MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 34, Number 5, 2015ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2015.0022

Monoclonal Antibody LpMab-9 Recognizes
O-glycosylated N-Terminus of Human Podoplanin
Mika K. Kaneko,1* Hiroharu Oki,1,2* Yasukazu Hozumi,3 Xing Liu,1,2
Satoshi Ogasawara,1 Michiaki Takagi,2 Kaoru Goto,3 and Yukinari Kato1

Podoplanin (PDPN) induces cell invasion and cancer metastasis, and its expression in cancer cells or cancerassociated fibroblasts has been reported to be involved in poor prognosis of several cancers including malignant
gliomas and lung cancers. PDPN is also expressed in normal cells such as lymphatic endothelial cells, lung typeI alveolar cells, and kidney podocytes. Many anti-PDPN monoclonal antibodies (MAbs) have been established;however, almost all anti-PDPN MAbs recognize a platelet aggregation-inducing (PLAG) domain, because the
PLAG domain is known to be highly immunogenic. Here, we developed and characterized LpMab-9, a novelanti-PDPN MAb. LpMab-9 reacted with LN319 glioblastoma cells, but did not react with LN319/PDPN knockout cells. LpMab-9 showed slight reaction with sialylated O-glycan-deficient PDPN. We identified the minimum epitope of LpMab-9 as Thr25–Asp31, which is the N-terminus of human PDPN, using Western blot
analysis. Furthermore, Thr25, Gly26, Gln27, and Pro28 were shown to be critical for LpMab-9-binding to
PDPN using flow cytometry. Antibody-overlay lectin microarray using LpMab-9 demonstrated that PDPN
reacts with sialic acid – core1 binders and sialo-mucin binders. Taken together, these results indicate that
LpMab-9 recognizes O-glycosylation of Thr25 in the N-terminus of PDPN. LpMab-9 could be useful for
uncovering the physiological function of O-glycosylated N-terminus of human PDPN.

IntroductionP

odoplanin (PDPN)/Aggrus is a platelet aggregationinducing glycoprotein.(1,2) Expression of PDPN has
been reported in malignant brain tumors, esophageal cancers,lung cancers, malignant mesotheliomas, bladder cancers,
testicular tumors, and osteosarcoma.(1,3–14) Moreover, PDPN
expression in cancer-associated fibroblasts (CAFs) is involved in poor prognosis of cancers.(15–19) We recently performed comparative crystallographic studies of PDPN in
complex with C-type lectin-like receptor-2 (CLEC-2).(20)The interaction with CLEC-2 was mainly observed at Glu47
and Asp48 in the PLAG domain and the a2-6 linked sialic
acid at Thr52 of PDPN. Anti-PDPN monoclonal antibodies(MAbs) with high sensitivity and specificity are necessary toinvestigate the physiological function of PDPN in cancers
and normal tissues. Almost all anti-PDPN MAbs such as
NZ-1 and D2-40 react with a platelet aggregation-inducing(PLAG) domain of human PDPN, because the PLAG domain
is highly immunogenic.(7,21–23)
We recently established the platform to produce cancerspecific MAbs (CasMabs).(24) The newly established LpMab-

2 MAb recognized both an aberrant O-glycosylation and a
Thr55-Leu64 peptide from human PDPN. Because LpMab-2
reacted with PDPN-expressing cancer cells but not withnormal cells, it is an anti-PDPN CasMab that is expected to
be useful for molecular targeting therapy against PDPNexpressing cancers. Using CasMab methods, we also developed an anti-PDPN MAb, LpMab-7, which is more sensitive
than NZ-1 MAb in immunohistochemistry.(25) We identified
the minimum epitope of LpMab-7 as Arg79–Leu83 of human
PDPN, which is different from PLAG domain. Immunohistochemical analysis using LpMab-7 showed that PDPN
expression at metastatic lesions was higher compared withprimary lesions.(26) We produced another anti-PDPN MAb,
LpMab-3, the epitope of which is a sialylated glycopeptide of PDPN.(27) We identified the minimum epitope of
LpMab-3 as Thr76–Glu81 of human PDPN. These results
demonstrate that anti-PDPN MAbs against non-PLAG domain are also useful for analyzing function or expressionof PDPN in cancers and normal tissues. Here, we developed and characterized a novel anti-PDPN MAb, LpMab-9,
the epitope of which is the O-glycosylated N-terminus ofhuman PDPN.

1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Department of Orthopaedic Surgery, 3Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine, Yamagata,Japan.
*These authors contributed equally to this work.
2

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LpMab-9 RECOGNIZES O-GLYCOSYLATED N-TERMINUS OF hPDPN
Materials and Methods
Cell lines

LN229, NCI-H226, HEK-293T, Chinese hamster ovary(CHO)-K1, glycan-deficient CHO cell lines (Lec1, Lec2, andLec8), and P3U1 were purchased from the American Type
Culture Collection (ATCC, Manassas, VA). Human lymphatic endothelial cells (LEC) were obtained from Cambrex(Walkersville, MD). The human glioblastoma cell line
LN319 was donated by Dr. Webster K. Cavenee (Ludwig
Institute for Cancer Research, San Diego, CA). LN229,
CHO-K1, Lec1, Lec2, and Lec8 were transfected with human
PDPN plasmids (LN229/hPDPN, CHO/hPDPN, Lec1/
hPDPN, Lec2/hPDPN, Lec8/hPDPN) using Lipofectamine
2000 (Thermo Fisher Scientific, Waltham, MA) according to
the manufacturer’s instructions.(24) LN319/PDPN knock-out(KO) cells were produced by transfecting CRISPR/Cas plasmids, which target PDPN (Sigma-Aldrich, St. Louis, MO),
using a Gene Pulser Xcell electroporation system (Bio-Rad
Laboratories, Philadelphia, PA). CHO-K1, Lec1, Lec2, Lec8,
NCI-H226, and P3U1 were cultured in RPMI 1640 medium(Nacalai Tesque, Kyoto, Japan), and LN229 and LN319 were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)(Nacalai Tesque), supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Life Technologies), 2 mM L-glutamine(Thermo Fisher Scientific), 100 U/mL of penicillin, and 100 mg/
mL of streptomycin (Thermo Fisher Scientific) at 37C in ahumidified atmosphere of 5% CO2 and 95% air. L-proline(0.04 mg/mL) was added for Lec1, Lec2, and Lec8. LEC wascultured in endothelial cell medium EGM-2MV supplemented
with 5% FBS (Cambrex).
Antibodies

LpMab-7 (mouse IgG1, kappa) and RcMab-1 (rat IgG2a,kappa) were developed previously in our labs.(7,21,24,28) AntiFLAG tag MAb (1E6) and anti-b-actin MAb (AC15) werepurchased from Wako Pure Chemical Industries and SigmaAldrich, respectively.
Production of PDPN mutants

The amplified human PDPN cDNA was subcloned into a
pcDNA3 vector (Thermo Fisher Scientific) and a FLAG
epitope tag was added at the C-terminus. Substitution ofamino acids to alanine in PDPN was performed using a
QuikChange Lightning site-directed mutagenesis kit (Agilent
Technologies, Santa Clara, CA).(24,29) CHO-K1 cells weretransfected with the plasmids using a Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories).
Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.)
injection of LN229/hPDPN cells (1 · 108) together with Imject Alum (Thermo Fisher Scientific). After several additional immunizations, a booster injection was given i.p. 2
days before spleen cells were harvested. The spleen cells werefused with P3U1 cells using GenomONE-CF (Ishihara Sangyo
Kaisha, Osaka, Japan). The hybridomas were grown in RPMI
medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific). The
culture supernatants were screened using enzyme-linked im-

311

munosorbent assay (ELISA) for binding to recombinant human PDPN purified from LN229/hPDPN cells.
Western blot analyses

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque).(30) The proteins were electrophoresed
on 5–20% polyacrylamide gels (Wako Pure Chemical Industries, Osaka, Japan) and were transferred onto a PVDF
membrane (EMD Millipore, Billerica, MA). After blocking
with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher
Scientific), the membrane was incubated with primary antibodies (1 mg/mL) and then with peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark; 1:1000 diluted),and developed with the Pierce Western Blotting Substrate Plus
(Thermo Fisher Scientific) using a Sayaca-Imager (DRC Co.,Tokyo, Japan).
Flow cytometry

Cell lines were harvested by brief exposure to 0.25% Trypsin/
1 mM EDTA (Wako Pure Chemical Industries).(31) Afterwashing with phosphate buffered saline (PBS), the cells weretreated with primary antibodies (1 mg/mL) for 30 min at 4C,
followed by treatment with Alexa Fluor 488 conjugated goatanti-mouse IgG or Oregon Green 488 conjugated goat antimouse IgG (Life Technologies). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Immunocytochemical analyses

Cultured cells were fixed with 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) for 15 min at room temperature. Cells were then treated with 10% normal goat serum
in PBS (NGS/PBS) to block non-specific binding sites, andwere incubated with 5 mg/mL of LpMab-7, LpMab-9, or
control (PBS) for 1 h at 4C in a moist chamber. They wereincubated with Alexa Flour 488 conjugated goat anti-mouse
IgG (dilution 1:200; Thermo Fisher Scientific) for 30 min atroom temperature. Cells were also treated with DAPI(Thermo Fisher Scientific) to stain the cell nuclei. They were
examined using confocal laser-scanning microscopy (LSM700;
Carl Zeiss, Jena, Germany). Then fluorescent images were
processed using image-processing software (Adobe Photoshop, Adobe Systems, San Jose, CA).
Affinity determination by surface plasmon resonanceTo determine the affinity, recombinant PDPN-Fc was immobilized on the surface of chips for analysis using the BIAcore 3000 system (GE Healthcare, Piscataway, NJ). The
running buffer was 10 mM HEPES, 150 mM NaCl, and
0.005% v/v Surfactant P20 (pH 7.4; GE Healthcare, BR-100368). LpMab-9 was passed over the biosensor chip, and theaffinity rate constants (association rate constant, kassoc, anddisassociation rate constant, kdiss) were determined by nonlinear curve-fitting using the Langmuir one-site binding modelof the BIAevaluation software (GE Healthcare). The affinity
constant (KA) at equilibrium was calculated as KA = kassoc/kdiss,and the dissociate constant (KD) was determined as 1/KA.
Lectin microarray

PDPN from CHO/hPDPN cells was solubilized using 1%
Triton-X100 in PBS (PBST) and was purified using a FLAG-312

tag system (Sigma-Aldrich). Then, 100 mL of purified PDPN
(31.25–2000 ng/mL) were applied to a lectin array (LecChip
v1.0; GlycoTechnica, Hokkaido, Japan), including triplicate
spots of 45 lectins in each of seven divided incubation bathson the glass slide. After incubation at 20C for 17 h, 4 mL ofhuman IgG (5 mg/mL; Sigma-Aldrich) was applied to eachwell. After incubation at 20C for 30 min, the glass slide waswashed three times with PBST; 60 mL of biotinylated
LpMab-9 (1 mg/mL) in PBS was applied to the array and thenincubated at 20C for 3 h. After washing three times with
PBST, Cy3-labeled streptavidin (Thermo Fisher Scientific)

KANEKO ET AL.

was added to the array and then incubated at 20C for 30 min.
The glass slide was scanned using a GlycoStation Reader
1200 (GlycoTechnica). Abbreviation of lectins are: LTL,
Lotus tetragolonobus lectin; PSA, Pisum sativum agglutinin;
LCA, Lens culinaris agglutinin; UEA I, Ulex europaeus agglutinin I; AOL, Aspergillus oryzae L-fucose-specific lectin;
AAL, Aleuria aurantia lectin; MAL I, Maackia amrensis
lectin I; SNA, Sambucus nigra lectin; SSA, Sambucus sieboldiana agglutinin; TJA-I, Trichosanthes japonica agglutinin I; PHA-L, Phaseolus vulgaris leucoagglutinin; ECA,
Erythrina cristagalli agglutinin; RCA120, Ricinus communis

FIG. 1. (A) Western blot analysis. The membrane was incubated with 1 mg/mL of LpMab-9, LpMab-7, and anti-b-actin, andthen with peroxidase-conjugated anti-mouse IgG; the membrane was detected using a Sayaca-Imager. (B) Flow cytometricanalysis. LN319 and LN319/PDPN-KO cells were treated with LpMab-9 and LpMab-7 (1 mg/mL) for 30 min at 4C, followed
by treatment with Alexa Fluor 488 conjugated goat anti-mouse IgG. Fluorescence data were collected using a Cell Analyzer
EC800. (C) Immunocytochemical analysis. LN319 and LN319/PDPN-KO cells were incubated with 5 mg/mL of LpMab-9,
LpMab-7, or PBS for 1 h at 4C in a moist chamber. They were incubated with Alexa Fluor 488 conjugated goat anti-mouse
IgG for 30 min at room temperature. Cells were also treated with DAPI to stain the cell nuclei. Scale bars, 20 mm.

LpMab-9 RECOGNIZES O-GLYCOSYLATED N-TERMINUS OF hPDPN

agglutinin 120; PHA-E, Phaseolus vulgaris erythroagglutinin;
DSA, Datura stramonium agglutinin; GSL-II, Griffonia
simplicifolia lectin II; NPA, Narcissus pseudonarcissus agglutinin; ConA, Concanavalin A; GNA, Galanthus nivalis
agglutinin; HHL, Hippeastrum hybrid lectin; ACG, Agrocybe
cylindricea galectin; TxLCI, Tulipa gesneriana lectin; BPL,
Bauhinia purpurea alba lectin; TJA-II, Trichosanthes japonica agglutinin; EEL, Euonymus europaeus lectin; ABA,
Agaricus bisporus agglutinin; LEL, Lycopersicon esculentum313

lectin; STL, Solanum tuberosum lectin; UDA, Urtica dioica
agglutinin; PWM, pokeweed mitogen; PNA, peanut agglutinin; WFA, Wisteria floribunda agglutinin; ACA, Amaranthus
caudatus agglutinin; MPA, Maclura pomifera agglutinin;
HPA, Helix pomatia agglutinin; VVA, Vicia villosa agglutinin; DBA, Dolichos biflorus agglutinin; SBA, soybean agglutinin; PTL I, Psophocarpus tetragonolobus lectin I; MAH,
Maackia amurensis hemagglutinin; WGA, wheat germ agglutinin; GSL-I, Griffonia simplicifolia lectin I.

FIG. 2. (A) Western blot analysis. The membrane was incubated with 1 mg/mL of LpMab-9, LpMab-7, and anti-b-actin,
and then with peroxidase-conjugated anti-mouse IgG; the membrane was detected using a Sayaca-Imager. (B) Flowcytometric analysis. CHO/hPDPN, Lec2/hPDPN, and Lec8/hPDPN were treated with LpMab-9 and LpMab-7 (1 mg/mL) for
30 min at 4C, followed by treatment with Oregon Green 488 conjugated goat anti-mouse IgG. Fluorescence data werecollected using a Cell Analyzer EC800. (C) Glycan profiling of PDPN using antibody-overlay lectin microarray. Purified
PDPN was applied to a lectin array including 45 lectins. After blocking, biotinylated LpMab-9 was applied, and Cy3-labeledstreptavidin was added. Signal was scanned using a GlycoStation Reader 1200.

314
Results
Production and characterization of novelanti-PDPN monoclonal antibody LpMab-9

To develop novel anti-PDPN MAbs, we immunized micewith LN229/hPDPN cells. The culture supernatants werescreened using ELISA for binding to recombinant human
PDPN purified from LN229/hPDPN cells. After limiting the

KANEKO ET AL.

dilution of the hybridomas, LpMab-9 (IgG1, kappa) was established. LpMab-9 reacted with LN319 cells, not with
LN319/PDPN knock-out (KO) cells in Western blot analysis(Fig. 1A), flow cytometry (Fig. 1B), and immunocytochemical analysis (Fig. 1C). LpMab-7, which was used as a positive control, reacted with PDPN of LN319 (Fig. 1A–C).
Furthermore, LpMab-9 detected endogenous PDPN, which isexpressed in a lymphatic endothelial cell (LEC) (Fig. 2A).

FIG. 3. Epitope mapping of LpMab-9 by Western blot and flow cytometry. (A) Western blot analysis. The membrane wasincubated with 1 mg/mL of LpMab-9, LpMab-7, 1E6 (anti-FLAG), RcMab-1 (a-IDH1), and AC-15 (anti-b-actin) and thenwith peroxidase-conjugated secondary antibodies. The membrane was detected using a Sayaca-Imager. (B) Point mutants ofhuman podoplanin were treated with LpMab-9 (1 mg/mL) for 30 min at 4C, followed by treatment with Alexa Fluor 488
conjugated goat anti-mouse IgG. Fluorescence data were collected using a Cell Analyzer EC800.

LpMab-9 RECOGNIZES O-GLYCOSYLATED N-TERMINUS OF hPDPN

LpMab-9 faintly reacted with Lec2/hPDPN (sialic acid deficient) and Lec8/hPDPN (O-glycan deficient) cells, although
it strongly reacted with CHO/hPDPN and Lec1/hPDPN (Nglycan deficient) in Western blot analysis (Fig. 2A). Flowcytometric analysis also showed that the reaction of LpMab-9
against Lec2/hPDPN and Lec8/hPDPN decreased comparedwith CHO/hPDPN, indicating that the epitope of LpMab-9
includes sialylated O-glycan of PDPN. In contrast, LpMab-7
strongly detected all types of PDPN (Fig. 2A, B). We next
performed lectin microarray analysis of PDPN. We previously established antibody-overlay lectin microarray.(7,32)
Here, we applied LpMab-9 to this system. As shown in Figure
2C, PDPN reacted with Jacalin, one of sialic acid – core1
binders and wheat germ agglutinin (WGA), one of sialomucin binders. We performed a kinetic analysis of theinteraction of LpMab-9 with a recombinant PDPN using
surface plasmon resonance (BIAcore). Determination of theassociation and dissociation rates from the sensorgrams revealed that a kassoc of 6.79 · 103 (mol/L-s)-1 and a kdiss of
2.21 · 10-3 s-1. The KA at binding equilibrium, calculated as
KA = kassoc/kdiss, was 3.07 · 106 (mol/L)-1, KD = 1/KA = 3.25 ·10-7 M.
Epitope mapping by Western blot analysis
and flow cytometry

To determine the LpMab-9 epitope, we first performed
Western blot analysis. LpMab-9 reaction was lost in point
mutations of Thr25-Asp30 (Fig. 3A). The faint band wasobserved in D31A. This epitope includes Thr25, the only Ser/Thr residue, indicating that Thr25 is O-glycosylated and isessential for LpMab-9 recognition. In contrast, LpMab-7
reaction was observed in all point mutants. LpMab-9 detected
only one band (40 kDa), although both LpMab-7 and antiFLAG MAb detected two bands (40 kDa and 35 kDa). We
next performed flow cytometric analysis using LpMab-9
against the same point mutants of PDPN. The results revealed
that LpMab-9 weakly reacted with T25A and did not reactwith G26A, Q27A, and P28A (Fig. 3B), indicating that the
TGQP sequence is the critical epitope of LpMab-9.
Discussion

LpMab-9 possesses a unique epitope, which is totallydifferent from previously reported anti-PDPN MAbs such as
NZ-1, D2-40, LpMab-2, LpMab-3, and LpMab-7.(21,24–27)
The epitope of LpMab-9 needs O-glycosylation of Thr25.
Previous reports showed that Thr25 is not O-glycosylated
because O-glycan was not detected using several methods,(24,33) indicating that site-specific MAbs against glycopeptides are more advantageous for detecting O-glycan
compared with mass spectrometry or Edman sequencing.
Therefore, LpMab-9 is useful for distinguishing Thr25glycosylated PDPN with Thr25-nonglycosylated one. The
staining patterns of LpMab-7 and LpMab-9 look slightly
different (Fig. 1C). We showed that LpMab-9 recognizes
only sialylated PDPN (Fig. 2A, B); therefore, not all fractionsof PDPN are detected by LpMab-9. In contrast, LpMab-7 canreact with all types of PDPN independently of glycosylation,
which might lead to the immunocytochemical difference.
Antibody-overlay lectin microarray was previously developed.(32) Specific and sensitive monoclonal antibodies arenecessary for this system. In this study, we applied LpMab-9

315

to this system for investigating the glycan profiling of PDPN.
Our previous study using NZ-1 showed that PDPN reacted
strongly with sialic acid – core1 binders (ABA, Jacalin,
ACA, and MPA), sialo-mucin binders (MAH and WGA), andalpha-GalNAc binder (HPA). In contrast, using LpMab-9,
PDPN reacted with only Jacalin and WGA (Fig. 2C). This
inconsistency may be derived from the different binding
epitopes and binding affinities of anti-PDPN MAbs. Both
LpMab-7 and anti-FLAG MAbs detect two bands (40 kDa
and 35 kDa); in contrast, LpMab-9 detected only one band
(40 kDa). Our previous study showed that another anti-PDPN
MAb, clone NZ-1, also detects only one band (40 kDa), although the epitope of NZ-1 does not include glycosylation.(7,24) Therefore, we cannot conclude that 35-kDa band isa non-glycosylated form. In the near future, we should determine what kind of post-translational modification is involved in this specific 35 kDa isoform of PDPN. The binding
affinity of LpMab-9 was shown to be low (KD, 3.25 · 10-7 M)
using BIAcore analysis. However, we previously demonstrated that KD of LpMab-9 is 1.6 · 10-8 M using flow cytometry against LN229/hPDPN, indicating that the binding
affinity of LpMab-9 against PDPN is much higher in cellbased assay.(24) We performed immunohistochemical analyses using LpMab-9 against several normal tissues andcancers; however, LpMab-9 did not stain any PDPN-positive
cells probably because of the low binding affinity of LpMab9 or the loss of sialic acid on PDPN in paraffin-embedded
tissues.
PDPN is highly expressed not only in cancer cells but also
in normal tissues.(34,35) The activation of CLEC-2 by PDPN
rearranges the actin cytoskeleton in dendritic cells to promote
efficient motility along stromal surfaces.(36) In contrast, thesignal from CLEC-2 to PDPN controls the contractility offibroblastic reticular cells and lymph node microarchitecture.(37)The physical elasticity of lymph nodes is maintained by
PDPN of stromal fibroblastic reticular cells.(38) PDPNCLEC-2 interaction is also important for embryonic bloodlymphatic vascular separation using platelet aggregation.(39)
To investigate these complicated functions of PDPN, different epitope-possessing anti-PDPN MAbs are necessary
because site-specific glycosylation is observed in pathological conditions. Until now, we established different epitopepossessing anti-PDPN MAbs such as NZ-1, LpMab-2,
LpMab-3, LpMab-7, and LpMab-9, and LpMab.(21,24–27,40,41)
Among those clones, LpMab-2, LpMab-3, and LpMab-9
detect site-specific O-glycosylation: LpMab-2 against Thr55/
Ser56, LpMab-3 against Thr76, and LpMab-9 against Thr25.
Different epitope-possessing anti-PDPN MAbs should befurther developed for analyzing function of PDPN in cancers
and normal tissues in the future.
Acknowledgments

We thank Yuta Tsujimoto, Takuro Nakamura, Noriko
Saidoh, and Kanae Yoshida for their excellent technical assistance. This work was supported in part by the Platform for
Drug Discovery, Informatics, and Structural Life Science(PDIS) from Japan Agency for Medical Research and Development, AMED (Y.K.); by the Basic Science and Platform Technology Program for Innovative Biological
Medicine from AMED (Y.K.); by the Regional Innovation
Strategy Support Program from the Ministry of Education,

316

Culture, Sports, Science and Technology (MEXT) of Japan(Y.K.); and by, and by JSPS KAKENHI Grant Number
25462242 (Y.K.) and Grant Number 26440019 (M.K.K.).
Author Disclosure Statement

The authors have no financial interests to disclose.
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Address correspondence to:
Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: April 13, 2015
Accepted: June 12, 2015