INFECTION AND IMMUNITY, Sept. 1997, p. 3556–3560 0019-9567/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 65, No. 9
Relevance of Immunoglobulin G Fc Receptor Polymorphism to Recurrence of Adult Periodontitis in Japanese Patients TETSUO KOBAYASHI,1 NOMDO A. C. WESTERDAAL,2 AKIRA MIYAZAKI,1 W.-LUDO VAN DER POL,2 TAKASHI SUZUKI,1 HIROMASA YOSHIE,1 JAN G. J. VAN DE WINKEL,2,3 AND KOHJI HARA1* Department of Periodontology, Niigata University School of Dentistry, Niigata, Japan,1 and Department of Immunology2 and Medarex Europe,3 University Hospital Utrecht, 3584 CX Utrecht, The Netherlands Received 19 December 1996/Returned for modification 3 February 1997/Accepted 13 June 1997
Polymorphonuclear neutrophil (PMN) phagocytic function has been shown to be impaired in some patients with periodontitis. PMN constitutively express members of two immunoglobulin G receptor (FcgR) classes: FcgRIIa (CD32) and FcgRIIIb (CD16). Both receptors exhibit genetically determined structural and functional biallelic polymorphisms, which have been shown to influence PMN phagocytic function. In this study, we assessed the relevance of these FcgR polymorphisms to susceptibility to adult periodontitis and recurrence rate. The distribution of FcgRIIa and FcgRIIIb genotypes of 100 Japanese patients with adult periodontitis during follow-up was compared to the distribution of genotypes in 105 race-matched healthy controls. No significant skewing of distributions of FcgRIIa and FcgRIIIb genotypes was observed between patients and controls. Notably, however, a significant overrepresentation of the FcgRIIIb-NA2 allotype was found in patients with disease recurrence (P < 0.05; odds ratio, 4.29; 95% confidence interval, 1.19 to 16.24). Moreover, the annual rate of recurrence was significantly higher in patients with the FcgRIIIb-NA2/NA2 and FcgRIIIbNA1/NA2 genotypes than in FcgRIIIb-NA1/NA1 individuals (P < 0.05). FcgRIIa-R/R131 individuals also exhibited higher recurrence rates, though the difference was not statistically significant (P 5 0.06). These results suggest that the FcgRIIIb-NA2 allotype represents a risk factor for recurrence of adult periodontitis. NA1/NA1 individuals (3, 23–25, 29). To determine whether FcgR allotypes influence incidence of adult periodontitis or clinical outcome, we compared the distributions of FcgRIIa and FcgRIIIb genotypes between Japanese patients and racematched healthy controls, as well as between patients with and without recurrence of disease.
Adult periodontitis, which is a major cause of tooth loss, is characterized by destruction of periodontal tissues. Some patients respond poorly to conventional treatment and suffer from recurrence, which results in progressive attachment loss (8, 16). This recurrence might be associated with compromised function of polymorphonuclear neutrophils (PMN) (15, 20). Polymorphisms of the receptors for the constant part of immunoglobulin G (IgG) (FcgR) have been shown to influence PMN phagocytic function (2, 22, 24, 25). Human PMN constitutively express members of two FcgR classes, FcgRIIa (CD32) and FcgRIIIb (CD16), both of which exhibit genetically determined structural and functional biallelic polymorphisms (4, 17, 18, 30). FcgRIIa exhibits either an arginine (FcgRIIa-R131) or a histidine (FcgRIIa-H131) at amino acid position 131 in the ligand binding site. This difference was found to be critical for the binding of human IgG2 complexes (22, 31). In vitro studies showed that PMN obtained from FcgRIIa-H/H131 individuals internalize human IgG2-opsonized particles more efficiently than those from FcgRIIa-R/R131 individuals (1, 2, 26, 27). Since IgG2 is considered an important subclass in the defense against encapsulated bacteria (11), the hypothesis that the FcgRIIa-R/R131 genotype constitutes a risk factor for such infections was put forward (1, 27). Furthermore, a role for this FcgRIIa polymorphism in the pathogenesis of localized juvenile periodontitis has been proposed (33). The two allelic forms (allotypes) of the PMN-specific FcgRIIIb differ by four amino acids and two potential glycosylation sites (10, 17, 18, 29). PMN from FcgRIIIb-NA2/NA2 individuals were shown to exhibit lower phagocytic capacities of human IgG1 and IgG3 complexes than those of FcgRIIIb-
MATERIALS AND METHODS Study subjects. One hundred Japanese patients with adult periodontitis (21) (48 male and 52 female; age range, 34 to 67 years; mean age, 47.8 years) referred to the Periodontal Clinic of the Niigata University Dental Hospital and 105 Japanese healthy volunteers (73 male and 32 female; age range, 23 to 63 years; mean age, 27.0 years) were included in the study. Informed consent was obtained from all participants. None of the participants had a history or current signs of systemic disease, nor had they used any medication 3 months prior to the study; none had been fitted with removable prosthodontic appliances. Clinical assessments. Clinical assessment of the patients was performed by the same periodontist at the first visit (baseline), the completion of treatment (follow-up start), and the latest recall appointment. Probing depth (from free gingival margin to bottom of pocket) and attachment level (from cement-enamel junction to bottom of pocket) of all teeth were assessed by using a Williams probe at six sites: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual. Measurements were recorded to the nearest millimeter, and
TABLE 1. Distribution of FcgR genotypes in Japanese patients with adult periodontitis and race-matched healthy controlsa No. (%) of subjects Subject group (n)
FcgRIIab
FcgRIIIbc
R/R131 R/H131 H/H131 NA1/NA1 NA1/NA2 NA2/NA2
Healthy con4 (3.8) 38 (36.2) 63 (60.0) 38 (36.2) trols (105) Periodontitis 6 (6.0) 38 (38.0) 56 (56.0) 37 (37.0) patients (100)
* Corresponding author. Mailing address: Department of Periodontology, Niigata University School of Dentistry, Gakko-cho 2-5274, Niigata, Japan. Phone: (81) 25-223-6161, ext. 4352. Fax: (81) 25-2233761. E-mail:
[email protected].
a b c
3556
55 (52.4)
12 (11.4)
43 (43.0)
20 (20.0)
FcgR genotypes were determined by PCR (5, 19). Frequency of genotype (3 3 2 contingency table): x2 5 0.69, P 5 0.71. Frequency of genotype (3 3 2 contingency table): x2 5 3.40, P 5 0.19.
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TABLE 2. Distribution of FcgR genotypes and alleles in Japanese adult periodontitis patients with and without disease recurrence No. (%) of subjects Genotype Patient subgroupa (n)
Without disease recurrence (15) With disease recurrence (85)
b
FcgRIIac
Allelic frequency (%) FcgRIIIbd
FcgRIIae
FcgRIIIbf
R/R131
R/H131
H/H131
NA1/NA1
NA1/NA2
NA2/NA2
R131
H131
NA1
NA2
0 (0) 6 (7.1)
6 (40.0) 32 (37.6)
9 (60.0) 47 (55.3)
10 (66.7) 27 (31.8)
4 (26.7) 39 (45.9)
1 (6.7) 19 (22.3)
20 26
80 74
80 55
20 45
Disease recurrence was defined as the presence of more than one diseased site with a loss of $2 mm in attachment level during follow-up. Determined by PCR (5, 19). Frequency of genotype (3 3 2 contingency table): x2 5 1.10, P 5 0.57. Odds ratio for risk of periodontitis recurrence in FcgRIIa-R/R131 and IIa-R/H131 compared with IIa-H/H131: 1.21 (95% CI, 0.35 to 4.27); x2 5 0.003, P 5 0.96. d Frequency of genotype (3 3 2 contingency table): x2 5 6.90, P 5 0.03). Odds ratio for risk of periodontitis recurrence in FcgRIIIb-NA2/NA2 and IIIb-NA1/NA2 compared with IIIb-NA1/NA1: 4.29 (95% CI, 1.19 to 16.24); x2 5 5.25, P 5 0.02. e Allelic frequency: odds ratio, 1.41 (95% CI, 0.69 to 2.88); x2 5 0.71, P 5 0.40. f Allelic frequency: odds ratio, 3.27 (95% CI, 1.67 to 6.46); x2 5 13.13, P 5 0.0003. a b c
every observation close to 0.5 mm was rounded to the lower whole number. All Japanese patients with adult periodontitis were treated by conventional periodontal therapy consisting of oral hygiene instruction, scaling, root planing, and periodontal surgery. When the patients were considered free from all periodontal lesions, they were monitored clinically at 3-month intervals for more than 1 year (mean, 38.9 months) posttherapy. The mean number of teeth in the patients was 25.7 (standard deviation 5 3.4) at the follow-up start. Recurrence of periodontitis was defined as having more than one diseased site with a loss of $2 mm in attachment level in the entire dentition during follow-up. Annual rate of disease recurrence of individual patients was also expressed as the percentage of diseased sites with a loss of $2 mm in attachment level in the entire dentition per year (7, 12, 35). Preparation of cells and sera. PMN were isolated from peripheral blood by sedimentation with 5% dextran (Nacalai Tesque Inc., Kyoto, Japan) solution (4 volumes of blood, 1 volume of dextran) for 20 min at 37°C. After removal of erythrocytes by isotonic cell lysis (with NH4Cl for 10 min, room temperature), PMN were washed with phosphate-buffered saline, resuspended in phosphatebuffered saline at 106 PMN/ml, and used immediately. Serum was removed after centrifugation of the clot, recentrifuged to eliminate residual erythrocytes, and stored at 270°C until used. MAbs. Anti-FcgRII (CD32) monoclonal antibody (MAb) IV.3 (IgG2b [13]) was obtained from Medarex (Annandale, N.J.). Anti-FcgRII MAb 41H.16 (IgG2a [6]) was kindly provided by B. M. Longenecker (University of Alberta, Edmonton, Canada). Anti-FcgRIII (CD16) MAb MG38 (IgG1 [28]) was obtained from Nichirei Co. (Tokyo, Japan), and anti-FcgRIII MAb GRM1 (IgG2a [10]) was obtained from Harlan Sera-lab Co. (Sussex, England). Determination of FcgRIIa and FcgRIIIb phenotypes and genotypes. FcgRIIa allotyping of PMN was performed by flow cytometry as described previously (26). In short, PMN were incubated with CD32 MAb IV.3, which reacts with both FcgRIIa allotypes (13), and MAb 41H.16, which selectively binds the FcgRIIaR131 allotype (6). PMN were labeled for single-fluorescence flow cytometry, using phycoerythrin-conjugated F(ab9)2 fragments of goat antimouse IgG (Caltag, San Francisco, Calif.) as a fluorescent label. PMN were analyzed with a FACScan flow cytometer and Consort 30 software (Becton Dickinson, San Jose, Calif.). PMN were gated according to their characteristic forward and sideward scatter pattern. Ten thousand cells were counted per sample. The FcgRIIIb-NA1 and -NA2 allotypes were determined by using allotype-specific CD16 MAb MG38, which recognizes FcgRIIIb-NA1 (28), and GRM1, which recognizes FcgRIIIb-NA2 (10). For genotyping, genomic DNA was isolated from peripheral blood (Easy-DNA kit; Invitrogen, San Diego, Calif.), and FcgRIIa and FcgRIIIb genotypes were determined by PCR as described before (5, 19). Coincidence rates of the results obtained by the two methods were 91.2% for FcgRIIa and 99.0% for FcgRIIIb. In the absence of coincidence, PCR results were used. Determination of serum IgG subclass levels. Serum IgG subclass concentrations were determined by one-step sandwich enzyme-linked immunosorbent assay (human IgG subclass profile kit; Zymed, San Francisco, Calif.). The optical density of each well was read at a 450-nm wavelength with a microplate reader (Multiskan Bichromatic; Labsystems, Helsinki, Finland). Six standard concentrations (1/500, 1/1,000, 1/2,000, 1/4,000, 1/8,000, and 1/16,000) were used in duplicate to construct the standard curve. Standard curves were prepared by graphing optical density value versus log concentration for each IgG subclass. The IgG subclass concentration of each test sample was calculated from the standard curve. Statistical analysis. The x2 test was used to compare the FcgRIIa and FcgRIIIb genotype distributions between Japanese patients with adult periodontitis and race-matched healthy controls and between patients with and without recurrence (3 3 2 contingency table). The same test was used to assess the role of the FcgRIIa-R131 and FcgRIIIb-NA2 allotypes as a risk factor for recurrence (2 3 2 contingency table; patients with versus patients without recurrence,
R/R131 and R/H131 versus H/H131, and NA2/NA2 and NA1/NA2 versus NA1/ NA1). Differences in annual rate of recurrence among the FcgRIIa or FcgIIIb genotypes were assessed by the Kruskal-Wallis test with pairwise comparisons, using Fisher’s multiple-comparison procedure with the StatView. The relevance of each FcgR genotype to the annual recurrence rate was determined by multifactorial analysis of variance (ANOVA) with the STATA. Serum IgG subclass levels and clinical parameters in FcgRIIa and FcgRIIIb genotypes were compared with the Kruskal-Wallis test (StatView). All risk rates were considered significant at 5% (P , 0.05).
FIG. 1. Relationship between FcgR genotypes and annual rate of disease recurrence. The annual rate of recurrence was expressed as the percentage of diseased sites with a loss of $2 mm in attachment level in the entire dentition per year. Each circle represents the annual recurrence rate for one subject. The mean and SE are indicated for each FcgR genotype (FcgRIIa-R/R131, -IIa-R/ H131, and -IIa-H/H131, 7.38% 6 3.53%, 3.95% 6 0.75%, and 2.59% 6 0.40%, respectively; FcgRIIIb-NA1/NA1, -IIIb-NA1/NA2, and -IIIb-NA2/NA2, 1.71% 6 0.32%, 4.34% 6 0.82%, and 4.48% 6 0.90%, respectively). p, significantly different from the annual recurrence rate for individuals with the FcgRIIIb-NA1/ NA1 genotype (Kruskal-Wallis test) (P , 0.05).
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TABLE 3. Relevance of FcgRIIa and FcgRIIIb to annual rate of recurrencea Analysis
Partial sum of squares
df
Mean square
F value
Model (FcgRIIa 1 FcgRIIIb) FcgRIIa FcgRIIIb Residual
0.0054 0.0015 0.0040 0.0323
4 2 2 95
0.0013 0.0007 0.0020 0.0003
3.96 0.005 2.15 0.122 5.86 0.004
a
P value
Data were generated via multifactorial ANOVA. r2 5 0.143.
RESULTS Distribution of FcgR genotypes in periodontitis patients and healthy controls. We first assessed the distribution of FcgRIIa and FcgRIIIb genotypes in Japanese patients with adult periodontitis and race-matched healthy controls. No skewing was found in the distribution of the FcgRIIa or FcgRIIIb genotypes between patients and controls (Table 1). Likewise, neither FcgRIIa nor FcgRIIIb allele frequency differed between the two groups (2 3 2 contingency table; for FcgRIIa, odds ratio, 1.18, 95% confidence interval [CI], 0.58 to 2.39, x2 5 0.11, P 5 0.74; for FcgRIIIb, odds ratio, 1.16, 95% confidence interval, 0.64 to 2.14; x2 5 0.15, P 5 0.70). Distribution of FcgR genotypes in periodontitis patients with and without disease recurrence. We next studied whether there was a relationship between FcgR genotypes and recurrence of periodontitis. Distributions of FcgRIIa and FcgRIIIb genotypes were compared between 85 Japanese adult periodontitis patients with and 15 Japanese adult periodontitis patients without disease recurrence. A significant skewing was observed in the distribution of the FcgRIIIb genotypes between these subgroups (Table 2). Likewise, the FcgRIIIb allele frequency differed between the two groups (Table 2). In the patients with recurrence, there was a relative overrepresentation of the FcgRIIIb-NA2 allele. Homozygosity for the FcgRIIIb-NA2 allele was found in 22.3% of the 85 patients with, compared to 6.7% in the 15 patients without, recurrence (Table 2). No significant skewing was observed in the distribution of the FcgRIIa genotypes between the two subgroups (Table 2). Relationship between FcgR genotypes and annual rate of disease recurrence. We compared the mean annual rates of disease recurrence among FcgRIIa and FcgRIIIb genotype groups in 100 Japanese patients with adult periodontitis. As shown in Fig. 1, the annual rate of recurrence was significantly higher in patients with the FcgRIIIb-NA2/NA2 (mean 6 standard error [SE], 4.48 6 0.90%) and FcgRIIIb-NA1/NA2 (4.34 6 0.82%) genotypes than in those with the IIIb-NA1/ NA1 genotype (1.71 6 0.32%) (P , 0.05). FcgRIIa genotypes were not linked to the annual rate of recurrence (R/R131,
7.38 6 3.53% [mean 6 SE]; R/H131, 3.95 6 0.75%; H/H131, 2.59 6 0.40%) (P 5 0.06). We next verified whether FcgRIIa and FcgRIIIb genotypes represent independent factors associated with the annual rate of recurrence. Multifactorial ANOVA was used to test which FcgR genotype was critical for recurrence rate. As shown in Table 3, the adequacy of our analysis model was statistically confirmed (P 5 0.005), supporting the hypothesis that recurrence rate can be predicted by the FcgR genotype. This test also confirmed that FcgRIIIb was and FcgRIIa was not relevant to the recurrence rate (P 5 0.004 for FcgRIIIb; P 5 0.122 for FcgRIIa). Relationship between FcgR genotypes and serum IgG subclass levels in periodontitis patients. We then evaluated whether IgG subclass levels vary between blood donors of the different FcgR genotypes. Serum levels of IgG subclasses were noted to vary considerably between individuals. We thus also calculated the proportion of each subclass relative to the total IgG content (11). IgG subclass levels and relative distributions did not differ between FcgRIIa and FcgRIIIb genotypes (Table 4). Clinical parameter values in each FcgR genotype group in periodontitis patients. We also sought to determine whether other confounding factors, such as differences in probing depth and/or attachment level at the time of initial examination, could account for differences in disease recurrence patterns. As shown in Table 5, no significant differences were observed in mean values of clinical parameters, including probing depth and attachment level, between FcgRIIa or FcgRIIIb genotypes. Furthermore, no significant difference in mean patient age or follow-up period was observed. DISCUSSION The pathogenesis of adult periodontitis remains poorly understood. The basis for disease recurrence may be associated with both genetic and environmental factors. In this study, we evaluated the role of leukocyte FcgR polymorphisms as heritable risk factors for the onset and recurrence of disease. FcgRIIa and FcgRIIIb distributions did not differ between Japanese patients and race-matched controls, indicating that FcgR genotypes do not, per se, constitute a risk factor for development of adult periodontitis. However, we noted a positive correlation between the FcgRIIIb-NA2 allotype and recurrence of adult periodontitis (Tables 1 and 2). The relevance of FcgR genotypes to the recurrence rate was evaluated by multifactorial ANOVA. Results showed that the recurrence rate could be predicted by the occurrence of the two FcgR genotypes (P 5 0.005). Our data showed FcgRIIIbNA2/NA2 or FcgRIIIb-NA1/NA2 patients to have a risk for recurrence of disease two times higher than that of FcgRIIIbNA1/NA1 patients (Fig. 1). To the best of our knowledge, this
TABLE 4. Relationship between FcgR genotypes and serum IgG subclass levels in Japanese patients with adult periodontitisa Mean serum concn (mg/ml) 6 SE (mean % 6 SE)
Genotype (n)
FcgRIIa-R/R131 (6) FcgRIIa-R/H131 (38) FcgRIIa-H/H131 (56) FcgRIIIb-NA1/NA1 (37) FcgRIIIb-NA1/NA2 (43) FcgRIIIb-NA2/NA2 (20) a
IgG1
IgG2
IgG3
IgG4
6,779 6 1,245 (63.8 6 7.1) 9,242 6 743 (68.6 6 2.5) 8,214 6 666 (63.4 6 2.2) 8,705 6 1,064 (65.1 6 3.0) 8,633 6 555 (65.6 6 2.2) 7,927 6 646 (65.6 6 3.6)
3,265 6 793 (30.3 6 6.4) 3,193 6 312 (25.4 6 2.2) 3,549 6 257 (29.9 6 1.9) 3,095 6 251 (28.5 6 2.5) 3,674 6 318 (28.5 6 2.0) 3,359 6 485 (27.2 6 3.4)
300 6 79 (2.7 6 0.6) 377 6 42 (3.1 6 0.3) 440 6 40 (3.6 6 0.3) 393 6 49 (3.5 6 0.3) 379 6 38 (2.9 6 0.3) 496 6 68 (4.0 6 0.5)
346 6 62 (3.1 6 0.3) 342 6 43 (2.9 6 0.4) 332 6 31 (3.2 6 0.5) 284 6 36 (3.0 6 0.6) 372 6 38 (3.0 6 0.4) 360 6 58 (3.3 6 0.9)
IgG subclass concentrations and FcgR genotypes were determined by ELISA and PCR (5, 19), respectively.
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TABLE 5. Clinical parameter values in each FcgR genotype of Japanese patients with adult periodontitisa Probing depth (mm) Genotype (n)
FcgRIIa-R/R131 (6) FcgRIIa-R/H131 (38) FcgRIIa-H/H131 (56) FcgRIIIb-NA1/NA1 (37) FcgRIIIb-NA1/NA2 (43) FcgRIIIb-NA2/NA2 (20) a b
Attachment level (mm)b
Baseline
Follow-up start
Baseline
Follow-up start
Follow-up period (mo)
3.08 6 0.22 3.00 6 0.10 2.95 6 0.07 2.90 6 0.10 3.04 6 0.08 3.00 6 0.11
2.49 6 0.16 2.45 6 0.05 2.37 6 0.05 2.35 6 0.06 2.46 6 0.05 2.42 6 0.09
3.28 6 0.30 3.14 6 0.11 3.18 6 0.09 3.07 6 0.12 3.22 6 0.09 3.26 6 0.13
2.69 6 0.19 2.79 6 0.08 2.70 6 0.07 2.67 6 0.10 2.76 6 0.07 2.79 6 0.09
28.1 6 6.7 37.2 6 4.4 41.2 6 3.9 47.0 6 5.0 34.5 6 3.7 33.2 6 6.1
Age (yr)
47.3 6 4.9 47.5 6 1.5 48.1 6 1.3 47.1 6 1.6 47.5 6 1.4 49.8 6 2.1
FcgR genotypes were determined by PCR (5, 19). Values represent means 6 SE. Distance from cement-enamel junction to bottom of pocket.
is the first study in which evidence for a role of the FcgRIIIbNA2 gene as an independent risk factor for disease has been presented. Only six Japanese patients (6.0%) exhibited the FcgRIIa-R/ R131 genotype in this study (Table 1). This finding is consistent with the results of previous epidemiological studies showing the FcgRIIa-H/H131 genotype to predominate in Japan (19, 23). Although no significant skewing was observed in the distribution of the FcgRIIa genotypes between Japanese patients with and without recurrence, it is noteworthy that all FcgRIIaR/R131 patients exhibited recurrence (Table 2). Moreover, the mean recurrence rate was approximately three times higher in the FcgRIIa-R/R131 individuals than in the FcgRIIa-H/H131 individuals, although this difference was not statistically significant (Fig. 1), likely due to the very small number of FcgRIIaR/R131 individuals in Japan. The hypothesis that FcgRIIa polymorphism is important for disease recurrence warrants testing in cohorts consisting of other ethnic populations. We also investigated whether confounding factors were responsible for the observed correlation. Serum IgG subclass levels were comparable between FcgR genotypes (14, 32, 34) (Table 4). Clinical parameters at baseline and at follow-up start that may influence progression of adult periodontitis were similar for all FcgR genotypes (Table 5). We also did not find differences in the distribution of disease recurrence between Japanese subjects with mild and those with severe adult periodontitis at baseline (data not shown). Smoking has also been proposed to constitute a risk factor for progression of periodontal disease (9, 15). A retrospective examination showed that the percentages of patients who were reported to be cigarette smokers did not differ significantly among the FcgRIIa and FcgRIIIb genotypes (data not shown). In conclusion, the NA1/NA2 polymorphism of FcgRIIIb was observed to be closely related to the recurrence of adult periodontitis. To our knowledge, this represents the first study documenting evidence for a role of the FcgRIIIb-NA2 allotype as a risk factor in human disease. More extensive studies in larger groups of patients, and also in other ethnic populations, should be undertaken to better assess the putative relevance of the FcgRIIa polymorphism to the pathophysiology of adult periodontitis. ACKNOWLEDGMENTS We are grateful to Hideo Miyazaki and Masatoshi Yano, Department of Preventive Dentistry, Niigata University School of Dentistry, for technical help with the statistical analysis. This work was supported by Grant-in-Aids for Scientific Research 07407054, 06454536, and 08672184 from the Ministry of Education, Science, Sports and Culture, Japan; Netherlands Organization for Scientific Research grants 950-10-624; and the Fund for Scientific Promotion of Tanaka Industries Co. Ltd., Niigata, Japan.
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