Lipopolysaccharide treatment suppresses spontaneously developing ankylosing enthesopathy in B10.BR male mice: The potential role of interleukin-10
© Capkova et al.; licensee BioMed Central Ltd. 2012
Received: 28 August 2011
Accepted: 15 June 2012
Published: 21 June 2012
Ankylosing enthesopathy (ANKENT) is an animal model of human ankylosing spondylitis. ANKENT is an inflammatory disease affecting the ankle and tarsal joints of the hind limbs in susceptible mouse strains. In the disease, the participation of intestinal microbiota components was suggested. Therefore, we attempted to increase the incidence of ANKENT by systemic administration of lipopolysaccharide (LPS), which is a component of bacterial cellular walls and stimulates inflammatory processes.
ANKENT occurrence, serum cytokine profiles, spleen cellular composition and in vitro cytokine response to LPS were analysed in LPS-treated and control LPS-untreated B10.BR male mice.
Contrary to expectations, LPS treatment decreased the incidence of ANKENT in LPS-treated group compared to control LPS-untreated group. Flow cytometry analysis of splenocytes showed an increased percentage of macrophages, dendritic cells and neutrophils and a decreased percentage of B cells, T cells and T helper cells in LPS-treated males following LPS administration. In addition, LPS-treated males had significantly elevated IL-6 and IL-10 serum levels. At 20–22 weeks after the final LPS application, splenocytes from LPS-treated mice were more susceptible to in vitro LPS stimulation than those of the controls and produced significantly higher levels of TNFα and IL-6.
Repeated systemic stimulation with microbial component lipopolysaccharide in early adulthood significantly reduced the incidence of ANKENT in B10.BR mice and this finding can support the “hygiene hypothesis”. In LPS-treated mice, the innate immunity parameters and the level of anti-inflammatory IL-10 cytokine were significantly increased. Nevertheless, the immunological mechanism of the LPS protective effect remains unclear.
Ankylosing spondylitis (AS) is a serious rheumatological disease resulting in patient disability. The disease affects mainly men and the first symptoms usually manifest themselves in early adulthood. A relevant genetic risk factor for AS is the HLA-B27 allele: 96% of AS patients carry the HLA-B27 gene [1, 2]. To study the causes of AS, various biological models were created [3–6].
Our animal model for AS shows disease of the ankle and tarsal joints in the hind paws of mice. The disease affects joints and entheses, which are the areas of insertion of ligaments, tendons or joint capsules into bone. Analogously to human spondyloarthropathies (SpA) [7, 8], enthesitis is a specific marker of the affected joints and the disease was classified as ank ylosing ent hesopathy (ANKENT) . The disease occurs spontaneously in some inbred mouse strains [10, 11] and almost exclusively in males . Its incidence is significantly increased in HLA-B27 mice , confirming an implication of the B27 allele in ANKENT development.
The disadvantage of the ANKENT model is the relative low frequency of the disease - about 10% B10.BR male mice under conventional (CV) and about 5% mice under specific pathogen free (SPF) conditions naturally develop ANKENT .
In previous studies, we found that germ-free mice remain ANKENT free  and their colonization with a mixture containing a low number of common intestinal bacteria (Bacteroides sp., Enterococcus sp., Staphylococcus sp., Veillonella sp.) alone results in ANKENT development . Because mucosal barrier impairment and the consequent increased penetration of commensal microbiota components were suggested to play a role in the disease development , we attempted to induce ANKENT and increase its frequency through the systemic administration of lipopolysaccharide (LPS). LPS, a component of bacterial cellular walls, is an efficient stimulator of the immune inflammatory response  and its intraperitoneal application increases intestinal permeability . A further aim of our work was to study the immunological profile of ANKENT-susceptible B10.BR mice during LPS administration and at the end of the observation period and to compare it to that of LPS-untreated control mice.
All experiments were approved by the Institutional Animal Care Committees of the Institute of Biotechnology and the Institute of Microbiology and were in accordance with the EC Directive 86/609 EEC for animal experiments.
ANKENT screening and evaluation
ANKENT is a progressive stiffening of the ankle and tarsal joints in the hind paws of mice. Joint stiffness was evaluated by gentle stretching once a week and a score between 0 (normal mobility) and 3 (completely immobile) was assigned. Scores of 2 and above were regarded as clearly positive for ANKENT. Joints of some ANKENT positive animals were also checked by radiography. The mice were observed until they reached the age of 10 months.
LPS and its administration
LPS was isolated from E. coli O83 and purified by phenol-water extraction . Twenty mg of LPS was dissolved in 20 ml of sterile PBS and the solution was administered immediately or aliquoted and stored at – 20 °C until used. Mice were intraperitoneally (i.p.) injected with LPS (100 μg LPS/0.1 ml PBS per mouse) four times at two-week intervals. At the beginning of LPS treatment the males were 3-31/2 months old. Control LPS-untreated mice were injected i.p. with 0.1 ml PBS according to the same schedule.
The following monoclonal antibodies with matching isotype controls were used: PE-conjugated anti-mouse CD19 for detection of B cells; FITC-conjugated anti-mouse CD3e, FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8a and PE-conjugated anti-mouse Foxp3 for detection of T cells and their subpopulations; PE-conjugated anti-mouse CD49b for detection of NK cells; PE-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse CD11c, and PE-Cy5 conjugated anti-mouse Ly-6 G for detection of leukocyte populations. All monoclonal antibodies were purchased from eBioscience, USA.
Phenotypic analysis of cells isolated from the spleen was performed using flow cytometry (FACS). Cells were resuspended in ice-cold FACS buffer (1% bovine serum albumin and 0.1% sodium azide in PBS) to a concentration of 2 x 107 cells/ml and pre-incubated with 1 μg of anti-mouse CD16/CD32 per 106 cells for 5 min on ice prior to staining. Antibodies were diluted to predetermined optimal concentrations in 50 μl of FACS buffer and dispensed into wells of a 96-well microtiter plate. Fifty μl of the cell suspension was added to each well and incubated for 20 min at 4 °C in darkness. Then, the cells were washed twice and resuspended in 100 μl of FACS buffer. Intracellular staining of mouse Foxp3 was performed using PE anti-mouse Foxp3 Staining Set according to the manufacturer’s protocol. Sample data were acquired using FACSCalibur flow cytometer (Becton Dickinson, USA) and analysed with FlowJo software (Tree Star, USA).
Blood samples were collected the third day after the second and the fourth LPS dose and at the end of experiment (Figure 1). Not treated blood was let stand for 2 h at room temperature, centrifuged and sera were collected and stored at −20 °C until cytokine analysis.
In vitro cytokine stimulation
Spleen cells were cultured at 2 x 106 cells/ml in a 96-well flat-bottom culture plate in complete RPMI-1640 medium. The cells were stimulated with 10 μg/ml LPS and incubated in 5% CO2 at 37 °C for 48 h.
Multiplex cytokine determination
Cytokine levels in sera and supernatants were detected using fluorescent beads coated with analyte-specific capture antibodies (BioSource, USA) according to the manufacturer’s protocols. The beads were analyzed using the Bio-Plex System (Bio-Rad Laboratories, Hercules, USA).
Statistical differences in ANKENT occurrence between LPS-treated and control groups were evaluated by Fisher’s exact test (two-tailed). P values less than 0.05 were considered statistically significant.
Data concerning the proportional representation of cell populations and cytokine levels are expressed as mean ± standard deviation (SD). Statistical differences between LPS-treated and control groups were examined using the Student’s t test. A p-value of <0.05 was considered statistically significant. Statistical significance was analyzed using Prism 5.0 statistical software (GraphPad Software, San Diego, CA).
Among LPS-treated males we found only one ANKENT positive male, i.e. 1.8% (1/55) incidence. On the other hand, ANKENT occurrence in the control LPS-untreated males was relatively high and joint disease developed in 14.2% (10/70) cases (Figure 1). Statistical evaluation by Fisher’s exact test demonstrated a significant difference in ANKENT incidence between LPS-treated and control males (p < 0.02).
Leukocyte subpopulations in the spleen
Lymphocyte subpopulations in the spleen
Cytokine levels in blood sera
In vitro cytokine response of spleen cells
Bacterial lipopolysaccharide (endotoxin) triggers acute inflammation, associated with activation of the innate immune system and production of multiple proinflammatory mediators [19, 20]. In this study, we attempted to utilize the proinflammatory capability of LPS to enhance the incidence of the naturally occurring inflammatory joint disease ANKENT in mice.
ANKENT develops almost exclusively in males and this hallmark of the disease is associated with hormonal and behavioural factors – similarly as in naturally occurring arthritis in DBA/1 male mice . The males caged together have a significantly higher risk of ANKENT development than males caged alone . During the whole experiment both LPS-treated and LPS-untreated males were caged under the same conditions and we have not observed any significant difference in behaviour between the groups.
Previously, we showed that germ-free mice do not suffer from the joint disease . However, after their colonization with a mixture containing common intestinal bacteria, some mice developed ANKENT . The close connection between the gut and SpA was repeatedly demonstrated in laboratory animals [23–27] and was also confirmed in humans [28–33]. Because LPS is an agent which when administered intraperitoneally enhances intestinal permeability, and leads to translocation of commensal microbiota from the gut , we expected a higher frequency of the disease in LPS-treated males. Surprisingly, LPS administration to ANKENT-susceptible B10.BR male mice in their early adulthood acted protectively: the incidence of ANKENT in LPS-treated males was significantly reduced compared to control LPS-untreated group. This unexpected result could be explained by an overactivation of innate immunity cells leading to an upregulation of negative regulatory pathways .
The second aim of our work was to study the immune response. The cellular infiltrate in joints and elevated IL-6 levels in the sera were demonstrated in ANKENT positive animals at the time when ANKENT became manifest [9, 11]. These signs disappeared after the period of acute inflammation. In this study we did not test mice in the acute stages of the disease, but we were interested in the immune response that precedes ANKENT onset. We compared populations of immune cells and cytokine levels in LPS-treated group, where ANKENT occurrence was suppressed, with the control LPS-untreated group, where ANKENT was manifest.
Throughout the course of LPS administration, significantly higher percentages of macrophages (CD11b+), dendritic cells (CD11c+), neutrophils (Ly6G+) and also natural killer cells (CD3-CD49b+) were found in LPS-treated males than in LPS-untreated ones. This LPS-induced expansion of innate immune cells in spleen was transient and was not present at the end of experiment when the frequencies of these populations were similar in both groups.
Just the opposite effect of LPS treatment was observed in lymphocyte subpopulations. During LPS administration the percentage of B cells, T cells and T helper cells in the spleen was significantly decreased in LPS-treated males and the decrease in frequency of B cells lasted throughout LPS treatment. At the end of the experiment, no differences were found between LPS-treated and LPS-untreated males. This suggests that cells of the adaptive immune system did not directly participate in the immune response to LPS treatment, and the suppression of adaptive immunity in LPS-treated males might be involved in the reduction of ANKENT incidence.
Specific cells of innate and adaptive immunity are associated with the production of cytokines. In LPS-treated males we found significantly increased serum levels of IL-6 and IL-10 during LPS administration, whereas the percentage of CD4+ T cells, which is an important source of these cytokines, was decreased. However, macrophages also produce IL-6 and monocytes, whose frequency was strongly elevated in LPS-treated males, are the main cellular source of IL-10.
The finding of significantly elevated IL-10 levels in sera of LPS treated males was discrepant with the in vitro response of LPS stimulated splenocytes, which did not secrete detectable IL-10. This contradiction between serum and in vitro spleen cell response to LPS stimulation could be explained by the fact that the major source of circulating IL-10 after LPS stimulation is liver .
No significant serum differences were found between LPS-treated and LPS-untreated males in IL-6 and IL-10 and in all other tested cytokines at the end of the experiment. However, IL-6 and TNFα were elevated in both groups. The elevation of these pro-inflammatory cytokines at the end of experiment could be explained by the presence of ANKENT-positive animals. We could only speculate that LPS treatment suppressed and delayed the onset of ANKENT and that cytokine data both from serum and from in vitro stimulation show the subclinical signs of disease. Nevertheless, at the end of the experiment we have detected only a single affected mouse in LPS-treated group. To evaluate the data in more detail we would need to match single cytokine data with individual mice. However, both LPS-treated as well as LPS-untreated control males were evaluated as a group.
We do not know the exact mechanism of LPS effect in ANKENT development, but the high level of anti-inflammatory cytokine IL-10 could contribute to homeostasis in LPS-treated males. IL-10, which is a potent anti-inflammatory agent, might suppress the onset of ANKENT by its effect on the secretion of pro-inflammatory cytokines including TNFα and IL-6. A regulation of IL-10 is connected with a function of pro-inflammatory TNFα. In mice, TNFα up-regulates LPS-induced IL-10 synthesis and neutralization of TNFα with anti TNFα antibody results in a significant reduction of LPS-inducible IL-10 production .
To better understand the immune mechanisms underlying the development of ANKENT, in vitro LPS stimulation of splenocytes was performed. In the course of LPS administration we observed an elevated level of IL-6 in supernatants of splenocytes from LPS-treated males. This result was consistent with the elevated IL-6 levels in blood sera from the LPS group. At the end of the experiment we found not only further increase in IL-6 levels but also increase in TNFα levels in both groups. The levels of both TNFα and IL-6 were significantly higher in LPS-treated group compared to control group. TNFα is directly produced by macrophages in response to LPS stimulation  and is also a typical marker for AS in man . The anti-TNF monoclonal antibodies that act by neutralizing TNF have proved highly effective in AS therapy, but also in the treatment of other spondyloarthropathies [38, 39].
Repeated systemic stimulation with the microbial component LPS caused an activation of the innate immune system followed by suppression of both arms of the immune system in the LPS-treated males. These changes correlated with the reduction of the ANKENT incidence.
Considerable increase in the anti-inflammatory IL-10 level in the LPS-treated males can be a potential mechanism that decreased the ANKENT incidence.
Our findings suggest that LPS stimulated development, expansion and function of the immune system so that LPS-treated males were less susceptible to ANKENT development than LPS-untreated controls.
Tumor necrosis factor α
Transforming growth factor β
We would like to thank Dr. S. Takacova for language help and advice on the preparation of this manuscript. This work was supported by Institutional Research Supports (AVOZ 50520701 and RVO 61388971), by grants from Czech Science Foundation (310/09/P182 and 304/11/1252) and by a grant from Ministry of Education, Youth and Sports of the Czech Republic (7E09091).
- Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD: Ankylosing spondylitis and HL-A 27. Lancet. 1973, 1: 904-907.View ArticlePubMedGoogle Scholar
- Schlosstein L, Terasaki PI, Bluestone R, Pearson CM: High association of an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med. 1973, 288: 704-706.View ArticlePubMedGoogle Scholar
- Breban M, Hacquard-Bouder C, Falgarone G: Animal models of HLA-B27-associated diseases. Curr Mol Med. 2004, 4: 31-40.View ArticlePubMedGoogle Scholar
- Khare SD, Luthra HS, David CS: Spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking beta 2-microglobulin: a model of human spondyloarthropathies. J Exp Med. 1995, 182: 1153-1158.View ArticlePubMedGoogle Scholar
- Nordling C, Karlsson-Parra A, Jansson L, Holmdahl R, Klareskog L: Characterization of a spontaneously occurring arthritis in male DBA/1 mice. Arthritis Rheum. 1992, 35: 717-722.View ArticlePubMedGoogle Scholar
- Taurog JD: Animal models of spondyloarthritis. Adv Exp Med Biol. 2009, 649: 245-254.View ArticlePubMedGoogle Scholar
- D'Agostino MA, Said-Nahal R, Hacquard-Bouder C, Brasseur JL, Dougados M, Breban M: Assessment of peripheral enthesitis in the spondylarthropathies by ultrasonography combined with power Doppler: a cross-sectional study. Arthritis Rheum. 2003, 48: 523-533.View ArticlePubMedGoogle Scholar
- Granfors K, Marker-Hermann E, de Keyser F, Khan MA, Veys EM, Yu DT: The cutting edge of spondylarthropathy research in the millennium. Arthritis Rheum. 2002, 46: 606-613.View ArticlePubMedGoogle Scholar
- Eulderink F, Ivanyi P, Weinreich S: Histopathology of murine ankylosing enthesopathy. Pathol Res Pract. 1998, 194: 797-803.View ArticlePubMedGoogle Scholar
- Capkova J, Ivanyi P: H-2 influence on ankylosing enthesopathy of the ankle (ANKENT). Folia Biol (Praha). 1992, 38: 258-262.Google Scholar
- Weinreich S, Eulderink F, Capkova J, Pla M, Gaede K, Heesemann J, van Alphen L, Zurcher C, Hoebe-Hewryk B, Kievits F, et al.: HLA-B27 as a relative risk factor in ankylosing enthesopathy in transgenic mice. Hum Immunol. 1995, 42: 103-115.View ArticlePubMedGoogle Scholar
- Capkova J, Ivanyi P, Rehakova Z: Sexual dimorphism, but not testosterone itself, is responsible for ankylosing enthesitis of the ankle in B10.BR (H-2k) male mice. Ann Rheum Dis. 2006, 65: 130-132.View ArticlePubMedPubMed CentralGoogle Scholar
- Rehakova Z, Capkova J, Stepankova R, Sinkora J, Louzecka A, Ivanyi P, Weinreich S: Germ-free mice do not develop ankylosing enthesopathy, a spontaneous joint disease. Hum Immunol. 2000, 61: 555-558.View ArticlePubMedGoogle Scholar
- Sinkorova Z, Capkova J, Niederlova J, Stepankova R, Sinkora J: Commensal intestinal bacterial strains trigger ankylosing enthesopathy of the ankle in inbred B10.BR (H-2(k)) male mice. Hum Immunol. 2008, 69: 845-850.View ArticlePubMedGoogle Scholar
- Tlaskalova-Hogenova H, Stepankova R, Hudcovic T, Tuckova L, Cukrowska B, Lodinova-Zadnikova R, Kozakova H, Rossmann P, Bartova J, Sokol D, et al.: Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004, 93: 97-108.View ArticlePubMedGoogle Scholar
- Munford RS: Sensing gram-negative bacterial lipopolysaccharides: a human disease determinant?. Infect Immun. 2008, 76: 454-465.View ArticlePubMedGoogle Scholar
- Mishima S, Xu D, Lu Q, Deitch EA: The relationships among nitric oxide production, bacterial translocation, and intestinal injury after endotoxin challenge in vivo. J Trauma. 1998, 44: 175-182.View ArticlePubMedGoogle Scholar
- Westphal O, Luderitz O, Bister F: Uber die Extraktion von Bakterien mit Phenol/Wasser. Z Naturforsch. 1952, 7: 148-155.Google Scholar
- Aird WC: The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 2003, 101: 3765-3777.View ArticlePubMedGoogle Scholar
- Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal. 2001, 13: 85-94.View ArticlePubMedGoogle Scholar
- Holmdahl R, Jansson L, Andersson M, Jonsson R: Genetic, hormonal and behavioural influence on spontaneously developing arthritis in normal mice. Clin Exp Immunol. 1992, 88: 467-472.View ArticlePubMedPubMed CentralGoogle Scholar
- Weinreich S, Capkova J, Hoebe-Hewryk B, Boog C, Ivanyi P: Grouped caging predisposes male mice to ankylosing enthesopathy. Ann Rheum Dis. 1996, 55: 645-647.View ArticlePubMedPubMed CentralGoogle Scholar
- Hacquard-Bouder C, Ittah M, Breban M: Animal models of HLA-B27-associated diseases: new outcomes. Joint Bone Spine. 2006, 73: 132-138.View ArticlePubMedGoogle Scholar
- Milia AF, Ibba-Manneschi L, Manetti M, Benelli G, Messerini L, Matucci-Cerinic M: HLA-B27 transgenic rat: an animal model mimicking gut and joint involvement in human spondyloarthritides. Ann N Y Acad Sci. 2009, 1173: 570-574.View ArticlePubMedGoogle Scholar
- Onderdonk AB, Richardson JA, Hammer RE, Taurog JD: Correlation of cecal microflora of HLA-B27 transgenic rats with inflammatory bowel disease. Infect Immun. 1998, 66: 6022-6023.PubMedPubMed CentralGoogle Scholar
- Rath HC, Herfarth HH, Ikeda JS, Grenther WB, Hamm TE, Balish E, Taurog JD, Hammer RE, Wilson KH, Sartor RB: Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats. J Clin Invest. 1996, 98: 945-953.View ArticlePubMedPubMed CentralGoogle Scholar
- Taurog JD, Richardson JA, Croft JT, Simmons WA, Zhou M, Fernandez-Sueiro JL, Balish E, Hammer RE: The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats. J Exp Med. 1994, 180: 2359-2364.View ArticlePubMedGoogle Scholar
- Ciccia F, Bombardieri M, Principato A, Giardina A, Tripodo C, Porcasi R, Peralta S, Franco V, Giardina E, Craxi A, et al.: Overexpression of interleukin-23, but not interleukin-17, as an immunologic signature of subclinical intestinal inflammation in ankylosing spondylitis. Arthritis Rheum. 2009, 60: 955-965.View ArticlePubMedGoogle Scholar
- de Vlam K, Mielants H, Cuvelier C, De Keyser F, Veys EM, De Vos M: Spondyloarthropathy is underestimated in inflammatory bowel disease: prevalence and HLA association. J Rheumatol. 2000, 27: 2860-2865.PubMedGoogle Scholar
- Faustini F, Zoli A, Ferraccioli GF: Immunologic and genetic links between spondylarthropathies and inflammatory bowel diseases. Eur Rev Med Pharmacol Sci. 2009, 13 (Suppl 1): 1-9.PubMedGoogle Scholar
- Jacques P, Mielants H, Coppieters K, De Vos M, Elewaut D: The intimate relationship between gut and joint in spondyloarthropathies. Curr Opin Rheumatol. 2007, 19: 353-357.View ArticlePubMedGoogle Scholar
- Palm O, Moum B, Ongre A, Gran JT: Prevalence of ankylosing spondylitis and other spondyloarthropathies among patients with inflammatory bowel disease: a population study (the IBSEN study). J Rheumatol. 2002, 29: 511-515.PubMedGoogle Scholar
- Rodriguez-Reyna TS, Martinez-Reyes C, Yamamoto-Furusho JK: Rheumatic manifestations of inflammatory bowel disease. World J Gastroenterol. 2009, 15: 5517-5524.View ArticlePubMedPubMed CentralGoogle Scholar
- Biswas A, Wilmanski J, Forsman H, Hrncir T, Hao L, Tlaskalova-Hogenova H, Kobayashi KS: Negative regulation of Toll-like receptor signaling plays an essential role in homeostasis of the intestine. Eur J Immunol. 2011, 41: 182-194.View ArticlePubMedGoogle Scholar
- Barsig J, Kusters S, Vogt K, Volk HD, Tiegs G, Wendel A: Lipopolysaccharide-induced interleukin-10 in mice: role of endogenous tumor necrosis factor-alpha. Eur J Immunol. 1995, 25: 2888-2893.View ArticlePubMedGoogle Scholar
- Zhang WJ, Wei H, Hagen T, Frei B: Alpha-lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway. Proc Natl Acad Sci U S A. 2007, 104: 4077-4082.View ArticlePubMedPubMed CentralGoogle Scholar
- Old LJ: Tumor necrosis factor (TNF). Science. 1985, 230: 630-632.View ArticlePubMedGoogle Scholar
- Oldfield V, Plosker GL: Golimumab: in the treatment of rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. BioDrugs. 2009, 23: 125-135.View ArticlePubMedGoogle Scholar
- Sieper J: Developments in the scientific and clinical understanding of the spondyloarthritides. Arthritis Res Ther. 2009, 11: 208-View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/110/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.