphism (C-A substitution at position -863) in the promoter region of the tumour necrosis factor-alpha (TNF-alpha) gene associated with reduced circulating levels of TNF-alpha. Hum Mol Genet 1999;8:1443–1449.[Abstract/Free Full Text]

This article has been cited by other articles:

				R. T. Sawyer, A. P. Fontenot, T. A. Barnes, C. E. Parsons, B. C. Tooker, L. A. Maier, M. M. Gillespie, E. B. Gottschall, L. Silveira, J. Hagman, et al. Beryllium-Induced TNF-{alpha} Production Is Transcription-Dependent in Chronic Beryllium Disease Am. J. Respir. Cell Mol. Biol., February 1, 2007; 36(2): 191 - 200. [Abstract] [Full Text] [PDF]

				J. Muller-Quernheim, K. I. Gaede, E. Fireman, and G. Zissel Diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients Eur. Respir. J., June 1, 2006; 27(6): 1190 - 1195. [Abstract] [Full Text] [PDF]

				R. T. Sawyer, C. E. Parsons, A. P. Fontenot, L. A. Maier, M. M. Gillespie, E. B. Gottschall, L. Silveira, and L. S. Newman Beryllium-Induced Tumor Necrosis Factor-{alpha} Production by CD4+ T Cells Is Mediated by HLA-DP Am. J. Respir. Cell Mol. Biol., July 1, 2004; 31(1): 122 - 130. [Abstract] [Full Text] [PDF]

				B. P. Barna, D. A. Culver, B. Yen-Lieberman, R. A. Dweik, and M. J. Thomassen Clinical Application of Beryllium Lymphocyte Proliferation Testing Clin. Vaccine Immunol., November 1, 2003; 10(6): 990 - 994. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Amicosante, M. Articles by Saltini, C. Search for Related Content PubMed PubMed Citation Articles by Amicosante, M. Articles by Saltini, C.