J Med Microbiol 54 (2005), 527-531; DOI: 10.1099/jmm.0.45936-0
© 2005 Society for General Microbiology
ISSN 0022-2615
Reduced interleukin-18 secretion in Brucella abortus 2308-infected murine peritoneal macrophages and in spleen cells obtained from B. abortus 2308-infected mice
Luis Fernández-Lago1,
Antonio Orduña2 and
Nieves Vizcaíno1
1Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain 2Departamento de Microbiología, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain
Correspondence Luis Fernández-Lago lrlago{at}usal.es
Received October 21, 2004
Accepted February 4, 2005
Th1 immune responses in which gamma interferon (IFN-
) production predominates are associated with protective immunity against intracellular bacteria. Following infection, interleukin-18 (IL-18) may contribute, in association with IL-12, to optimal IFN- production. In this study, the secretion of IL-18 following intracellular infection with virulent Brucella abortus 2308 in CD-1 cultured peritoneal macrophages and splenocyte cultures was investigated. The production of IL-18 was reduced in both CD-1 mouse peritoneal macrophages infected with B. abortus 2308 and splenocyte cultures obtained from B. abortus 2308-infected mice at 3, 6 and 10 days post-infection (p.i.). In contrast, splenocyte cultures obtained from B. abortus 2308-infected mice at 3 days p.i. secreted significant amounts of IFN-. Stimulation of these cells with recombinant IL-18 (rIL-18) and/or rIL-12 did not significantly increase IFN- secretion at the splenocyte level. These data suggest that once the infection has been established, B. abortus 2308 selectively limits IL-18 secretion without affecting endogenous IFN- production.
Abbreviations: HK, heat-killed; i.p., intraperitoneal; p.i., post-infection; S-LPS, smooth lipopolysaccharide.
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INTRODUCTION
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Brucella abortus is a facultative intracellular bacterium and is one of the causative agents of brucellosis in animals and humans (Fernández-Lago et al., 1999). Professional phagocytes are the first target of B. abortus invasion and the bacteria are able to survive and multiply within these cells (Pizarro-Cerdá et al., 1998). Resistance to B. abortus depends on the effective generation of a T-cell-mediated response (Araya et al., 1989) as well as on an effective activation of macrophages by gamma interferon (IFN-)-producing CD4 T-lymphocytes (Zhan et al., 1993). IFN- is a key mediator in conferring protection against Brucella spp. infections both in vivo (Zhan & Cheers, 1995) and in vitro (Jiang & Baldwin, 1993), and an elevated IFN- production, mainly in the early phase of the infection, could be crucial in their control (Pasquali et al., 2002). In consequence, cytokines secreted in this initial response, which can affect IFN- secretion, can strongly affect the outcome of infection.
Interleukin-18 (IL-18) is a proinflammatory cytokine produced by several cell types, including activated macrophages, dermal keratinocytes, osteoblasts, adrenal cortex cells and intestinal epithelial cells (Gerdes et al., 2002), that is synthesized as a biologically inactive 24 kDa precursor protein. Cleavage to an 18 kDa active mature protein, mediated by the interleukin-1 (IL-1)-converting enzyme, also called caspase-1, is essential for IL-18 to become biologically active (Ghayur et al., 1997). Although IL-18 alone does not induce significant IFN- production, it synergistically enhances IL-12-stimulated IFN- production (Yoshimoto et al., 1998) and promotes cell-mediated immunity (Takeda et al., 1998). Besides its IFN- inducing effect, IL-18 has a direct proinflammatory effect on T and natural killer cells, enhancing proliferation and cytotoxicity and stimulating the production of cytokines such as tumour necrosis factor alpha (TNF-
), IL-1ß, IL-6 and IL-8 (Netea et al., 2000).
Previous studies have demonstrated the role of IL-18 in the host's response to infection. Endogenously synthesized IL-18 has been demonstrated to be required in mice for an adequate host defence against mycobacterial infections (Sugawara et al., 1999), disseminated candidiasis (Stuyt et al., 2004), pneumococcal pneumonia (Lauw et al., 2002), and Pseudomonas aeruginosa (Schultz et al., 2003), Yersinia enterocolitica (Bohn et al., 1998), Salmonella typhimurium (Mastroeni et al., 1999), Leishmania major (Ohkusu et al., 2000) and Listeria monocytogenes (Neighbors et al., 2001) infections. Recently, it has been shown that only a combined administration of recombinant IL-18 and IL-12 induces protection against B. abortus 2308 infection in mice, and this effect is possibly due to an increase in IFN- production in the early phases of infection (Pasquali et al., 2001, 2002). However, at present the dynamics of endogenous IL-18 production in response to a B. abortus infection have not been thoroughly investigated.
The aim of the present study was to investigate the capacity of B. abortus 2308 to stimulate the secretion of IL-18 by cultured CD-1 peritoneal macrophages and splenocyte cultures, and to evaluate the role of this cytokine in IFN- production in response to B. abortus 2308 infection.
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METHODS
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Mice.
Female outbred CD-1 mice (Charles River, Barcelona, Spain) were purchased at 8 weeks of age and kept for 1 week before use. The animals were housed in microisolator cages in Horsefall units, and were cared for in accordance with standard guidelines.
Bacteria and infection of animals.
Virulent B. abortus 2308, used as the challenge strain, was cultured on tryptic soy agar (TSA; BBL) enriched with 0.3 % (w/v) yeast extract (YE; Difco) for 72 h at 37 °C under a 5 % (v/v) CO2 atmosphere in air. To infect the mice, the bacteria were suspended in sterile PBS (pH 7.4) and adjusted turbidimetrically to the desired concentration. The exact dose was established retrospectively by viable cell counting. Mice were infected by the intraperitoneal (i.p.) route with 0.2 ml PBS containing 5 x 105 c.f.u. of the B. abortus 2308 virulent strain. Control groups received 0.2 ml PBS. At the indicated time in each experiment, five mice per group were killed by cervical dislocation.
Reagents.
B. abortus 2308 smooth lipopolysaccharide (S-LPS) and culture supernatant extracts were obtained as described previously (Vizcaíno et al., 1991; Joubier-Maurin et al., 2001). The S-LPS contained, as a percentage of dry weight, 1.5 % protein and 1.1 % 2-keto-3-deoxyoctulosonic acid. S-LPS from Escherichia coli O157 was obtained from Sigma. Recombinant mouse IL-18 and IL-12 were obtained from Pharmingen.
Isolation and infection of CD-1 mouse peritoneal macrophages.
Macrophages were isolated as described previously (Fernández-Lago et al., 1999), with some modifications. Briefly, CD-1 mice were inoculated i.p. with 0.3 ml incomplete Freund's adjuvant (Sigma). Three days later, the mice were killed and peritoneal cells were harvested by washing with cold RPMI (Gibco) with 10 % (v/v) fetal calf serum (FCS; Gibco). The peritoneal cells thus collected were washed twice by centrifugation at 1200 g for 5 min, and resuspended in RPMI, 20 mM HEPES (Life Technologies) supplemented with 2 mM L-glutamine (Life Technologies), 10–5 M ß-mercaptoethanol and 10 % (v/v) FCS. The cell suspensions were adjusted to 106 cells ml–1, seeded on 24-well plates (1 ml per well), and macrophages were allowed to adhere for 90 min at 37 °C under a 5 % (v/v) CO2 atmosphere. Non-adhering cells were washed off and adhering cells were washed three times with 1 ml of the same medium. The number and viability of the macrophages obtained by this procedure were determined by morphological examination and trypan blue exclusion (Fernández-Lago et al., 1999). For macrophage infection, bacteria from overnight cultures were opsonized at 37 °C for 1 h in PBS containing a subagglutinating dilution (1 : 200) of the monoclonal antibody BmE10-5, which reacts specifically with the S-LPS of Brucella spp. (Vizcaíno et al., 1991). Opsonized B. abortus 2308 cells were then centrifuged, washed and diluted in RPMI supplemented as described above. For infection, macrophage cells were incubated with 1 ml of the opsonized bacterial suspension at a m.o.i. of 30 : 1 (bacteria : macrophage) for 1 h. After incubation, non- phagocytosed bacteria were washed off. Following this, 1 ml RPMI supplemented as described above and containing 50 µg gentamicin ml–1 was added to the macrophages to kill any remaining extracellular bacteria. The cells were then cultured in the same medium with gentamicin (50 µg ml–1) at 37 °C under a 5 % (v/v) CO2 atmosphere in air for 48 h, and culture supernatants were obtained from each well, filtered through 0.45 µm pore-size filters, and kept at –70 °C until used for cytokine analysis. Supernatants obtained at the same time from uninfected macrophage cultures were used as negative controls. Uninfected macrophage cultures (five wells per sample) were also incubated, in the same medium with gentamicin (50 µg ml–1), in the presence of either purified S-LPS (20 µg ml–1) or a 1/10 dilution of culture supernatant obtained from B. abortus 2308, and S-LPS from E. coli O157 (1 µg ml–1; Sigma). Macrophage supernatants were also obtained at 48 h from each well, filtered, and kept at –70 °C until assayed.
Splenocyte cultures.
Infected CD-1 mice were killed on days 3, 6 and 10 p.i. The spleens were removed aseptically, and individual cell suspensions were prepared by gentle teasing through cell strainers (Becton Dickinson). Erythrocytes were lysed by adding lysis buffer (0.155 M NH4Cl, 10 mM KHCO3, 10 mM EDTA, pH 7.4) to the pellet for 3 min. The cells were then washed three times in RPMI, 20 mM HEPES supplemented with 2 mM L-glutamine, 10–5 M ß- mercaptoethanol and 10 % (v/v) FCS. Spleen cells (106) were cultured in 24-well plates (0.5 ml per well) and stimulated with 0.5 ml heat-killed (HK) B. abortus 2308 at 107 cells ml–1. Cultures were incubated at 37 °C under a 5 % (v/v) CO2 atmosphere in air. Supernatants were harvested at 48 h, filtered through a 0.45 µm pore-size filter, and frozen at –70 °C for use in cytokine assays. Controls including cultures of splenocytes obtained from uninfected mice or from infected mice stimulated with the mitogen concanavalin A (5 µg per well; Sigma) were also established. In some experiments, splenocytes obtained at 3 days p.i. from mice infected with 5 x 105 c.f.u. B. abortus 2308 were cultured, once stimulated with 107 HK B. abortus 2308 cells, in the presence of recombinant IL-18 (10 ng ml–1) and/or rIL-12 (10 ng ml–1). Supernatants were also removed after 48 h, filtered, and tested for IFN- production.
Cytokine ELISA.
Cytokine contents in macrophage and splenocyte supernatants were determined by ELISA, using commercial kits for IL-18 (biologically active and non-active forms) and IFN- (OptEIA; Pharmingen). Protocols were as recommended by the manufacturer. Known concentrations of each cytokine were used to generate standard curves in each assay and used as references to calculate cytokine concentrations. Samples were assayed in duplicate and values are expressed as means ± SD. The lowest limit of detection for IL-18 and IFN- was 30 pg ml–1.
Statistical methods.
All experiments in this study were performed at least twice. Experimental values are given as means ± SD. The data obtained were analysed by one-way analysis of variance, comparing the mean values with the Fisher's protected LSD (least significant differences, P < 0.05).
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RESULTS AND DISCUSSION
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IL-18 secretion is reduced in B. abortus 2308-infected macrophages
To investigate the effect of B. abortus 2308 infection on macrophage-derived IL-18 secretion, CD-1 mouse elicited-peritoneal macrophage cultures were used. As shown in Fig. 1, 24 h culture supernatants of uninfected macrophages constitutively secreted IL-18. A significant (P < 0.05) increase in the secretion of this cytokine was observed when cultured peritoneal macrophages were exposed to S-LPS from E. coli O157 (1 µg ml–1) (Fig. 1). In contrast, when infected with opsonized B. abortus 2308 for 90 min, followed by removal of extracellular bacteria, supernatants of these macrophage cultures showed no IL-18 activity (< 30 pg ml–1; Fig. 1). These results demonstrate that B. abortus 2308 infection induces a reduction of IL-18 secretion in mice at the macrophage level. It has previously been demonstrated that Brucella culture supernatants contain a protein factor(s) that is able to inhibit the secretion in LPS-activated human macrophages of other macrophage-derived cytokines, such as TNF- (Joubier-Maurin et al., 2001). Accordingly, we also analysed the capacity of both the culture supernatants (1 : 10 dilution) and the S-LPS (20 µg ml–1) obtained from B. abortus 2308 to induce the secretion of IL-18 mediated by CD-1 mouse elicited-peritoneal macrophages. As shown in Fig. 1, neither B. abortus 2308 culture supernatants nor B. abortus 2308 S-LPS had a significant effect on IL-18 secretion. Similar results were obtained upon using different concentrations of both preparations (data not shown). These findings suggest, as happens with wild-type Salmonella dublin infections (Elhofy & Bost, 1999), that this decrease in IL-18 synthesis in mouse macrophages is possibly dependent on the intracellular replication of B. abortus 2308, although the mechanism responsible for this remains to be elucidated.
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Fig. 1. B. abortus 2308-infected peritoneal macrophages secrete reduced amounts of IL-18. Cultured peritoneal macrophages were uninfected, infected with virulent B. abortus 2308 (Ba-infected), or treated with S-LPS from E. coli (LPS-Ec; 1 µg ml–1), S-LPS from B. abortus 2308 (LPS-Ba; 20 µg ml–1) or a B. abortus 2308 culture extract (Extract-Ba; 1 : 10 dilution). After 2 days of culture, supernatants were harvested and assayed for IL-18 by ELISA. Results are expressed as means ± SD (five wells per point). *, Statistically different from uninfected macrophage cultures (P < 0.05).
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IFN- production in B. abortus 2308-infected splenocyte cultures is not dependent on the presence of IL-18
In subsequent experiments we analysed the effect that the reduction in IL-18 secretion, observed in cultured macrophages as a result of B. abortus 2308 infection, had on IFN- secretion in B. abortus 2308-infected mice at the splenocyte level. As shown in Table 1, a 48 h splenocyte culture supernatant obtained from uninfected CD-1 mice constitutively expressed IL-18. A significant decrease (P < 0.05) in the secretion of this cytokine was observed in 48 h splenocyte cultures obtained from B. abortus 2308-infected mice on days 3, 6 and 10 p.i., and stimulated in vitro with 107 HK B. abortus 2308 cells. In contrast, a statistically significant (P < 0.05) increase in IFN- secretion by the splenocyte cultures was observed on days 3, 6 and 10 p.i. (Table 1). These results show that the limited secretion of IL-18, as a result of B. abortus 2308 infection, does not significantly affect IFN- production, and therefore that IL-18 would not be involved in B. abortus-induced IFN- secretion at the splenocyte level. To investigate this further, splenocytes were obtained from B. abortus 2308-infected mice at 3 days p.i. and, once stimulated with 107 HK B. abortus cells, were treated with rIL-12 (10 ng ml–1) and/or rIL-18 (10 ng ml–1) or concanavalin A (5 µg ml–1). The levels of IFN- were then measured in the culture supernatants 48 h after stimulation. As shown in Table 2, the addition to splenocyte cultures of rIL-12, rIL-18 or both cytokines simultaneously did not significantly affect IFN- secretion as compared to untreated cells. A significant (P < 0.05) increase in IFN- production was only seen in the concanavalin A-treated splenocyte cultures (Table 2). It may therefore be concluded that once the infection has been established, the exogenous addition of IL-12, IL-18 or both cytokines simultaneously does not contribute to IFN- production in response to B. abortus 2308 infection. Thus it is possible that both cytokines are active, inducing a protective endogenous IFN- production only when pharmacologically effective levels of them in a biologically active form are present in the first stages of infection (Pasquali et al., 2002).
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Table 1. Production of IL-18 and IFN- in stimulated spleen cells from B. abortus 2308-infected mice CD-1 mice were i.p.-infected with 5 x 105 c.f.u. B. abortus 2308. Mice, five per group, were killed at 3, 6 and 10 days after infection. Data are mean values of five animals ± SD. An asterisk indicates that the results are statistically significant (P < 0.05) as compared to those for the uninfected control group.
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Table 2. Effect of IL-18 and IL-12 on IFN- production by stimulated spleen cells from B. abortus 2308-infected mice CD-1 mice were i.p.-infected with 5 x 105 c.f.u. B. abortus 2308. Mice, five per group, were killed at 3 days after infection. Data are mean values of five animals ± SD.
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ACKNOWLEDGEMENTS
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This work was financed by project G03/204 Red para la Investigación de la Brucelosis, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain.
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REFERENCES
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|---|
Araya, L. N., Elzer, P. H., Rowe, G. E., Enright, F. M. & Winter, A. J. (1989). Temporal development of protective cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus. J Immunol 143, 3330–3337.[Abstract]
Bohn, E., Sing, A., Zumbihl, R., Brielfeldt, C., Okamura, H., Kurimoto, M., Heesemann, J. & Autenrieth, I. B. (1998). IL-18 (IFN--inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice. J Immunol 160, 299–307.[Abstract/Free Full Text]
Elhofy, A. & Bost, K. (1999). Limited interleukin-18 response in Salmonella-infected murine macrophages and in Salmonella-infected mice. Infect Immun 67, 5021–5026.[Abstract/Free Full Text]
Fernández-Lago, L., Rodríguez-Tarazona, E. & Vizcaíno, N. (1999). Differential secretion of interleukin-12 (IL-12) subunits and heterodimeric IL-12p70 protein by CD-1 mice and murine macrophages in response to intracellular infection by Brucella abortus. J Med Microbiol 48, 1065–1073.[Abstract]
Gerdes, N., Sukhova, G. K., Libby, P., Reynolds, R. S., Young, J. L. & Schonbeck, U. (2002). Expression of interleukin (IL)-18 and functional IL-18 receptor on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for atherogenesis. J Exp Med 195, 245–257.[Abstract/Free Full Text]
Ghayur, T., Banerjee, S., Hugunin, M. & 11 other authors (1997). Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386, 619–626.[CrossRef][Medline]
Jiang, X. & Baldwin, C. L. (1993). Effects of cytokines on intracellular growth of Brucella abortus. Infect Immun 61, 124–134.[Abstract/Free Full Text]
Joubier-Maurin, V., Boigegrain, R. A., Cloeckaert, A. & 8 other authors (2001). Major outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages. Infect Immun 69, 4823–4830.[Abstract/Free Full Text]
Lauw, F. N., Branger, J., Florquin, S., Speelman, P., van Deventer, S. J. H., Akira, S. & van der Poll, T. (2002). IL-18 improves the early antimicrobial host response to pneumococcal pneumonia. J Immunol 168, 372–378.[Abstract/Free Full Text]
Mastroeni, P. S., Clare, S., Khan, S., Harrison, J. A., Hormaeche, C. E., Okamura, H., Kurimoto, M. & Dougan, G. (1999). Interleukin 18 contributes to host resistance and gamma interferon production in mice infected with virulent Salmonella typhimurium. Infect Immun 67, 478–483.
Neighbors, M., Xu, X., Barrat, F. J. & 7 other authors (2001). A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extend beyond its effects on interferon gamma production. J Exp Med 194, 343–354.[Abstract/Free Full Text]
Netea, M. G., Kullberg, B. J., Verschueren, I. & van der Meer, J. W. (2000). Interleukin-18 induces production of proinflammatory cytokines in mice: no intermediate role for the cytokines of the tumor necrosis factor family and interleukin-1ß. J Immunol 30, 3057–3060.
Ohkusu, K., Yoshimoto, T., Takeda, K., Ogura, T., Kashiwamura, S.-I., Iwakura, Y., Akira, S., Okamura, H. & Nakanishi, K. (2000). Potentiality of interleukin-18 as a useful reagent for treatment and prevention of Leishmania major infection. Infect Immun 68, 2449–2456.[Abstract/Free Full Text]
Pasquali, P., Adone, R., Gasbarre, L. C., Pistola, C. & Ciuchini, F. (2001). Mouse cytokine profiles associated with Brucella abortus RB51 vaccination or B.abortus 2308 infection. Infect Immun 69, 6541–6544.[Abstract/Free Full Text]
Pasquali, P., Adone, R., Gasbarre, L. C., Pistoia, C. & Ciuchini, F. (2002). Effect of exogenous interleukin-18 (IL-18) and IL-12 in the course of Brucella abortus 2308 infection in mice. Clin Diag Lab Immunol 9, 491–492.[Abstract/Free Full Text]
Pizarro-Cerdá, J., Moreno, E., Sanguedolce, V., Mege, J. L. & Gorvel, J. P. (1998). Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun 66, 2387–2392.[Abstract/Free Full Text]
Schultz, M. J., Knapp, S., Florquin, S., Pater, J., Takeda, K., Akira, S. & van der Poll, T. (2003). Interleukin-18 impairs the pulmonary host response to Pseudomonas aeruginosa. Infect Immun 71, 1630–1634.[Abstract/Free Full Text]
Stuyt, R. J., Netea, M. G., van Krieken, J. H., van der Meer, J. W. & Kullberg, B. J. (2004). Recombinant interleukin-18 protects against disseminated Candida albicans infection in mice. J Infect Dis 189, 1524–1527.[CrossRef][Medline]
Sugawara, I., Yamada, H., Kaneko, H., Mizuno, S., Takeda, K. & Akira, S. (1999). Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted mice. Infect Immun 67, 2585–2589.[Abstract/Free Full Text]
Takeda, K., Tsutsui, H., Yoshimoto, T., Adachi, O., Yoshida, N., Kishimoto, T., Okamura, H., Nakanishi, K. & Akira, S. (1998). Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8, 383–390.[CrossRef][Medline]
Vizcaíno, N., Chordi, A. & Fernández-Lago, L. (1991). Characterization of smooth Brucella lipopolysaccharides and polysaccharides by monoclonal antibodies. Res Microbiol 142, 971–978.[Medline]
Yoshimoto, T., Nagai, N., Ohkusu, K., Ueda, H., Okamura, H. & Nakanishi, K. (1998). IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN- production. J Immunol 161, 3400–3407.[Abstract/Free Full Text]
Zhan, Y. & Cheers, C. (1995). Endogenous interleukin-12 is involved in resistance to Brucella abortus infection. Infect Immun 63, 1387–1390.[Abstract]
Zhan, Y., Yang, J. & Cheers, C. (1993). Cytokine response of T-cell subsets from Brucella abortus-infected mice to soluble Brucella proteins. Infect Immun 61, 2841–2847.[Abstract/Free Full Text]
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INTRODUCTION
METHODS
