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[Cancer Research 66, 3287-3293, March 15, 2006]
© 2006 American Association for Cancer Research

Immunology

Human Leukocyte Antigen-A2–Restricted CTL Responses to Mutated BRAF Peptides in Melanoma Patients

Rajasekharan Somasundaram1, Rolf Swoboda1, Laura Caputo1, Laszlo Otvos1, Barbara Weber2, Patricia Volpe2, Patricia van Belle3, Susan Hotz3, David E. Elder3,6, Francesco M. Marincola7, Lynn Schuchter4,6, DuPont Guerry4,6, Brian J. Czerniecki5,6 and Dorothee Herlyn1,6

1 The Wistar Institute; 2 Abramson Family Cancer Research Institute, University of Pennsylvania Cancer Center; 3 Department of Pathology, 4 Hematology-Oncology Division, Department of Medicine, and 5 Division of Surgical Oncology, Department of Surgery, Hospital of the University of Pennsylvania; 6 Melanoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania; and 7 Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, NIH, Bethesda, Maryland

Requests for reprints: Dorothee Herlyn, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3962; Fax: 215-898-0980; E-mail: dherlyn{at}wistar.org.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutated BRAF (BRAFV600E) is a potential immunotherapeutic target for melanoma because of its tumor specificity and expression in the majority of these lesions derived from different patients. BRAFV600E is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Demonstration of patients' T cell responses to BRAFV600E would suggest the feasibility of active specific immunotherapy targeting the mutation in these patients. In the present study, BRAFV600E peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2–positive melanoma patients. Four of five patients with BRAFV600E-positive lesions showed lymphoproliferative responses to BRAFV600E peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 was restricted in three patients. Lymphocytes from these three patients were cytotoxic against HLA-A2–matched BRAFV600E-positive melanoma cells. None of the four patients with BRAFV600E-negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (~50%) of HLA-A2 among melanoma patients renders HLA-A2–restricted BRAFV600E peptides attractive candidate vaccines for these patients. (Cancer Res 2006; 66(6): 3287-93)


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BRAF alleles were identified as somatic mutations in 70% of melanomas, the majority of all types of nevi, and a minority of other cancers including lung, colon, and ovary carcinomas, but not in normal cells (1–3). The BRAF mutations were located in exons 11 or 15, with BRAFV600E (formerly BRAFV599E) representing nearly all (92%) the BRAF alleles in melanoma. BRAFV600E has oncogenic activity (in vitro transformation of NIH/3T3 cells, as shown in colony formation assays) through activation of the mitogen-activated protein kinase pathway (1). Because of its tumor specificity and expression in the majority of melanomas (1–3), BRAFV600E is a potential immunotherapeutic target for melanoma. BRAFV600E is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Importantly, anchors for human leukocyte antigen (HLA) class I and II molecules are expressed in close vicinity to the mutated epitope (4, 5), suggesting that both CD8+ HLA class I-restricted CTL and CD4+ HLA class II-restricted helper T cells may be induced by vaccines of BRAFV600E. Melanoma patients' CD4+ T lymphocytes proliferated in vitro following stimulation with BRAFV600E peptides and proliferation was HLA class II (DPB1*0401 or DRB1*0404)-dependent (5). However, it is unclear from this study whether the T cells were specific for the mutated epitope of BRAFV600E. Additionally, HLA class I (B*2705)-dependent CTL responses specific for BRAFV600E and unrelated to wild-type BRAF have been described in melanoma patients (4). The HLA restriction element (B*2705) used by the patients' T cells for BRAFV600E recognition is expressed by a low fraction of individuals [3.2%; ref. (6)] and therefore, immunotherapy based on targeting the B*2705-restricted BRAFV600E epitope would be limited to a minority of melanoma patients.

In the current study, we provide evidence for HLA-A*0201 binding sites on 9-mer and 10-mer BRAFV600E peptides. This HLA type is expressed by ~50% of melanoma patients and 28% of healthy individuals (7). We show that four of five melanoma patients with BRAFV600E-positive lesions developed lymphoproliferative responses to stimulation with BRAFV600E peptide. These responses were specific for the mutated epitope (absence of responses to wtBRAF peptide) and were HLA-A2–dependent in three patients. Proliferating lymphocytes were cytotoxic against HLA-A2/BRAFV600E–positive melanoma cells. These studies suggest the feasibility of vaccinating HLA-A2–positive melanoma patients with the BRAFV600E peptides.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients. Nine HLA-A*0201-positive melanoma patients (two with primary melanoma and seven with metastatic disease; see Table 1 ) and five HLA-A*0201-positive normal healthy donors were included in this study. All human blood samples were obtained under informed consent using a protocol approved by the Institutional Review Boards of the Hospital of the University of Pennsylvania and the Wistar Institute.


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  		Table 1. Patients' HLA type and BRAF status

 
Cell lines. Melanoma cell lines WM35, WM278, and WM793 were established from three patients' primary melanomas (8). WM3248, WM3456, and WM3457 were established from patients' metastatic melanomas as previously described (8). All melanoma cells were maintained in MCDB153-L15 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 2% fetal bovine serum.

Reagents. The following monoclonal antibodies (mAb) were used: HLA class I–specific mAb W6/32 and HLA class II–specific mAb B33.1 (obtained from Dr. B. Perussia, Thomas Jefferson University and Dr. G. Trinchieri, The Wistar Institute); mAb MA2.1 to HLA-A2 and -B57 (17) and mAb KS1 to HLA-A2 (obtained from Dr. S. Ferrone, Roswell Park Cancer Institute, Buffalo, NY; ref. 9); FITC or phycoerythrin-labeled anti-CD4, CD8, and CD25 mAb; FITC-conjugated mAb GB11 to granzyme B and phycoerythrin-conjugated mAb {delta}G9 to perforin (BD PharMingen, San Diego, CA). BRAFV600E or wtBRAF peptides (see Table 2 ) were synthesized, high-pressure liquid chromatography purified and encapsulated in poly(DL-lactide-co-glycolide; PLG) microspheres (10).


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  		Table 2. HLA-A*0201-binding peptides of BRAFV600E and human wtBRAF

 
Detection of BRAFV600E and wtBRAF. DNA isolated from fresh or paraffin-embedded tissue blocks or cell lines of patients with melanoma were used for the detection of BRAFV600E or wtBRAF (2). Tumor tissue contained <2% normal cells as determined histopathologically. Genomic DNA was screened for mutations using heteroduplex and sequence analysis. BRAF exon 15 was amplified by PCR using fluorescence-labeled primer and heteroduplexes were screened for the presence of BRAFV600E or wtBRAF mutation as described earlier (2).

T cell proliferation assay. This assay was done as described (11). Briefly, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation of heparinized blood and cryopreserved. Adherent monocytes (5 x 104 per well of a 96-well round-bottomed microtiter plate; Corning, NY) were pulsed for 8 hours with BRAFV600E, wtBRAF, or control peptides (25 µg/mL) in PLG microspheres (1 µg/mL; see Table 2). All preparations were in T cell medium [RPMI 1640 with GlutaMAX medium (Life Technologies-Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated human AB serum (Gemini Bioproducts, Calabasas, CA), 10 mmol/L HEPES and 5 x 10–5 mol/L 2-ß mercaptoethanol (both from Sigma Chemical)]. After the incubation, excess peptides were removed and the peptide-pulsed monocytes were cocultured with PBMC (105 cells/well) in T cell medium supplemented with L-arginine (116 mg/L; Life Technologies-Invitrogen) and L-asparagine (36 mg/L; Life Technologies-Invitrogen). Proliferative responses of the PBMC were determined on day 5 by [3H]thymidine incorporation assay. All determinations were done in triplicate.

Generation of anti-BRAF V600E CTL. Growing T cells from BRAFV600E peptide-stimulated lymphocytes were periodically restimulated in T-cell medium with BRAFV600E peptide and 20 units/mL of recombinant interleukin 2 (a gift from the Biological Resources Branch, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD). This process was repeated every 7 days until day 56 when lymphocytes were harvested and tested for cytolytic activity.

Cytotoxicity assay. The cytolytic activity of T cells was tested in a standard 6-hour 51Cr-release assay (12).

Blocking of lymphocyte proliferation or CTL activity with anti-HLA class I or class II antibodies. Peptide-pulsed monocytes (proliferation assay) or tumor targets (CTL assay) were incubated with anti-HLA class I mAb W6/32 (IgG2a, 10 µg/mL), anti-HLA class II mAb B33.1 (IgG2a, 10 µg/mL), anti-HLA-B17/A2 mAb MA2.1 (IgG1, 10 µg/mL), or anti-HLA-A2 mAb KS1 (IgG1, 10 µg/mL). Mouse IgG at similar concentrations were used as controls (12). CTL and lymphocyte proliferation blocking assays were done as previously described (12).

Cytokine measurements. Supernatants obtained from PBMC stimulated with peptide-pulsed monocytes after 2 to 4 days were tested for the presence of IFN-{gamma} and interleukin 4. All cytokine determinations were done using ELISA kits (Endogen, Rockford, IL).

Phenotyping of lymphocytes. Cultured lymphocytes were incubated with saturating concentrations (5 µg/mL) of FITC or phycoerythrin-labeled anti-CD4, -CD8, or -CD25 mAb in RPMI 1640 supplemented with 5% human AB serum for 1 hour at 4°C. Antibody binding was analyzed in a cytofluorograph.

Statistical analyses. Differences between experimental and control values were analyzed for significance by Student's two-sided t test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BRAFV600E status of melanoma cells. BRAFV600E status was determined by PCR of genomic DNA obtained from fresh or paraffin-embedded tissue blocks or cell lines of melanoma patients. Five of nine melanoma samples, including four tissues (CS, 3457, 3463, 3502) and one cell line (35) were positive for BRAFV600E (Table 1). All five samples also expressed wtBRAF. It is unclear whether in the four tissue samples (CS, 3457, 3463, 3502) and in the other exclusively wtBRAF-positive tissue samples (RP, 3495), wtBRAF is expressed by tumor cells or normal cells present in the tumor tissues. However, in three melanoma cell lines (35, 3445, and 3456), wtBRAF is exclusively expressed by tumor cells. These results are consistent with our earlier observations (1, 2) and the observations of other investigators (5, 13, 14).

Identification of putative HLA-A*0201 anchor residues in BRAFV600E peptides. MHC class I binding algorithm (SYFPEITHI) was used to analyze BRAFV600E amino acid fragments 582 to 623 for potential HLA-A*0201 binding residues. Two putatively HLA-A*0201–binding peptides (residues 597-605 and 597-606) were identified (Table 2). Two algorithms predicting proteasome cleavage (PAProc and FRAGPREDICT) did not reveal any possible cleavage sites within the identified peptide fragments. Two wtBRAF peptides corresponding to the two BRAFV600E peptides and one control peptide with BRAF-unrelated sequence, but expressing HLA-A*0201 anchors are also shown in Table 2. All the peptides were encapsulated in PLG microspheres and used in the lymphoproliferation assays.

In vitro lymphocyte proliferative response to stimulation with BRAFV600E and wtBRAF peptides. Nine HLA-A*0201–positive melanoma patients and five HLA-A*0201–positive normal healthy donors were analyzed for their lymphoproliferative responses to stimulation with HLA-A*0201 binding BRAFV600E and wtBRAF peptides (Figs. 1 and 2 ). Four patients (35, CS, 3457, and 3463) had significant lymphoproliferative responses to stimulation with BRAFV600E peptides. In one of the four patients (CS), the wtBRAF peptide also induced a low but statistically significant lymphoproliferative response. Similarly, we have observed lymphoproliferative responses in patients with breast cancer following stimulation of the lymphocytes not only with mutated, but also with wild-type peptides of epidermal growth factor receptor (EGFR; ref. 15). All four patients (35, CS, 3457, and 3463) with lymphoproliferative responses to BRAFV600E peptide stimulation expressed BRAFV600E on tumor cells. The lymphocytes of patient 3457 responded to stimulation with 9-mer, but not 10-mer BRAFV600E peptide. Thus, a difference of just one amino acid has a profound effect on lymphocyte stimulation by BRAFV600E peptide, in agreement with the results reported by other investigators (16). Patient 3502 showed no lymphoproliferative response to BRAFV600E peptide stimulation despite the presence of BRAFV600E mutation in tumor cells.


Figure 1
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  		Figure 1. Proliferative responses of PBMC to stimulation with HLA-A*0201-binding peptides of BRAFV600E and wtBRAF. Adherent monocytes (5 x 104/well) were pulsed for 8 hours with peptides (25 µg/mL) in PLG microspheres (1 µg/mL). At the end of incubation, excess peptides were removed and the monocytes were cultured with PBMC (1 x 105) for 5 days. Proliferative responses were determined by standard [3H]thymidine incorporation assay. Columns, mean cpm (triplicate determinations) of [3H]thymidine incorporation; bars, SD; *, P < 0.01, significantly different from the values obtained with control peptides.

 

Figure 2
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  		Figure 2. Absence of lymphoproliferative responses to BRAFV600E peptide stimulation in healthy donors. Proliferation assay was done using HLA-A*0201–positive healthy donor lymphocytes as described in Fig. 1.

 
CD markers and cytokine secretion. Proliferating lymphocytes from patients 35, 3457, and 3463 predominantly expressed the cytotoxic T cell marker CD8 (80%, 62%, and 49% of the cells positive, respectively). The lymphocytes of patient CS could not be phenotyped because of the lack of a sufficient number of cells. Lymphocytes from all three patients stained positive for granzyme B. Supernatants from lymphocytes which proliferated to stimulation with BRAFV600E peptide contained high amounts of IFN-{gamma} (>300 pg/mL) but not interleukin 4 (<20 pg/mL). WtBRAF peptide did not induce IFN-{gamma} release in any of the patients' lymphocytes. Patient CS could not be included in those studies because a sufficient number of lymphocytes were not available. This finding is consistent with the absence of lymphoproliferative responses to stimulation with wtBRAF (see Fig. 1).

Blocking of lymphocyte proliferative response to BRAFV600E peptide stimulation by anti-HLA class I and anti-HLA-A2 antibodies. The patients' (35, 3457, and 3463) lymphoproliferative responses to stimulation with BRAFV600E peptide were significantly (P < 0.01) blocked by anti-HLA class I and anti-HLA-A2 antibodies, indicating that the proliferative responses are HLA-A2–restricted (Fig. 3 ). The anti-HLA class II antibody showed no effect, presumably because the peptide used for lymphocyte stimulation lacked HLA class II binding epitopes (data not shown).


Figure 3
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  		Figure 3. Inhibition of lymphoproliferative responses to BRAFV600E peptide stimulation by anti-HLA class I and anti-HLA-A2 antibodies. Monocytes were pulsed with peptides as described in Fig. 1. At the end of the incubation period, excess peptides were removed and the peptide-pulsed monocytes were incubated with anti-HLA class I, anti-HLA class II, or anti-HLA-A2 antibodies or control normal mouse immunoglobulins (all antibodies at 10 µg/mL) for 1 hour at room temperature. At the end of incubation, monocytes were cultured with PBMC to determine lymphoproliferative responses as described in Fig. 1. a-l, values with the same letters differ significantly from each other.

 
Absence of healthy donor lymphocyte responses to BRAFV600E peptide stimulation. None of the five HLA-A*0201–positive healthy donors had lymphoproliferative responses to stimulation with BRAFV600E or wild-type peptides (Fig. 2). In one donor (024), lymphocytes showed significant (P < 0.05) proliferative responses to stimulation with control peptide (PLG-encapsulated EGFR peptide) when compared with unstimulated T cells (Fig. 2), but there was no proliferative response to stimulation with BRAFV600E or wtBRAF peptides.

Cytolytic activity of short-term T cell lines. Short-term T cell lines (8 weeks in culture) obtained from patient 35 (80% CD8+), patient 3457 (62% CD8+), and patient 3463 (49% CD8+) were tested for cytotoxic activity against autologous or allogeneic WM35, WM278, and WM3457 melanoma cells (all cell lines are HLA-A2+, wtBRAF+, and BRAFV600E+), allogeneic WM3456 melanoma cells (HLA-A2+, wtBRAF+, and BRAFV600E–), allogeneic WM793 melanoma cells (HLA-A2–, wtBRAF+, and BRAFV600E+), and WM3248 (HLA-A2–, wtBRAF+, and BRAFV600E–). All three short-term T cell lines lysed WM35, WM278, and WM3457 cells, but not WM3456, WM793, or WM3248 cells, at an effector-to-target (E:T) ratio as low as 12.5 (Fig. 4 ). The cytotoxic activity of proliferating lymphocytes from patient CS could not be determined because of the lack of a sufficient number of lymphocytes. Thus, CTL activity is dependent on both HLA-A2 and BRAFV600E expression by melanoma cells.


Figure 4
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  		Figure 4. Cytotoxic activity of short-term T cell lines obtained from patients 35, 3457, and 3463. Short-term (8 weeks) T cell lines were established by stimulating PBMCs of patients 35, 3457, and 3463 with autologous adherent monocytes pulsed with BRAFV600E peptide (patients 35 and 3463, peptides 597-606; patient 3457, peptides 597-605; 25 µg/mL in PLG microspheres). After 7 days, growing lymphocyte cultures were harvested and restimulated with peptide and 20 units/mL of natural human interleukin 2. This process was repeated every 7 days until day 56 when lymphocytes were harvested and tested for cytotoxic activity against melanoma cells in standard 51Cr-release assay.

 
Blocking of CTL activity by anti-HLA class I and anti-HLA-A2 antibodies. Cytotoxic activity of short-term CTL line from patient 35 against autologous WM35 melanoma cells (Fig. 5A ) and allogeneic WM278 melanoma cells (HLA-A2+ BRAFV600E+; Fig. 5B) was blocked in the presence of anti-HLA class I mAb W6/32 and anti-HLA-A2 mAb KS1, indicating that the T cells recognize antigen in an HLA-A2–restricted manner.


Figure 5
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  		Figure 5. Inhibition of cytotoxic activity of short-term 35 T cell line (predominantly CD8+) in the presence of anti-HLA class I and anti-HLA-A2 mAb. Cytotoxic activity of short-term 35 T cell line was determined as described in Fig. 4. WM35 (A) and WM278 (B) tumor targets were incubated with anti-HLA class I (W6/32) or anti-HLA-A2 (KS1) or control mouse immunoglobulin antibodies for 1 hour at room temperature before the addition of T cells at E:T of 20. a-f, values with the same letters differ significantly from each other.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An important question for the consideration of immunotherapies targeting mutated epitopes in cancer patients is whether the epitope expressed by the patients' tumors is immunogenic. Our studies have shown the immunogenicity of BRAFV600E in melanoma patients and suggests that the mutant is a potential immunotherapeutic target for these patients. We have shown previously that mutated EGFR (EGFR-vIII) expressed by breast cancer patients' tumors is immunogenic in 50% of the patients (15). Patients had both humoral and cellular immune responses specific for the mutated epitope. Furthermore, circulating antibodies to mutated p21ras, bcr-abl, and p53 have been described in cancer patients (17–19). The mutated tumor-associated epitopes recognized by cancer patients' T cells usually are individual-specific (20–22), and therefore, they do not provide immunotherapeutic targets for a larger population of patients. In contrast, BRAFV600E mutation is expressed by 70% of specimens derived from melanoma patients (3), and the peptides described here have vaccine potential for HLA-A2+ melanoma patients (~50% of patients; ref. 7). These peptides are more specific than the peptides currently used for the immunization of melanoma patients (23).

The feasibility of vaccinating against tumors by targeting mutated proteins has been shown in the mutated p53, ras, and EGFR-vIII systems. Thus, the induction of an idiotypic network against mutated p53 has resulted in tumor growth inhibition in mice (24). Mutated ras protein/peptide vaccines have induced CTL in mice and cancer patients and have inhibited the growth of established tumors in mice (25, 26). A peptide of EGFR-vIII has protected mice against challenge with tumor cells and this effect may have been mediated by CTL and/or antibodies (27). Furthermore, anti-idiotypic antibodies mimicking the mutated epitope of EGFR-vIII have inhibited melanoma growth in mice through the induction of mutation-specific antibodies (28).

Our results show a positive correlation between BRAFV600E mutation status in melanoma lesions and immune responses to the mutated epitope. Thus, three out of five patients with BRAFV600E-positive lesions showed lymphoproliferative responses to stimulation with BRAFV600E, but not wtBRAF peptide. One patient with a BRAFV600E-positive lesion showed lymphoproliferative responses to stimulation with both BRAFV600E and wtBRAF peptides. It is possible that the uncloned lymphocyte population reacts with both mutated and wtBRAF epitopes or with wild-type epitopes only, although immunologic tolerance of lymphocytes to wtBRAF sequences would be expected. However, lymphocyte reactivity with normal tissue antigens has been shown in cancer patients (11, 29). Only one of the five patients with BRAFV600E-positive lesions showed the absence of lymphoproliferative responses to stimulation with BRAFV600E or wtBRAF peptides. None of the four patients with BRAFV600E-negative lesions had a lymphoproliferative response to stimulation with BRAFV600E or wtBRAF peptide. In one patient (3463), the primary lesion expressed BRAFV600E (Table 1), whereas the metastatic lesion did not (data not shown). Lymphoproliferative responses to BRAFV600E peptide stimulation were positive at the time of metastatic occurrence. The lymphoproliferative response to BRAFV600E peptide stimulation of this patient most likely was elicited by BRAFV600E expressed by the primary lesion. Thus, the loss of the BRAFV600E genotype during progression from primary to metastatic melanoma in patients with BRAFV600E-specific T cell responses suggests an active immune selection of nonmutated melanoma clones by the tumor-bearing host (4).

Our results indicate that the lymphoproliferative responses to BRAFV600E are HLA-A2 restricted as anti-HLA-A2 antibody inhibited these responses in three patients tested. CTL responses to BRAFV600E peptide stimulation were observed in the same three patients. These responses also were HLA-A2 restricted. To our knowledge, this is the second report of CTL responses elicited by BRAFV600E peptides (4). However, in the previous report (4), CTL were obtained by stimulation of lymphocytes with a modified BRAFV600E peptide and the CD8+ T cells were enriched to show CTL activity, whereas in the present study, we obtained CD8+ CTL responses to unmodified BRAFV600E peptide in nonenriched CD8+ T cells. Furthermore, the low population frequency (3.2%) of the HLA restriction element (B*2705) used by the lymphocytes for peptide recognition in the previous study (4) limits the potential clinical utility of the peptide as vaccine for melanoma patients, whereas, in our study, the high prevalence of HLA-A2 among melanoma patients (50% of patients are HLA-A2 positive; ref. 7) renders the described HLA-A2–binding peptides attractive candidate vaccines for these patients. Immunotherapy of melanoma patients is a promising strategy as lymphocytic infiltration of melanoma tissues in vivo is associated with prognostically favorable disease outcome (30–32) and melanoma patients generally are not immunosuppressed by their tumors.


   Acknowledgments
 
Grant support: NIH grants CA25874, CA93372, and CA10815, and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jeffrey Faust and Alistaire Acosta for FACS analysis, and Marion Sacks for editorial assistance.

Received 6/ 2/05. Revised 12/13/05. Accepted 1/13/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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