Positive correlation between circulating cathelicidin antimicrobial peptide (hCAP18/LL-37) and 25-hydroxyvitamin D levels in healthy adults
© Dixon et al.; licensee BioMed Central Ltd. 2012
Received: 6 September 2012
Accepted: 19 October 2012
Published: 24 October 2012
Transcription of the cathelicidin antimicrobial peptide (CAMP) gene is induced by binding of the bioactive form of vitamin D, 1,25-dihydroxyvitamin D, to the vitamin D receptor. Significant levels of the protein hCAP18/LL-37 are found in the blood and may protect against infection and/or sepsis. We hypothesized that serum vitamin D levels may modulate the circulating levels of hCAP18. Only three studies have shown a positive correlation between circulating 25-hydroxyvitamin D and hCAP18 levels. Here we provide additional evidence for such a correlation in healthy, middle-aged adults.
Serum levels of 25-hydroxyvitamin D [25(OH)D] and plasma levels of hCAP18 were determined in 19 healthy middle-aged (mean of 50.1 years) adult men and women. Plasma hCAP18 concentrations correlated with serum 25(OH)D concentrations in subjects with 25(OH)D levels ≤ 32 ng/ml (r = 0.81, p < 0.005) but not in subjects with concentrations > 32 ng/ml (r = 0.19, p = 0.63).
We conclude that plasma hCAP18 levels correlate with serum 25(OH)D levels in subjects with concentrations of 25(OH)D ≤ 32 ng/ml as opposed to those with concentrations > 32 ng/ml and that vitamin D status may regulate systemic levels of hCAP18/LL-37.
KeywordsVitamin D 25-hydroxyvitamin D Cathelicidin hCAP18 LL-37 Immunity Serum Plasma CAMP Infection
CAMP gene expression is important in host defense as mice and humans lacking hCAP18/LL-37 are susceptible to bacterial infections in numerous tissues including the skin, eye, urinary tract, colon and lung[1–11]. Also, increasing hCAP18/LL-37 in these tissues helps reduce and clear infection[12–14]. Collectively, these data strongly implicate expression of the CAMP gene in maintaining adequate host defense. In addition, the LL-37 peptide has additional biological activities that include neutralizing LPS, chemoattraction of leukocytes and promotion of wound healing and angiogenesis.
The human cathelicidin antimicrobial peptide (CAMP) gene encodes an 18 kDa propeptide (hCAP18) that is packaged in secondary granules of neutrophils and proteolytically cleaved to generate the mature antimicrobial peptide, LL-37, that kills invading pathogens[16–19]. The CAMP gene is regulated at the level of transcription by the bioactive form of vitamin D, 1,25(OH)2D, and the vitamin D receptor[20–22]. The hCAP18/LL-37 protein is expressed by a wide array of immune cells[23, 24] and tissues that are constantly exposed to microbes including the skin, mouth, airways, intestine and colon and is secreted in saliva, sweat, semen and circulates at high levels in the plasma[25–30]. In vitro administration of 1,25(OH)2D induces CAMP gene expression in immune and other epithelial barrier cells[20–22, 31, 32]. It has been shown that adequate levels of serum 25(OH)D are required for expression of hCAP18/LL-37 by immune-activated macrophages, but the relationship between serum 25(OH)D and plasma hCAP18/LL-37 levels has been unclear[34, 35].
The biological importance of circulating hCAP18/LL-37 is not well understood, but it may play a role in protecting against infection and/or the development of sepsis. Recently we found that dialysis patients with the lowest levels of hCAP18 were at a greater than 2-fold risk of death of infectious causes, but a correlation between 25(OH)D levels and hCAP18 was not observed. Also, in a study of patients with bone disease we did not find a correlation[34, 35]. On the other hand, severely ill (sepsis and non-sepsis) patients were found to have lower 25(OH)D and LL-37 than healthy subjects and a positive correlation between vitamin D levels and LL-37 was shown in all populations. More recently, Bhan and colleagues demonstrated that 25(OH)D levels positively correlate with both baseline cathelicidin levels and changes in cathelicidin levels after high-dose ergocalciferol treatment. Interestingly, the positive correlation only existed at 25(OH)D levels <32 ng/ml, but not when the levels were >32 ng/ml. The relationship between vitamin D status and circulating hCAP18 levels has been inconsistent; therefore, we conducted a prospective study in healthy middle-aged adults to test the hypothesis that serum vitamin D modulates circulating hCAP18 levels.
Materials and methods
Characteristics of subjects enrolled (n = 19)
Mean Vitamin D intake (IU)
Mean serum 25(OH)D (ng/ml)
602.6 ± 362.3
33.4 ± 13.9
To determine circulating 25-hydroxyvitamin D levels a modified technique for vitamin D extraction and measurement was developed[38, 39]. Briefly, 100 μl of serum was added to 400 μl of a 2:1 methanol:chloroform solution containing deuterated 25-hydroxyvitamin D3 as an internal standard (40 ng/ml final concentration). To achieve phase separation, 750 μl of ddH2O was added, mixed by vortexer, and centrifuged at 15,000 g for 2 min. The aqueous phase and debris between the two phases were removed, discarded, and the remaining solution dried to completion. The pellet was dissolved in 100 μl methanol and analyzed by injecting 10 μl into an Agilent HPLC (Series 6410, Model G6410A, Santa Clara, CA, USA). 25(OH)D3, 25(OH)D2 and the internal standard were detected on an Agilent tandem mass spectrometer (Series 6410, Model G6410A, Santa Clara, CA, USA) using atmospheric pressure chemical ionization (APCI) detection. Concentrations of 25(OH)D3, 25(OH)D2 and the internal standard were determined relative to authentic standards and corrected for recovery. Average recovery was 92.2%. Intra-day coefficient of variation was 4.7% and inter-day coefficient of variation was 9.4%. The detection limit was determined to be less than 1 ng/ml of all analytes. The results were directly compared with two external, FDA-approved, clinical laboratories (Quest Diagnostics and ZRT Laboratory, Beaverton, OR) and levels determined by our assay matched their results (data not shown).
Determination of circulating protein levels
The levels of hCAP18 (ng/ml) in each plasma sample were determined using a non-commercial ELISA with a detection limit of 0.084 ng/ml and an intra- and inter-assay coefficient of variation of ≤6.3%.
Data was checked for normality with a Kolmogorov-Smirnov test prior to all statistical analyses. Relationships between variables were examined with a Pearson Product Moment Linear correlation. All statistical analyses were performed with SysStat software (SigmaPlot 10.0, SigmaStat 3.5, Chicago, IL). Statistical Significance was set at p < 0.05 (for correlation coefficients, n = 10, r ≥ 0.632; n = 9, r ≥ 0.666).
With the discovery that bio-active forms of vitamin D induce the expression of the CAMP gene, it has been hypothesized that vitamin D status may affect the levels of circulating hCAP18[20, 34]. In a study of dialysis patients, those with the lowest circulating levels of hCAP18 were at a greater than two-fold increased risk of death of infectious causes, but a correlation between serum 25(OH)D and hCAP18 levels was not observed.
More recently, Bhan and colleagues discovered a positive correlation in healthy individuals (mean age 39) at 25(OH)D levels <32 ng/ml, but not when levels were higher. In their study, the 25(OH)D and cathelicidin concentrations were rank transformed, leaving a possible question as to the biological validity and interpretability of the association. On the other hand, Jeng and colleagues observed a positive association between vitamin D status and plasma LL-37 in sepsis, critically ill and healthy patients without transforming the data. Interestingly, 100% of the sepsis patients (mean age 54.0 ±17.1), 92% of the critically ill patients (mean age 56.1 ±15.9) and 66.5% of the healthy individuals (mean age 46.5 ±6.1) had serum 25(OH)D levels below 30 ng/ml. The authors did not apply a cut-off for analysis, but the majority of their subjects were already below 32 ng/ml, and it is possible that analysis of subjects >32 ng/ml 25(OH)D would have lacked the statistical power to discern such a trend. Similarly, in a study by Alvarez-Rodriguez and colleagues of 71 healthy individuals with about two-thirds of participants below 32 ng/ml serum 25(OH)D, a positive correlation between serum 25(OH)D and LL-37 levels was observed. On the other hand, no relationship between maternal 25(OH)D serum levels and LL-37 were detected in cord-blood samples or in patients with active pulmonary tuberculosis, but again these studies did not apply a cut-off in the analysis. Interestingly, Alvarez-Rodriguez and colleagues showed that LL-37 levels decreased with age; however, it also has been reported that LL-37 levels increase with age[43, 44]. A correlation with age could not be examined in this study due to the limited numbers of subjects.
This study confirms the findings of the three earlier studies described above[36, 37, 40] and further confirms those of Bhan and colleagues that plasma hCAP18 levels correlated with serum 25(OH)D in subjects with 25(OH)D concentrations ≤ 32 ng/ml as opposed to those with concentrations > 32 ng/ml. These studies suggest that there is a threshold for the biological effects of vitamin D, as measured by 25(OH)D, at or around 32 ng/ml particularly with regard to hCAP18/LL-37 blood levels. To determine the exact threshold (and optimal circulating level of 25(OH)D) for each downstream effect of vitamin D, further research needs to be conducted. Studies with much larger subject numbers and with a broad range of serum 25(OH)D levels would allow testing of different thresholds to determine if the cutoff is higher or lower than 30–32 ng/ml. This study did not show a correlation at a cutoff of 40 ng/ml 25(OH)D, but we could not test lower cutoffs due to insufficient subject numbers.
Given the nature of the relationship between serum 25(OH)D and hCAP18/LL-37 levels, it is possible that vitamin D supplementation or exposure to sunlight to synthesize vitamin D may provide a means to raise systemic levels of hCAP18/LL-37, thus enhancing protection against infection and/or sepsis. In support of this, in vivo supplementation of individuals with serum 25(OH)D levels <32 ng/ml resulted in an increase in hCAP18 levels in those individuals showing the greatest increase in serum 25(OH)D. In another study, supplementation of normal and atopic dermatitis (AD) patients with 4,000 IU oral vitamin D3 (cholecalciferol) for 21 days resulted in a statistically significant increase in cathelicidin expression in the AD lesions. It was not determined if supplementation improved immunologic outcomes in these studies; therefore, future randomized controlled trials are needed to establish an immune enhancing role for vitamin D supplementation and to determine optimal circulating levels.
We thank Charlotte Horn and Deborah Hobbs for technical assistance and Mary Fantacone for critically reading the manuscript. This project was supported by USANA Health Sciences Inc., Salt Lake City, UT, and the National Institute of Allergy and Infectious Diseases at the National Institutes of Health [5R01AI065604-06 to A.F.G.].
- Nizet V, Ohtake T, Lauth X, Trowbridge J, Rudisill J, Dorschner RA, Pestonjamasp V, Piraino J, Huttner K, Gallo RL: Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature. 2001, 414 (6862): 454-457. 10.1038/35106587.PubMedView Article
- Huang LC, Reins RY, Gallo RL, McDermott AM: Cathelicidin-deficient (Cnlp −/− ) mice show increased susceptibility to Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci. 2007, 48 (10): 4498-4508. 10.1167/iovs.07-0274.PubMedPubMed CentralView Article
- Chromek M, Slamova Z, Bergman P, Kovacs L, Podracka L, Ehren I, Hokfelt T, Gudmundsson GH, Gallo RL, Agerberth B, et al.: The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Nat Med. 2006, 12 (6): 636-641. 10.1038/nm1407.PubMedView Article
- Iimura M, Gallo RL, Hase K, Miyamoto Y, Eckmann L, Kagnoff MF: Cathelicidin mediates innate intestinal defense against colonization with epithelial adherent bacterial pathogens. J Immunol. 2005, 174 (8): 4901-4907.PubMedView Article
- Yu FS, Cornicelli MD, Kovach MA, Newstead MW, Zeng X, Kumar A, Gao N, Yoon SG, Gallo RL, Standiford TJ: Flagellin stimulates protective lung mucosal immunity: role of cathelicidin-related antimicrobial peptide. J Immunol. 2010, 185 (2): 1142-1149. 10.4049/jimmunol.1000509.PubMedPubMed CentralView Article
- Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, Gallo RL, Leung DY: Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med. 2002, 347 (15): 1151-1160. 10.1056/NEJMoa021481.PubMedView Article
- Putsep K, Carlsson G, Boman HG, Andersson M: Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet. 2002, 360 (9340): 1144-1149. 10.1016/S0140-6736(02)11201-3.PubMedView Article
- Gombart AF, Koeffler HP: Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP(epsilon). Curr Opin Hematol. 2002, 9 (1): 36-42. 10.1097/00062752-200201000-00007.PubMedView Article
- Yang YH, Zheng GG, Li G, Zhang B, Song YH, Wu KF: Expression of LL-37/hCAP-18 gene in human leukemia cells. Leuk Res. 2003, 27 (10): 947-950. 10.1016/S0145-2126(03)00020-1.PubMedView Article
- An LL, Ma XT, Yang YH, Lin YM, Song YH, Wu KF: Marked reduction of LL-37/hCAP-18, an antimicrobial peptide, in patients with acute myeloid leukemia. Int J Hematol. 2005, 81 (1): 45-47. 10.1532/IJH97.A10407.PubMedView Article
- Steinstraesser L, Oezdogan Y, Wang SC, Steinau HU: Host defense peptides in burns. Burns. 2004, 30 (7): 619-627. 10.1016/j.burns.2004.05.013.PubMedView Article
- Bals R, Weiner DJ, Moscioni AD, Meegalla RL, Wilson JM: Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide. Infect Immun. 1999, 67 (11): 6084-6089.PubMedPubMed Central
- Jacobsen F, Mittler D, Hirsch T, Gerhards A, Lehnhardt M, Voss B, Steinau HU, Steinstraesser L: Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections. Gene Ther. 2005, 12 (20): 1494-1502. 10.1038/sj.gt.3302568.PubMedView Article
- Raqib R, Sarker P, Bergman P, Ara G, Lindh M, Sack DA, Nasirul Islam KM, Gudmundsson GH, Andersson J, Agerberth B: Improved outcome in shigellosis associated with butyrate induction of an endogenous peptide antibiotic. Proc Natl Acad Sci USA. 2006, 103 (24): 9178-9183. 10.1073/pnas.0602888103.PubMedPubMed CentralView Article
- Gombart AF: The vitamin D-antimicrobial peptide pathway and its role in protection against infection. Future Microbiol. 2009, 4 (9): 1151-1165. 10.2217/fmb.09.87.PubMedPubMed CentralView Article
- Agerberth B, Gunne H, Odeberg J, Kogner P, Boman HG, Gudmundsson GH: FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc Natl Acad Sci USA. 1995, 92 (1): 195-199. 10.1073/pnas.92.1.195.PubMedPubMed CentralView Article
- Larrick JW, Hirata M, Balint RF, Lee J, Zhong J, Wright SC: Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein. Infect Immun. 1995, 63 (4): 1291-1297.PubMedPubMed Central
- Cowland JB, Johnsen AH, Borregaard N: hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules. FEBS Lett. 1995, 368 (1): 173-176. 10.1016/0014-5793(95)00634-L.PubMedView Article
- Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS, Hiemstra PS, Borregaard N: Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood. 2001, 97 (12): 3951-3959. 10.1182/blood.V97.12.3951.PubMedView Article
- Gombart AF, Borregaard N, Koeffler HP: Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3. FASEB J. 2005, 19 (9): 1067-1077. 10.1096/fj.04-3284com.PubMedView Article
- Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q, Liao J, Tavera-Mendoza L, Lin R, Hanrahan JW, Mader S, et al.: Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immunol. 2004, 173 (5): 2909-2912.PubMedView Article
- Weber G, Heilborn JD, Chamorro Jimenez CI, Hammarsjo A, Torma H, Stahle M: Vitamin D induces the antimicrobial protein hCAP18 in human skin. J Invest Dermatol. 2005, 124 (5): 1080-1082. 10.1111/j.0022-202X.2005.23687.x.PubMedView Article
- Agerberth B, Charo J, Werr J, Olsson B, Idali F, Lindbom L, Kiessling R, Jornvall H, Wigzell H, Gudmundsson GH: The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood. 2000, 96 (9): 3086-3093.PubMed
- Sorensen O, Arnljots K, Cowland JB, Bainton DF, Borregaard N: The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood. 1997, 90 (7): 2796-2803.PubMed
- Frohm Nilsson M, Sandstedt B, Sorensen O, Weber G, Borregaard N, Stahle-Backdahl M: The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6. Infect Immun. 1999, 67 (5): 2561-2566.PubMedPubMed Central
- Bals R, Wang X, Zasloff M, Wilson JM: The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA. 1998, 95 (16): 9541-9546. 10.1073/pnas.95.16.9541.PubMedPubMed CentralView Article
- Murakami M, Ohtake T, Dorschner RA, Gallo RL: Cathelicidin antimicrobial peptides are expressed in salivary glands and saliva. J Dent Res. 2002, 81 (12): 845-850. 10.1177/154405910208101210.PubMedView Article
- Malm J, Sorensen O, Persson T, Frohm-Nilsson M, Johansson B, Bjartell A, Lilja H, Stahle-Backdahl M, Borregaard N, Egesten A: The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa. Infect Immun. 2000, 68 (7): 4297-4302. 10.1128/IAI.68.7.4297-4302.2000.PubMedPubMed CentralView Article
- Andersson E, Sorensen OE, Frohm B, Borregaard N, Egesten A, Malm J: Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes. Hum Reprod. 2002, 17 (10): 2529-2534. 10.1093/humrep/17.10.2529.PubMedView Article
- Sorensen O, Cowland JB, Askaa J, Borregaard N: An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma. J Immunol Methods. 1997, 206 (1–2): 53-59.PubMedView Article
- Gombart AF, O’Kelly J, Saito T, Koeffler HP: Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues. J Steroid Biochem Mol Biol. 2007, 103 (3–5): 552-557.PubMedView Article
- Schauber J, Dorschner RA, Yamasaki K, Brouha B, Gallo RL: Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli. Immunology. 2006, 118 (4): 509-519.PubMedPubMed Central
- Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, et al.: Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006, 311 (5768): 1770-1773. 10.1126/science.1123933.PubMedView Article
- Gombart AF, Bhan I, Borregaard N, Tamez H, Camargo CA, Koeffler HP, Thadhani R: Low plasma level of cathelicidin antimicrobial peptide (hCAP18) predicts increased infectious disease mortality in patients undergoing hemodialysis. Clin Infect Dis. 2009, 48 (4): 418-424. 10.1086/596314.PubMedView Article
- Adams JS, Ren S, Liu PT, Chun RF, Lagishetty V, Gombart AF, Borregaard N, Modlin RL, Hewison M: Vitamin d-directed rheostatic regulation of monocyte antibacterial responses. J Immunol. 2009, 182 (7): 4289-4295. 10.4049/jimmunol.0803736.PubMedPubMed CentralView Article
- Jeng L, Yamshchikov AV, Judd SE, Blumberg HM, Martin GS, Ziegler TR, Tangpricha V: Alterations in vitamin D status and anti-microbial peptide levels in patients in the intensive care unit with sepsis. J Transl Med. 2009, 7: 28-10.1186/1479-5876-7-28.PubMedPubMed CentralView Article
- Bhan I, Camargo CA, Wenger J, Ricciardi C, Ye J, Borregaard N, Thadhani R: Circulating levels of 25-hydroxyvitamin D and human cathelicidin in healthy adults. J Allergy Clin Immunol. 2011, 127 (5): 1302-1304. 10.1016/j.jaci.2010.12.1097. e1. Epub 2011 Feb 9. No abstract available. PMID: 21310475PubMedPubMed CentralView Article
- WJ BEaD: A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959, 37: 911-917. 10.1139/o59-099.View Article
- Jones G: Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography. Clin Chem. 1978, 24 (2): 287-298.PubMed
- Alvarez-Rodriguez L, Lopez-Hoyos M, Garcia-Unzueta M, Amado JA, Cacho PM, Martinez-Taboada VM: Age and low levels of circulating vitamin D are associated with impaired innate immune function. J Leukoc Biol. 2012, 91 (5): 829-838. 10.1189/jlb.1011523.PubMedView Article
- Mandic Havelka A, Yektaei-Karin E, Hultenby K, Sorensen OE, Lundahl J, Berggren V, Marchini G: Maternal plasma level of antimicrobial peptide LL37 is a major determinant factor of neonatal plasma LL37 level. Acta Paediatr. 2010, 99 (6): 836-841. 10.1111/j.1651-2227.2010.01726.x.PubMedView Article
- Yamshchikov AV, Kurbatova EV, Kumari M, Blumberg HM, Ziegler TR, Ray SM, Tangpricha V: Vitamin D status and antimicrobial peptide cathelicidin (LL-37) concentrations in patients with active pulmonary tuberculosis. Am J Clin Nutr. 2010, 92 (3): 603-611. 10.3945/ajcn.2010.29411.PubMedPubMed CentralView Article
- Jiang H, Schiffer E, Song Z, Wang J, Zurbig P, Thedieck K, Moes S, Bantel H, Saal N, Jantos J, et al.: Proteins induced by telomere dysfunction and DNA damage represent biomarkers of human aging and disease. Proc Natl Acad Sci USA. 2008, 105 (32): 11299-11304. 10.1073/pnas.0801457105.PubMedPubMed CentralView Article
- Jiang H, Chen W, Qu L, Chen Y, He Q, Wang H, Wu J, Shou Z, Ju Z, Chen J: ELISA for aging biomarkers induced by telomere dysfunction in human plasma. J Biomed Biotechnol. 2010, 2010: 121947-PubMedPubMed CentralView Article
- Hata TR, Kotol P, Jackson M, Nguyen M, Paik A, Udall D, Kanada K, Yamasaki K, Alexandrescu D, Gallo RL: Administration of oral vitamin D induces cathelicidin production in atopic individuals. J Allergy Clin Immunol. 2008, 122 (4): 829-831. 10.1016/j.jaci.2008.08.020.PubMedPubMed CentralView Article
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.