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Experimental Therapeutics, Preclinical Pharmacology |
Department of Pathology, Sapporo Medical University School of Medicine, Chuo-ku Sapporo 060-8543 [Y. H., T. T., A. M., Y. N., K. K., H. I., N. S.]; Hokkaido Prefectural Haboro Hospital, Haboro 078-4197 [T. S.] Department of Surgery, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543 [K. H.]; and Department of Otolaryngology, Wakayama Medical College, Wakayama 641-8510 [Y. H., J. Y., N. Y.], Japan
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ABSTRACT |
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Ex3 in various cancer cells, immortalized cells, and normal adult tissues. It was demonstrated that two splicing variants were detected in various types of cancer cells as well as survivin, and their expression was more restricted to cancer cells as compared with survivin expression. To identify HLA-A24-restricted T-cell epitopes from survivin and the variant proteins, three peptides were selected from amino acid sequence of these proteins, based on the HLA-A24-binding motif. Peptide binding assay to HLA-A24 revealed that only one peptide designated as survivin-2B80–88 (AYACNTSTL) was capable of binding to HLA-A24. By stimulating peripheral blood lymphocytes with the peptide-pulsed antigen-presenting cells, CTLs were successfully induced in vitro from five of five HLA-A24-positive cancer patients. The CTLs showed significant cytotoxicity against HLA-A24-positive survivin-2B-positive cancer cells. These data suggest that survivin-2B80–88 may be a potent T-cell epitope eliciting CTL response against a splicing variant survivin-2B, which is specifically expressed in many kinds of cancer cells. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Apoptosis is a physiological suicide program that is critical for development and maintenance of tissue homeostasis. Cells with genetic abnormality can be eliminated by apoptosis. Therefore, diminished or disordered apoptosis results in accumulation of genetic mutation, which may lead to tumor initiation, progression, and therapeutic resistance. Survivin was identified as a member of the IAP family with single baculovirus IAP repeat domain (11) . Survivin has a capability to inhibit caspase-3, -7, and -9 in cells receiving an apoptotic stimulus (12) . Survivin is present during fetal development but undetectable in terminally differentiated normal adult tissues. Importantly, survivin is abundantly expressed in transformed cell lines and in most of common cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer, bladder cancer, breast cancer, melanoma, neuroblastoma, hepatocellular carcinoma, and diffuse large B-cell lymphoma (13) . The overexpression of survivin in tumors correlates with resistance to a variety of apoptotic stimuli, including chemotherapeutic agents and radiation therapy, and is closely associated with poor clinical prognosis.
Mahotka et al. (14)
reported that two novel splice variants of survivin were expressed in renal cell carcinoma lines. One is survivin-Ex3 lacking exon 3 of survivin, and the other is survivin-2B retaining a part of intron 2 as a cryptic exon. In their transfection experiments, survivin-Ex3 conserved antiapoptotic potential, and survivin-2B also had significant antiapoptotic potential, although their physiological functions have not been clarified. Using the RT-PCR method, we show here that both survivin-Ex3 and survivin-2B are expressed in various types of tumor cell lines as well as survivin but not in normal tissues. On the basis of this finding, we hypothesized that survivin and its two splicing variants might become target molecules of CTLs. Three peptides were determined from survivin-2B amino acid sequence according to the binding motif for HLA-A*2402, the most common allele in the Japanese population and also frequent in Caucasians. One of these peptides, survivin-2B80–88, was capable of inducing CTL response in the context of HLA-A*2402, when pulsed with APCs. CTLs were induced in vitro from both cancer patients and normal healthy donors, and the CTLs exerted cytotoxic activity against survivin-2B-expressing tumor cell lines in an HLA-A24-restricted manner. We demonstrate for the first time the HLA-A24-restricted CTL epitope derived from the survivin splicing variant, which may serve as a widely applicable antigen for cancer immunotherapy of HLA-A24-positive patients.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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RT-PCR Analysis of the Expression of Survivin and Its Splicing Variants.
A set of total RNA from normal human adult tissues was purchased from Clontech (human total RNA master panel). Total RNA was isolated from cultured cells by using ISOGEN reagent (Nippon Gene, Tokyo, Japan). The cDNA mixture was synthesized from 1 µg of total RNA by reverse transcription using Superscript II and oligo(dT) primer (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocols. PCR amplification was performed in 50 µl of PCR mixture containing 1 µl from the cDNA mixture, KOD Plus DNA polymerase (Toyobo, Osaka, Japan), and 50 pmol of primers. The PCR mixture was initially incubated at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 57°C (or 64°C for specific detection of survivin-2B or survivin-Ex3) for 30 s, and extension at 68°C for 1 min. Primer pairs used for RT-PCR analysis were 5'-TCAAGGACCACCGCATCTCTAC-3' and 5'-GCACTTTCTTCGCAGTTTCCTC-3' as a forward and a reverse primer, respectively. Expected sizes of PCR products for survivin, survivin-Ex3, and survivin-2B were 355, 236, and 424 bp, respectively. For specific detection of survivin-2B and survivin-Ex3 expression, the following reverse primers were used: 5'-GTGCTGGTATTACAGGCGTAAG-3' for survivin-2B with an expected PCR product of 221 bp; and 5'-TGGTTTCCTTTGCATGGGG-3' for survivin-Ex3 with an expected PCR product of 195 bp. As an internal control glyceraldehyde-3-phosphate dehydrogenase was detected by using a forward primer 5'-ACCACAGTCCATGCCATCAC-3' and a reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' with an expected PCR product of 452 bp. The PCR products were visualized with ethidium bromide staining under UV light after electrophoresis on 1.0% agarose gel. Nucleotide sequence of the PCR products was confirmed by direct sequencing using ABI Genetic analyzer PRIM 310 and an AmpliCycle sequencing kit (Perkin-Elmer, Foster City, CA).
Cloning of HLA-A*2402 cDNA.
Total RNA from LHK-2 cells was extracted and cDNA mixture was synthesized from the RNA by reverse transcription reaction using Superscript II and oligo(dT) primer as described previously. PCR was performed using 1 µl of the reverse transcription mixture, Pfu DNA polymerase (Stratagene, La Jolla, CA), and 50 pmol of forward and reverse primers in a total reaction volume of 50 µl. Nucleotide sequences of the forward and reverse primers are 5'-GACTCAGATGATATCCAGACGCCGAGGATGGCCGTCATG-3' and 5'-CGCGGATCCGCGGCCGCAGGGAGCACAGGTCAGCGTGGGAA-3', which contain HLA-A gene-specific sequence flanked by EcoRV or BamHI restriction sequence, respectively. The amplification protocol consisted of denaturation for 15 s at 98°C, annealing for 45 s at 58°C, and extension for 4 min at 72°C for a total of 30 cycles, using a GeneAmp PCR system model 2400 (Perkin-Elmer). The PCR product was purified and cloned into pIRESpuro mammalian expression vector (Clontech, Palo Alto, CA). Nucleotide sequence of the insert was analyzed by ABI Genetic analyzer PRIM 310 using AmpliCycle sequencing kit (Perkin-Elmer).
Synthetic Peptides.
Three peptides survivin85–93 (AFLSVKKQF), survivin92–101 (QFEELTLGEF), and survivin-2B80–88 (AYACNTSTL) were derived from the amino acid sequence of survivin or survivin-2B (11
, 14) , based on the HLA-A24-binding motifs. Three peptides used as control peptides were EBV LMP2-derived peptide (TYGPVFMSL) and HIV env-derived peptide (RYLRDQQLLGI), which have been shown to become CTL epitopes in the context of HLA-A*2402 previously (15
, 16)
, and F4.2 peptide (YSWMDISCWI) that was identified as HLA-A31-bound gastric cancer peptide (4)
. All of the peptides were synthesized and purchased from Sigma Genosis (Ishikari City, Japan). Peptides were dissolved in DMSO and stored at -80°C before use.
Cytokines.
Human recombinant IL-2 and IL-4 were kind gifts from Takeda Pharmaceutical (Osaka, Japan) and Ono Pharmaceutical (Osaka, Japan), respectively. Human recombinant granulocyte/macrophage-colony stimulating factor was a kind gift from Novartis Pharmaceuticals (Basel, Switzerland). Human recombinant IL-7 was purchased from Life Technologies, Inc.
Peptide Binding Assay.
Peptide binding affinity to HLA-A24 was assessed by HLA-A24 stabilization assay as described previously (16)
, based on the findings that MHC class I molecules could be stabilized on the cell surface in the presence of binding peptides. RMA-S A*2402/Kb cells are TAP-deficient mouse RMA-S cells transfected with a chimeric MHC class I cDNA consisting of 
1 and 2 domains derived from human HLA-A*2402 molecule and 3, transmembrane, and intracellular domains derived from mouse H-2Kb molecule (kindly provided from Dr. H. Takasu, Sumitomo Pharmaceutical, Osaka, Japan). After incubation of cells in culture medium at 26°C for 18 h, cells (2 x 105) were washed with PBS and suspended with 1 ml of Opti-MEM (Life Technologies, Inc.) containing 3 µg/ml of 2-microglobulin with or without 100 µg of peptide, followed by incubation at 26°C for 3 h and then at 37°C for 3 h. After washing with PBS, the cells were incubated with anti-HLA-A24 mAb (c7709A2.6, kindly provided by Dr. P. G. Coulie, Ludwig Institute for Cancer Research, Brussels Branch) at 4°C for 30 min, followed by incubation with FITC-conjugated rabbit antimouse IgG at 4°C for 30 min. The cells were then suspended with 1 ml of PBS containing 1% formaldehyde and analyzed by FACScan (Becton Dickinson, Mountain View, CA). Binding affinity was evaluated by comparing mean fluorescence intensity of HLA-A24 expression in the presence of peptide pulsation to mean fluorescence intensity in the absence of the peptide.
Preparation of APCs and CD8-positive T Cells from PBMCs.
PBMCs were isolated from four healthy volunteers and five cancer patients (patient 1, colon cancer; patient 2, breast cancer; patient 3, esophagus cancer; patients 4 and 5, gastric cancer) by standard density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). PBMCs were incubated in AIM-V medium (Life Technologies, Inc.) supplemented with 2-mercaptoethanol (50 µM) and HEPES (10 mM) for 2 h at 37°C in a culture flask to separate adherent cells and nonadherent cells. Adherent cells were then cultured in the presence of IL-4 (1000 units/ml) and granulocyte/macrophage-colony stimulating factor (1000 units/ml) in AIM-V medium for 7 days to generate monocytes-derived DCs. The adherent cells containing DCs were used for APCs. CD8-positive T lymphocytes were isolated from nonadherent cells by the MACS separation system (Miltenyi Boitech, Bergish Blabach, Germany) using anti-CD8 mAb coupled with magnetic microbeads according to the manufacturer’s instruction. To obtain PHA-stimulated blasts, CD8-negative nonadherent PBMCs were cultured in AIM-V medium containing 1 µg/ml of PHA (WAKO Chemicals, Osaka, Japan) and 100 units/ml of IL-2 for 3 days, followed by washing and cultivation in the presence of IL-2 (100 units/ml) for 4 days.
CTL Induction Using Autologous DCs and PHA-Blasts.
CTL induction was performed according to a procedure described previously with a slight modification (17
, 18)
. Briefly, autologous DCs were incubated at room temperature for 2 h in AIM-V with ß2-microglobulin (2.5 µg/ml) and peptide (50 µg/ml). DCs were then irradiated (100 Gy) and washed with AIM-V medium. On day 1, 1 x 105 peptide-pulsed DCs/well were plated on 24-well plates and cultured with 1 x 106 CD8+ T cells in 2 ml of AIM-V supplemented with recombinant IL-7 (10 ng/ml). On day 7, 5 x 105 PHA-blasts were pulsed with 50 µg/ml of peptides, irradiated (100 Gy), washed once, and then added to each well. On day 8, IL-2 was added to each well at a concentration of 50 units/ml. The peptide stimulation using PHA-blasts as APCs was repeated every 7 days. During CTL induction, cells were fed with fresh AIM-V medium supplemented with IL-2 (50 units/ml) every 3–4 days. On day 28, the cytotoxic activity of T cells was assessed by conventional 6-h 51Cr release assay.
Cytotoxicity Assay.
The lytic activity of CTLs was tested by a 51Cr release assay as described previously (19)
. Briefly, target cells were labeled with 100 µCi of 51Cr for 1 h at 37°C, washed three times, and resuspended in AIM-V medium. Then 51Cr-labeled target cells (2000 cells/well) were incubated with various numbers of effector cells for 6 h at 37°C in 96-well microtiter plates. Radioactivity of the culture supernatant was measured by gamma counter. The percentage of cytotoxicity was calculated as following: % cytotoxicity = (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100. In some cases, target cells (LHK-2, LNY-1, 888-mel, and 1353-mel) were treated with 100 units/ml of IFN-
for 48–72 h before the assay. For peptide-pulsed target cells, C1R-A*2402 or C1R-A*31012 cells were incubated with 1 µg/ml of peptide at room temperature for 1 h before the assay.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. A variety of tumor cell lines and immortalized cells were tested, including two adenocarcinoma lines, four squamous cell carcinoma lines, four melanoma lines, one renal cell carcinoma line, one bladder carcinoma line, and four EBV-transformed B-cells. Our study demonstrated the expression of survivin in all of the cancer cell lines and EBV-transformed B-cells tested (Fig. 2A)
, which was consistent with previous reports. A low level of survivin expression was also detected in PBMCs. For detection of two splicing variants of survivin (survivin-2B and survivin-Ex3), RT-PCR analysis was performed using the variant-specific primer sets. It was shown that both variant forms of survivin were detected in all of the tumor cell lines and EBV-transformed B-cells but not detected in PBMCs (Fig. 2A)
. Expression of survivin and the splicing variants was also assessed in normal adult tissues including stomach, small intestine, large intestine, spleen, lung, kidney, prostate, pancreas, heart, and thymus. As shown in Fig. 2B
, low levels of survivin expression were detected in all of these normal tissues. However, no expression of the two splicing variants was detected, except in the thymus. Thus, our data indicate that the two splicing variants, as compared with survivin, have a more restricted expression profile to cancer cells.
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Ex3 was screened for possible HLA-A24-bound nona- or decamer peptide, based on the HLA-A24-specific anchor motif (20)
. The motif consists of Y, F, M, or W at position 2 and F, L, I, W, or M at the COOH-terminal residue. As shown in Fig. 1B
, two peptides, survivin85–93 (AFLSVKKQF) and survivin92–101 (QFEELTLGEF) from the exon 3-encoded region, and one peptide, survivin-2B80–88 (AYACNTSTL) from exon 2B-encoded region, were determined as possible CTL epitopes presented by HLA-A24. Then the peptides were tested for binding affinity to HLA-A*2402 by using RMA-S cells transfected with HLA-A*2402/Kb chimera gene. It is known that MHC class I molecules become unstable in the absence of binding peptides. Because RMA-S cells lack TAP molecules, which transport peptides into the endoplasmic reticulum, the MHC class I level on the cell surface of RMA-S cells is low as compared with wild-type RMA cells. In the presence of binding peptides, MHC class I is stabilized, leading to up-regulation on the cell surface. As shown in Fig. 3
, within the three survivin peptides only survivin-2B-derived peptide survivin-2B80–88 was capable of increasing the HLA-A24 level on RMA-A A*2402/Kb cells. EBV (LMP2) peptide and HIV (env) peptide, which have been reported to be HLA-A24-presentable CTL epitopes, increased the HLA-A24 level as well, whereas F4.2 peptide, an HLA-A31-bound peptide, failed. These data indicate that survivin-2B80–88, but not survivin85–93 or survivin92–101, has a significant binding affinity to HLA-A*2402 that is comparable with that of HIV (env) peptide.
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, CTLs recognizing survivin-2B80–88 peptide were induced from PBMCs of all of the five HLA-A24-positive cancer patients. Increased E:T ratio resulted in increased cytotoxic activity when 1 µg/ml of peptide was pulsed onto C1R-A*2402 target cells. However, the CTLs could not show cytotoxic activity against peptide-pulsed C1R-A*31012 cells or K562 cells, indicating that CTLs recognized the peptide in the context of HLA-A*2402. It was also possible to induce CTLs from two of four HLA-A24-positive healthy volunteers (Fig. 5)
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, all of the CTLs induced from PBMCs of five cancer patients exerted significant cytotoxic activity against LNY-1-A24 cells but not against HLA-A24-negative LNY-1 cells. These data indicate that CTLs induced by survivin-2B80–88 ex vivo are capable of recognizing endogenously processed survivin-2B peptide in tumor cells in the context of HLA-A*2402. Furthermore, CTLs from patients 3 and 5 were tested for their cytotoxic activity against endogenous HLA-A24-expressing cancer cells, such as 888-mel melanoma cells (HLA-A1 and HLA-A24) and LHK-2 cells (HLA-A2 and HLA-A24). Both of these cells express survivin-2B detected by RT-PCR (Fig. 2A)
. As shown in Fig. 6B
, CTLs from patients 3 and 5 showed significant cytotoxic activity against HLA-A24-positive tumor cells but not against HLA-A24-negative tumor cells including LNY-1, 1353-mel, and K562 cells. These data strongly indicate that CTLs induced by survivin-2B80–88 peptide could kill tumor cells expressing survivin-2B and HLA-A24 and therefore suggest that the peptide might be useful for eliciting antitumor immune response in HLA-A24-positive cancer patients.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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In the present study, we focused on an IAP family protein survivin that is preferentially expressed in many cancer cells and tissues. Thus far, there have been several reports showing that survivin expressed in tumor cells could elicit both humoral and cellular immune responses in cancer patients. Anti-survivin antibodies were detected in sera of lung cancer patients and colon cancer patients (23 , 24) . HLA-A2-restricted CTL responses against survivin-derived peptides were shown (9) in melanoma patients and chronic lymphocytic leukemia patients by ELISPOT assay (10) . Tumor-infiltrating T cells reactive to survivin-derived peptides were also detected in situ by immunohistochemical staining of tumor tissues using FITC-conjugated multimeric peptide/MHC complexes (25) . These findings clearly demonstrate that survivin protein expressed in tumor cells is immunogenic to cancer patients.
Initially, it was reported that survivin was expressed specifically in cancer cells but not in normal tissues. However, it was revealed that survivin was a crucial element for mitosis of cells, and even noncancerous cells expressed survivin during mitosis (13)
. Our study actually demonstrated the low level of survivin expression in PBMCs and most of normal tissues (Fig. 2)
. These findings raise a possible limitation as to cancer-specific immunotherapy using survivin-derived peptides, because CTLs may injure normal proliferating cells as well as cancer cells. A similar drawback is also pointed out for hTERT-derived peptides, because hTERT expression is required for the maintenance of stem cells. Intriguingly, it was shown that two splicing variants were produced from a survivin gene (14)
. Survivin-Ex3 lacks exon 3 of survivin and contains frame-shift exon 4 and an extra-open reading frame consisting of the 3'-untranslated region. Survivin-2B contains a cryptic exon named exon 2B between exon 2 and exon 3 (Fig. 1A)
. Therefore, the variant proteins encoded by these splicing variants should contain a unique amino acid sequence to each variant protein, which may serve as CTL epitopes eliciting antitumor immune responses. Our study showed that both splicing variants were expressed in a variety of cancer cells as well as survivin. Importantly, the expression of splicing variants was more specific to cancer cells except for thymus (Fig. 2)
than survivin expression. In addition, it is possible that immune escape of tumor cells by loss of the expression of these variants may bring a disadvantage to tumor cells, because the splicing variant proteins conserve antiapoptotic potential in tumor cells as well as survivin. Taken together, it is speculated that the survivin variants may have the capability to become ideal tumor rejection antigens.
We demonstrated HLA-A24-restricted CTL responses against survivin-2B-derived peptide for the first time. The CTLs induced by the mixed lymphocytes peptide culture in the presence of DCs exerted a significant cytotoxic activity against survivin-2B-expressing cancer cells in the context of HLA-A24, indicating that survivin-2B80–88 (AYACNTSTL) might become a good candidate of peptide vaccines for HLA-A24-positive patients.
In the current study, CTLs were induced from PBMCs of two of four HLA-A24-positive healthy volunteers as well as cancer patients. Because survivin-2B is expressed in EBV-transformed B cells (Fig. 2A)
and PHA-blasts (data not shown), it is possible to speculate that previous viral infection might trigger off the sensitization of T cells, leading to increased CTL precursor frequency.
In general, CTLs are supposed to exert cytotoxicity on target cells by inducing apoptotic cell death. Because survivin is a member of IAP that inhibits caspase activation, one may ask how cancer cells with high survivin expression are sensitive to CTL-mediated cytotoxicity. Recently, it was reported that granzyme B, a component of cytotoxic granules, could directly cleave DFF45/ICAD protein, causing apoptosis of target cells without activation of caspase-3 (26) . Hence, overexpression of survivin may not be able to protect tumor cells from CTL-mediated apoptosis. In contrast, overexpression of survivin is correlated with resistance to chemotherapeutics or radiation therapy (13) . In this context, immunotherapy targeting survivin and its splicing variants may be advantageous to patients who suffer chemoresistant or radiation-resistant cancers.
In conclusion, we demonstrated the expression of survivin and its two splicing variants survivin-2B and survivin-Ex3 in a wide variety of cancer cells. Survivin-2B-derived peptide could induce CTL response in the context of HLA-A*2402. This peptide may serve as a widely applicable cancer antigen for HLA-A24-positive patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Pathology, Sapporo Medical University School of Medicine, South-1 West-17, Chou-ku Sapporo 060-8556, Japan. Phone: 81-11-613-8374; Fax: 81-11-643-2310; E-mail: nsatou{at}sapmed.ac.jp 
2 The abbreviations used are: TAA, tumor-associated antigen; IAP, inhibitor of apoptosis protein; RT-PCR, reverse transcription-PCR; IL, interleukin; mAb, monoclonal antibody; APC, antigen-presenting cell; PBMC, peripheral blood mononuclear cell; DC, dendritic cell; PHA, phytohemagglutinin; hTERT, human telomerase reverse transcriptase subunit.
Received 2/12/02; accepted 3/14/02.
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REFERENCES |
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Ex3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res., 59: 6097-6102, 1999.This article has been cited by other articles:
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T. Kurotaki, Y. Tamura, G. Ueda, J. Oura, G. Kutomi, Y. Hirohashi, H. Sahara, T. Torigoe, H. Hiratsuka, H. Sunakawa, et al. Efficient Cross-Presentation by Heat Shock Protein 90-Peptide Complex-Loaded Dendritic Cells via an Endosomal Pathway J. Immunol., August 1, 2007; 179(3): 1803 - 1813. [Abstract] [Full Text] [PDF]
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 S Abdul-Rasool, S H Kidson, E Panieri, D Dent, K Pillay, and G S Hanekom An evaluation of molecular markers for improved detection of breast cancer metastases in sentinel nodes. J. Clin. Pathol., March 1, 2006; 59(3): 289 - 297. [Abstract] [Full Text] [PDF]
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S. Idenoue, Y. Hirohashi, T. Torigoe, Y. Sato, Y. Tamura, H. Hariu, M. Yamamoto, T. Kurotaki, T. Tsuruma, H. Asanuma, et al. A Potent Immunogenic General Cancer Vaccine That Targets Survivin, an Inhibitor of Apoptosis Proteins Clin. Cancer Res., February 15, 2005; 11(4): 1474 - 1482. [Abstract] [Full Text] [PDF]
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R. Xiang, N. Mizutani, Y. Luo, C. Chiodoni, H. Zhou, M. Mizutani, Y. Ba, J. C. Becker, and R. A. Reisfeld A DNA Vaccine Targeting Survivin Combines Apoptosis with Suppression of Angiogenesis in Lung Tumor Eradication Cancer Res., January 15, 2005; 65(2): 553 - 561. [Abstract] [Full Text] [PDF]
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