Tocotrienols are good adjuvants for developing cancer vaccines
© Hafid et al; licensee BioMed Central Ltd. 2010
Received: 24 October 2008
Accepted: 6 January 2010
Published: 6 January 2010
Dendritic cells (DCs) have the potential for cancer immunotherapy due to their ability to process and present antigens to T-cells and also in stimulating immune responses. However, DC-based vaccines have only exhibited minimal effectiveness against established tumours in mice and humans. The use of appropriate adjuvant enhances the efficacy of DC based cancer vaccines in treating tumours.
In this study we have used tocotrienol-rich fraction (TRF), a non-toxic natural compound, as an adjuvant to enhance the effectiveness of DC vaccines in treating mouse mammary cancers. In the mouse model, six-week-old female BALB/c mice were injected subcutaneously with DC and supplemented with oral TRF daily (DC+TRF) and DC pulsed with tumour lysate from 4T1 cells (DC+TL). Experimental mice were also injected with DC pulsed with tumour lysate and supplemented daily with oral TRF (DC+TL+TRF) while two groups of animal which were supplemented daily with carrier oil (control) and with TRF (TRF). After three times vaccination, mice were inoculated with 4T1 cells in the mammary breast pad to induce tumour.
Our study showed that TRF in combination with DC pulsed with tumour lysate (DC+TL+TRF) injected subcutaneously significantly inhibited the growth of 4T1 mammary tumour cells as compared to control group. Analysis of cytokines production from murine splenocytes showed significant increased productions of IFN-γ and IL-12 in experimental mice (DC+TL+TRF) compared to control, mice injected with DC without TRF, mice injected with DC pulsed with tumour lysate and mice supplemented with TRF alone. Higher numbers of cytotoxic T cells (CD8) and natural killer cells (NK) were observed in the peripheral blood of TRF adjuvanted DC pulsed tumour lysate mice.
Our study show that TRF has the potential to be an adjuvant to augment DC based immunotherapy.
There are several types of vaccines used for prevention of infectious diseases, such as attenuated microorganisms, recombinant proteins and DNA vaccines. Currently, studies are being carried out in developing vaccines for tumours i.e. to activate the T-helper-1 (Th1) or cell-mediated arm of the immune system [1, 2]. The cell-mediated arm of the immune system is crucial in protecting the host against the onset, development and spread of tumour. There are several ways to activate the cell-mediated arm of the immune system such as use of adjuvants, recombinant cytokines and nutritional supplements.
Breast carcinoma is the most common cancer in female and responsible for the largest numbers of cancer-related deaths in women. There are numerous studies looking at different approaches to inhibit or retard the growth of breast cancer in animal models and clinical trials [3–6]. One of these approaches involves the use of dendritic cells (DC), which are well-known for their potent ability as professional antigen-presenting cells (APC). These cells possess unique properties that allow these cells to elicit primary and boost secondary immune responses as well as to regulate the type of T cell- mediated immune response that is induced [7–9]. Several studies have demonstrated that tumour antigen-pulsed DC cells are capable of inducing the generation and proliferation of T-helper (Th) and cytotoxic T-lymphocytes (CTL) cells via by presenting tumour peptides on their major histocompatibility complex (MHC) class I and class II molecules respectively to mediate tumour immunity [1, 10, 11].
Nutrition is an important aspect to maintain a healthy and vigilant immune system. Hence, certain nutritional products or supplement may provide a boost for host immune system to fight tumour and this could improve the outcome of treatment. We have previously shown that vitamin E, namely the tocotrienol-rich fraction isolated from palm oil is effective in inhibiting the growth of human mammary cancer cell line in culture [5, 12, 13]. In this study, investigation on the effects of TRF supplementation, a non-toxic natural compound from palm oil to improve the efficacy of vaccines against breast cancer in our BALB/C mouse model has been carried out.
Inbred female BALB/c mice (six to eight week-old) were obtained from the Institute for Medical Research (IMR), Kuala Lumpur and housed at the Animal Maintenance Facility of the same institute. Animals were maintained on commercially available pellet diet and water ad libitum. The soda bedding was changed every three-to-four days. All experiments with animals were performed in accordance with the guidelines approved by the Ethics Committee of the Institute for Medical Research (IMR), Malaysia.
Murine 4T1 cell line, a spontaneously metastatic tumour cells derived from mammary gland tumour of BALB/c mice was purchased from the American Type Culture Collection (ATCC, Rockville USA). The 4T1 cells are comparable to human stage IV breast cancer . These cells are poorly immunogenic and express surface MHC class I but not MHC class II molecules. The tumour cells were cultured in 25 ml cell culture flasks (Nunc, Denmark) as recommended by the ATCC.
Medium and cytokines
Complete medium (CM) consisted of RPMI 1640 supplemented with 10% heat-inactivated foetal bovine serum, 1% Glutamine and 1% Penicillin/streptomycin. Recombinant mouse cytokines were purchased from Chemicon (USA) and used at the following concentrations: granulocyte macrophage colony-stimulating factor (GM-CSF) 10 ηg/ml; interleukin (IL)-4, 10 ηg/ml; recombinant mouse tumour necrosis factor (TNF)-α, 20 ηg/ml.
Generation of bone marrow-derived DC
Murine bone marrow (BM) cells were harvested by flushing the marrow cavities of femur and tibia bones of six-to-eight week-old BALB/c mice with medium under aseptic condition. Erythrocyte-depleted mouse bone marrow cells were cultured in CM supplemented with GM-CSF and IL-4 [6, 15] in a humidified 5% CO2 incubator (Heraeus, Germany). The medium was changed every three to four days. On day six, TNF-α was added to the DC cultures to induce maturation. The DC is harvested between day seven to nine. Cell differentiation was monitored by inverted microscopy. The expression of FITC-CD11c (BD PharMingen, USA), FE-mouse MHC class II and FITC-mouse CD86 (eBiosciences, USA) were analysed after seven to nine days of culture using a flow cytometer (FACS Callibur; BD Biosciences).
The 4T1 cells, BM-derived DC and mouse splenocytes were plated at 1 × 105 cells/well in a microtitre plate and incubated at 37°C in a humidified 5% CO2 incubator (Heraeus, Germany) for 24 hours. The culture medium was replaced with fresh culture medium containing different concentrations (2 to 30 μg/ml) of TRF in 0.1% ethanol (final concentration v/v). Triplicate wells were used for each treatment. Control cells were incubated in complete RPMI 1640 medium containing just 0.1% ethanol (v/v). The cells were incubated at 37°C, 5% CO2 incubator (Heraeus, Germany) for 72 hours. After 72 hours, cell proliferation was determined using MTT cell proliferation assay according to the manufacturer's recommendations (Chemicon, USA). Percentage cell viability at different concentrations of TRF treatment was calculated based on the proliferation of the untreated control cells.
Preparation of tumour cell lysate and antigen pulsing of DC
The 4T1 cells were cultured in the presence or absence on 8 μg/ml of TRF in T25 culture flasks overnight. The TRF-treated confluent 4T1 cells were harvested in a 15 ml tube (Falcon, USA). The cells were then resuspended in 1 ml complete medium. Tumour lysate (TL) was prepared by subjecting these cells three-to-five cycles of freezing in liquid nitrogen and thawing at 65°C. The cell lysates were spun at 2000 rpm for 5 min to remove particulate cellular debris. Untreated 4T1 cells were used as control.
Inductions of IFN-γ and IL-12 released in DC culture in vitro
Freshly generated DC were plated at 1 × 106 cells/well in a 24-well plate overnight to allow the DC to adhere to the culture well. The medium was changed on the following day and 1 ml of tumour lysate preparation from TRF treated or untreated 4T1 cells were added to each well. After 72 h, the culture supernatants were collected. The amounts of IFN-γ and IL-12 in the culture supernatants were determined by a commercial ELISA according to manufacturer's recommendations (BD Biosciences, USA).
Preparation of tumour lysate-pulsed DC for injection into mice
The freshly generated DC were incubated for 18 hours with tumour lysate from 4T1 cells and cultured in the presence or absence of TRF at a 1:1 ratio (DC:4T1). Following this, the TL-pulsed DCs were collected, washed three times with PBS, and resuspended in complete medium. Cell count was adjusted to 10 × 106 cells/ml in medium and this preparation was used for injection into mice.
Ability of DC vaccinations and TRF supplementation to inhibit tumour growth in BALB/c mice
Five groups of five mice each were used for this study. The mice in DC+TL group were injected sub-cutaneously (s.c) in the left flank with 0.1 ml of 1 × 106 DC pulsed tumour lysate from 4T1 cells. The mice in DC+TRF group were injected sub-cutaneously (s.c.) in the left flank with 0.1 ml of 1 × 106 fresh DC. These mice were supplemented daily with oral TRF oil. The mice in DC+TL+TRF group were injected sub-cutaneously (s.c.) in the left flank with 0.1 ml of 1 × 106 DC pulsed tumour lysate and supplemented daily with TRF oil. The mice in TRF and control groups have not been treated with any DC injection but were supplemented with TRF and carrier oil daily. These injections were repeated on day 7 and 14. On day 28, the mice in all five groups were injected with a single s.c. containing 1 × 103 (0.05 ml) 4T1 cells in the right flank of their mammary breast pad. The mice were monitored daily for tumour growth and their body weight was recorded every week. Once tumour was palpable, the diameter of the tumour was measured using a calliper (Mitutoyo, Japan) as described previously . After the mice were sacrificed, blood and spleen samples were collected for various analyses. The experiment was repeated twice.
Analysis of whole blood by flow-cytometry
When the mice were sacrificed, blood was collected by heart puncture into heparinised tubes. The blood was stained with a number of monoclonal antibodies, including FITC-anti-mouse CD8α (BD PharMingen, USA) and FITC- Pan-NK cells-DX5 (BD PharMingen, USA) for flow-cytometry (FACSCallibur, Becton Dickinson, USA) analysis.
IFN-γ and IL-12 secretion assay in splenocytes culture of experimental mice
For measurement of T-cell proliferation, the spleen was removed aseptically when the mice were sacrificed. The splenocytes (1 × 105 cells/well) were cultured for 72 h in culture medium supplemented with 10 μg/ml Concanavalin A (Con A) in a humidified CO2 incubator (Heraeus, Germany) at 37°C. After 72 h, the splenocyte culture was transferred into a sterile 1.5 ml tube and centrifuged (200 g × 5 min) to recover the culture supernatant. The amounts of IFN-γ and IL-12 in the culture supernatants were determined using a commercial ELISA kit (BD Biosciences, USA).
The statistical significance of differential findings between experimental groups and control were determined using Student's t test. Significant values were indicated when two-tailed P values < 0.05.
Characterisation of DC by using CD11c
TRF induces cell death in 4T1 tumour cells
IFN-γ an IL-12 released by DC after incubated with tumour lysate from 4T1
Pre-treatment with tumour lysate pulsed DC and TRF supplementation can inhibit tumour growth in BALB/c mice and enhance production of IFN-γ splenic leucocytes
The number of BALB/c mice developed tumour in each group.
Week after inoculation
Number developed tumour
(n = 5)
(n = 5)
DC + TL
(n = 5)
DC + TRF
(n = 5)
DC + TL +TRF
(n = 5)
Immune regulation detected in the peripheral blood of treated mice groups
We have used a murine model of breast cancer where tumour growth in syngeneic female BALB/c mice is induced by a single s.c. injection of 1 × 103 4T1 cells in the right flank of the mice. The 4T1 tumour model closely resembles human breast cancer because its poor immunogenicity and ability to spontaneously metastasise to lungs, liver, bone marrow and brain [16, 17]. This model was used to evaluate the efficacy of oral supplementation of TRF to enhance the therapeutic benefits of DC immunotherapy in treating breast cancer in a mouse model.
Our results showed that TRF on its own, in both the in-vitro or in-vivo methods used in this study can significantly inhibit the growth of 4T1 cells or tumours induced by these cells. This study corroborates with previous reports of other tumour models [12, 18, 19]. In this study, we show for the first time, that the combination of using two therapeutic approaches i.e. three injections of tumour-lysate pulsed DC prior to inoculation of tumour cells and daily TRF supplementation can significantly inhibit the growth of tumour as well as improve the overall survival of mice induced with tumour.
The MTT assay showed that 2 μg/ml TRF could inhibit the proliferation of 4T1 tumour cells in vitro. However, TRF at this concentration did not exhibit any anti-proliferative effect of DC or murine splenocytes (Fig. 3b &3c). The IC50 value of TRF for 4T1 cells was determined to be 8 μg/ml. Although there was a slight increase in the CD11c expression by TRF-treated DCs compared to control, this increase was not statistically significant. The DC+TL+TRF treatment yielded significantly higher production of IFN-γ by the DC. In addition, the DC pulsed with tumour lysate from 4T1 cells could enhance the productions of IFN-γ and IL-12 by T-helper-1 cells when these cells were cultured in the presence of 8 μg/ml TRF. These findings show that TRF is a potent compound that can induce the immune system to release cytokines that promote cell mediated immune response.
In the animal model, mice that were injected with DC pulsed with tumour lysate from 4T1 cells and supplemented daily with oral TRF showed marked reduction in tumour onset and growth. Previous reports by Nesaretnam and co-workers [12, 13, 19–21] have shown that tocotrienol on its own can inhibit growth of human breast cancer cells in vitro [12, 19] as well as in athymic nude mice . The combination of tumour lysate pulsed-DC and TRF supplementation observed in this study could inhibit the growth of breast tumour in the mouse model. As shown in Fig. 5 and Table 1, the incidence of tumour in the DC+TL+TRF group showed smaller tumour burden compared to control group and DC+TL group, TRF group and DC+TRF group. The splenocytes from the DC+TL+TRF group produced the highest amount of IFN-γ (1346 pg/ml) compared to the DC+TL group (700 pg/ml), control untreated group (520 pg/ml), TRF group (760 pg/ml) or DC+TRF group (900 pg/ml). The IL-12 amount produced in splenocytes culture from experimental mice showed similar pattern as IFN-γ. There was a significant higher production of IFN-γ and IL-12, which are the two signature cytokines for Th1 response, by splenocytes from the DC+TL+TRF group which also suggest that the combination therapy has been effective in enhancing a cell-mediated immune response in these mice. Interferon-γ also promotes class-switching to IgG isotype, which is a key component of cell mediated immunity . This cytokine also upregulates the expression of class II MHC molecules and B7 co-stimulatory molecules on antigen presenting cells. For IL-12, it is expressed specifically in macrophages and dendritic cells [17, 23] and plays a central role in mediating cell mediated immunity, promoting differentiation of CD4+ T cells to the Th1 subset and of CD8+ T cells into mature cytotoxic T lymphocytes (CTLs) . IL-12 is also a potent stimulator of NK cells as well as enhancing cytocidal anti tumour immune responses [25, 26]. All these actions could serve to amplify T-cell responses . Our findings are in agreement with that reported by Ramanathapuram and co-workers , who showed that combination therapy using alpha-tocopheryl succinate (α-TOS) and DCs (α-TOS+DC) increased IFN-γ production by CD4+ and CD8+ T lymphocytes. The α-TOS is an esterified analogue of vitamin E used as an adjuvant to demonstrate the inhibitions of 3LL tumours in vitro and in C57BL/6 mice model. In our study, we used tocotrienol-rich fraction (TRF), which contains the natural isomers of vitamin E. The TRF can be found abundantly in palm oil was used as an adjuvant to develop cancer vaccine in this study because TRF could inhibit the growth of murine mammary cancer (4T1) and human breast cancer (MCF-7 & MDA) cells. In addition, when the DC were co-cultured with TRF, there was a significant increase in the production of IFN-γ by the DC. These are important properties for an adjuvant to be used in a tumour model to have as it could promote Th1 immune responses.
Both NK cells and CD8+ T-cells have a crucial role in the recognition and removal of tumour cells [27–29]. The NK cell activation induced by tumour cells can directly promote anti-tumour responses. Although activation of CD8+ T-cells is more complex, it is important for the development of tumour-specific memory T-cells, which is responsible for long-term protection against the same tumour . The percentages of NK cells and CD8+ T-cells increased in the TRF supplemented group compared to the control and DC+TL only group. Thus, the combine therapy of using DC+TL and daily oral TRF supplementation can promote tumour-specific immune responses in this mouse model of breast cancer.
In conclusion, this study reports on the ability of TRF to act as an adjuvant that promotes tumour-specific cell-mediated immune response in mice. The daily TRF supplementation augments the protective effect provided by three vaccinations of DC pulsed with tumour lysate from 4T1 cells in this mouse model. Further studies are needed to investigate in detail the effectiveness of TRF adjuvanted DC immunotherapy.
- 4T1 cell:
Mouse mammary cancer cell
American Type Cell Culture Collection
cluster of differentiation 8
cluster of differentiation 11c
- Con A:
Enzyme Linked Immuno Sorbent Assay
granulocyte macrophage colony-stimulating factor
major histocompatibility complex
- NK cell:
natural killer cell
- s.c :
tumour necrosis factor
- Shuler G, Steinman R: Dendritic cells as adjuvants for immune-mediated resistance to tumors. J Exp Med. 1997, 186: 1183-1187. 10.1084/jem.186.8.1183.View Article
- Ramanathapuram LV, Kobie JJ, Bearss D, Payne CM, Trevor KT, Akporiaye ET: α-tocopheryl succinat sensitizes established tumors to vaccination with nonmatured dendritic cells. Cancer Immunology Immunotherapy. 2004, 53 (7): 580-588. 10.1007/s00262-004-0499-7.View ArticlePubMed
- Candido KA, Shimizu K, McLaughlin JC, Kunkel R, Fuller JA, Redman BG, Thomas EK, Nickoloff TK, Mule JJ: Local administration of dendritic cells inhibits established breast tumor growth: Implication for apoptosis-inducing agents. Cancer Research. 2001, 61: 228-236.PubMed
- Field RC, Shimizu K, Mule JJ: Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc Natl Acad Sci USA. 1998, 95: 9484-9487. 10.1073/pnas.95.4.1647.View Article
- Guthrie N, Gapor A, Chambers AF, Carroll KK: Inhibition of proliferation of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination. Journal of Nutrition. 1997, 127 (3): 544S-548S.PubMed
- Prasad KN, Kumar B, Yan XD, Hanson AJ, Cole WC: α-Tocopherol succinate, the most effective form of vitamin E adjuvant cancer treatment: A review. Journal of the American College of Nutrition. 2003, 22 (2): 108-117.View ArticlePubMed
- Brossart P, Bevan MJ: Presentation of exogenous protein antigens on major histocompatibility complex class I molecules by dendritic cells: pathway of presentation and regulation by cytokines. Blood. 1997, 90: 1594-1599.PubMed CentralPubMed
- Steinman RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1999, 9: 271-296. 10.1146/annurev.iy.09.040191.001415.View Article
- Shimizu K, Field C, Giedlin M, Mule JJ: Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines. Proc Natl Acad Sci USA. 1999, 96: 2268-2273. 10.1073/pnas.96.5.2268.PubMed CentralView ArticlePubMed
- Porgador A, Snyder D, Gilboa E: Induction of antitumour immunity using bone marrow-generated dendritic cells. JImmunol. 1996, 156: 2918-2926.
- Sato M, Chamoto K, Nishimura T: A novel tumor-vaccine cell therapy using bone marrow-derived dendritic cell type 1 and antigen-specific Th1 cells. International Immunology. 2003, 15 (7): 837-843. 10.1093/intimm/dxg081.View ArticlePubMed
- Nesaretnam K, Stephen R, Dils R, Darbre P: Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status. Lipids. 1998, 33 (5): 461-9. 10.1007/s11745-998-0229-3.View ArticlePubMed
- Nesaretnam K, Dorasamy S, Darbre PD: Tocotrienols inhibit growth of ZR-75-1 breast cancer cells. International Journal of Food Sciences and Nutrition. 2000, 51: S95-103. 10.1080/096374800750049611.View ArticlePubMed
- Pulaski BA, Terman DS, Kabo S, Muller E, Ostrand-Resenberg S: Cooperativity of staphylococcal aureus enterotoxin B superanti-gen, major histocompatibility complex class H, and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model. Cancer Res. 2000, 60: 2710-2715.PubMed
- Rakhmilevich AL, Imboden M, Hao Z, Macklin MD, Roberts T, Wright KM, Albertini MR, Yang NS, Sondel PM: Effective particle-mediated vaccination against mouse melanoma by co-administration of plasmid DNA encoding gp100 and granulocyte-macrophage colony-stimulating factor. Clinical Cancer Research. 2001, 7: 952-961.PubMed
- Ramanathapuram LV, Hahn T, Dial SM, Akporiaye ET: Chemo-Immunotherapy of Breast Cancer using vesiculated α-Tocophyrl succinate in combination with Dendritic Cell Vaccination. Nutrition and Cancer. 2005, 53 (2): 177-193. 10.1207/s15327914nc5302_7.View ArticlePubMed
- Wu RS, Kobie JJ, Besselsen DG, Fong TC, Mack VD: Comparative analysis of IFN-gamma B7.1 and anti sense TGF-beta gene transfer on the tumorigenicity of poorly immunogenic metastatic mammary carcinoma. Cancer Immunology Immunotherapy. 2001, 50: 229-240. 10.1007/s002620100197.View ArticlePubMed
- Takahashi K, Loo G: Disruption of mitochondria during tocotrienol-induced apoptosis in MDA-MB-231 human breast cancer cells. Biochemical Pharmacology. 2004, 67 (2): 315-24. 10.1016/j.bcp.2003.07.015.View ArticlePubMed
- Nesaretnam K, Guthrie N, Chambers AF, Carroll KK: Effect of tocotrienols on the growth of a human breast cancer cell line in culture. Lipids. 1995, 30 (12): 1139-43. 10.1007/BF02536615.View ArticlePubMed
- Nesaretnam K, Ambra R, Selvaduray KR, Radhakrishnan A, Canali R, Virgili F: Tocotrienol-rich fraction from palm oil and gene expression in human breast cancer cells. Annals of the New York Academy of Sciences. 2004, 1031: 143-57. 10.1196/annals.1331.014.View ArticlePubMed
- Nesaretnam K, Ambra R, Selvaduray KR, Radhakrishnan A, Reimann K, Razak G, Virgili F: Tocotrienol-rich fraction from palm oil affects gene expression in tumors resulting from MCF-7 cell inoculation in athymic mice. Lipids. 2004, 39 (5): 459-467. 10.1007/s11745-004-1251-1.View ArticlePubMed
- Abbas AK, Lichtman AH: Basic Immunology, Functions and Disorders of Immune System. Elsevier Saunders Publishing, Updated 2006-2007, 2
- Zhu C, Rao K, Xiong H, Gagnidze K, Li F, Horvath C, Plevv S: Activation of the murine IL-12p40 promoter by functional interactions between NFAT and ICSBP. Journal of Biol Chem. 2003, 10: 1074-
- Barber DL, Wherry EJ, Ahmed R: Cutting edge: rapid in vivo killing by memory CD8 T cells. J Immunol. 2003, 171: 27-View ArticlePubMed
- Puddu P, Fantuzzi L, Borghi P, Varano B, Rainaldi G, Guillemard E, Malorni W, Nicaise P, Wolf SF, Belardelli F, Gessani S: IL-12 induces IFN-gamma expression and secrection in mouse peritoneal macrophages. The journal of Immunology. 2003, 159 (7): 3490-3497.
- Walzer T, Dalod M, Robbins SH, Zitvogel L, Viver E: Natural killer cells and dendritic cells. Blood Journal. 2005, 106: 2252-2258. 10.1182/blood-2005-03-1154.View Article
- Drakes M, Blanchard T, Czinn S: Bacterial probiotic modulation of dendritic cells. Infection and Immunity. 2004, 3299-3309. 10.1128/IAI.72.6.3299-3309.2004.
- Kawarada Y, Ganss R, Garbi N, Sacher B, Arnold T, Hammerling G: NK- and CD8T cell-mediate eradication of established tumours by peritumoural injection of CpG-containing oligodeoxynucleotides. J Immunol. 2001, 167 (1): 5247-5253.View ArticlePubMed
- Lawrence CW, Bracial TJ: Activation, Differentiation, and Migration of Naive Virus-Specific CD8+ T Cells during Pulmonary Influenza Virus Infection. J Immunol. 2004, 173: 1209-1218.View ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/5/prepub
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