The regenerating gene (Reg) was isolated originally as a gene specifically over-expressed in regenerating pancreatic islets and constitute a growth factor family. Reg gene product (Reg) is important in the pathophysiology of various human inflammatory diseases. Recently, the possible involvement of human REG in the regeneration of salivary ductal epithelial cells of patients with primary Sjögren's syndrome (SS) was reported. However, the expression of the REG family genes in minor salivary glands (MSG) and the occurrence of anti-REG Iα autoantibodies in SS patients were obscured. In this study, we examined the expression of REG family genes in the MSG of SS and screened anti-REG Iα autoantibodies in SS. The mRNA levels of REG family genes in MSG were quantified using real-time reverse transcription–polymerase chain reaction (RT–PCR) and REG Iα expression in the MSG was analysed by immunohistochemistry. The mRNA level of REG Iα in the MSG of SS patients was significantly higher than that of control. REG Iα protein was expressed highly in SS ductal epithelial cells. Anti-REG Iα autoantibodies in the sera were found in 11% of SS. All the MSG in the anti-REG Iα autoantibody-positive group showed REG Iα expression, whereas only 40% showed REG Iα expression in the anti-REG Iα autoantibody-negative group. The anti-REG Iα autoantibody-positive group showed significantly lower saliva secretion and a higher ratio of grade 4 (by Rubin–Holt) in sialography. These data suggest strongly that autoimmunity to REG Iα might play a role in the degeneration of MSG ductal epithelial cells in primary SS.
Primary Sjögren's syndrome (SS) is a chronic autoimmune disease of unknown aetiology, characterized by lymphocytic infiltration of salivary and lacrimal glands, leading to xerostomia and xerophthalmia, and characterized by the presence of a variety autoantibodies directed against organ- and non-organ-specific autoantigens. It is known that the production of autoantibodies is an antigen-driven immune response, as certain autoantibodies are disease-specific, contain multiple epitopes and the autoimmune response is perpetuated and augmented via intra- and intermolecular spreading against the same or other autoantigens. It is unknown whether or not any of the autoantibodies has a direct pathogenic potential or they merely participate in a secondary response to salivary glands that are already damaged by another process.
The regenerating gene, Reg, was isolated originally as a growth factor from a cDNA library of rat regenerating pancreatic islets [1-3]. Reg gene expression has also been identified outside the pancreas. Subsequently, many Reg-related proteins have been identified in human and other animals. The Reg family genes constitute a multi-gene family consisting of four subtypes . In the human, five functional REG genes, i.e. REG Iα [1, 2], REG Iβ , REG III , HIP/PAP [7, 8] and REG IV , have been isolated.
Reg family proteins function as acute phase reactants, lectins, anti-apoptotic factors and growth agents and include growth factors. These proteins are involved primarily in cell proliferation and differentiation, inflammation, diabetes and carcinogenesis [4, 10].
Types I and II Reg proteins are expressed in regenerating islets [4, 11, 12]. Type III Reg proteins have been suggested to be involved in cellular proliferation in intestinal cells, hepatic cells and neuronal cells. Importantly, mouse Reg III was shown to be a Schwann cell mitogen that accompanies the regeneration of motor neurones , and Reg protein functions as a neurotrophic factor for motor neurones . It was reported that REG I protein was expressed in ductal epithelial cells in the minor salivary glands (MSG) of patients with SS . However, which REG family gene(s) were expressed in MSG in SS patients was obscured.
Autoantibodies against REG (αREG) were found in some diabetic patients [16, 17]. However, the occurrence of αREG was obscured in SS patients. The presence of αREG might compromise regeneration of damaged ductal epithelial cells, and the expression of REG could be a key event in autoimmunity. This hypothesis is supported by the fact that the αReg retarded β cell proliferation in vitro .
It has been reported that the expression of Reg family genes was regulated by several factors such as nicotinamide [18, 19], glucocorticoids [18, 20], nutrient factors , interleukin (IL)-6 [12, 18], IL-8 , IL-11 , IL-22  interferon (IFN)-γ , IFN-β  and cytokine-induced neutrophil chemoattractant-2 (CINC-2)β . We examined the mRNA levels of IL-6 and IL-8, major proinflammatory cytokines produced in MSG in SS patients [27-30], in MSG specimens. We also examined mRNA levels of IL-6 receptor and gp130 in MSG specimens.
In the present study, we found over-expression of REG Iα, IL-6 and IL-8 mRNAs in MSG of SS patients and possible involvement of autoimmunity to REG in patients with primary Sjögren's syndrome.
Materials and methods
This study was approved by the Nara Medical University Hospital. After informed consent, a total of 117 patients with primary SS were enrolled into the study at Nara Medical University Hospital during 2001 to 2009. All the patients fulfilled the diagnostic criteria for definite SS proposed by the Research Committee on SS of the Ministry of Health and Welfare of the Japanese Government (1999) , and the diagnosis was also based on the diagnostic criteria proposed by the American–European Consensus Group criteria for SS . The enrolment procedure and study protocol were in compliance with the Declaration of Helsinki. We determined serum levels of amylase, immunoglobulin (Ig)G, HbA1c, SS-A/SS-B autoantibodies, anti-nuclear antibody (ANA) titre and rheumatoid factor. We also determined the sialographic staging based on the criteria of Rubin and Holt, the histological grade of MSG, the presence of keratoconjunctivitis sicca, abnormality of the tear production as determined by Schirmer's test (<5 mm after 5 min) and abnormality of saliva production, as determined by Saxon's test. SS-A/SS-B autoantibodies were measured with an enzyme-linked immunosorbent assay (ELISA) kit (MBL, Tokyo, Japan). The histological features of the labial salivary gland biopsy were evaluated according to Greenspan's histopathological grading , and we classified grade 4 with germinal centres as grade 5. The presence of keratoconjunctivitis was evaluated by fluorescence staining test or Rose Bengal test according to the van Bijsterveld score. The relationships among these clinicopatholocal factors and the occurrence of αReg were analysed.
MSG tissue specimens
MSG tissue was obtained from 53 patients with primary SS (one male, 52 females; mean age 55 ± 2·0 years, range 15–80 years) from the 117 Japanese SS patients, and also from 25 healthy Japanese controls (13 males, 12 females; mean age 39 ± 3·7 years, range 6–76 years). The tissue specimens were fixed with 10% formalin/phosphate-buffered saline (PBS) and embedded in paraffin.
Serum samples were collected from 117 Japanese with primary SS patients (two males, 115 females; mean age 56 ± 1·4 years, range 9–83 years), including 53 patients who kindly provided MSG tissue specimens, and 271 healthy Japanese controls (81 males, 190 females; mean age 33 ± 0·63 years, range 20–64 years), were divided into aliquots and stored at −80°C until used in screening of αReg.
MSG tissue was obtained from 23 Japanese patients with primary SS and from 25 healthy Japanese controls for analysing mRNA by RT–PCR. Total RNA was isolated from formalin-fixed, paraffin-embedded (FFPE) MSG tissue specimens using the RNeasy FFPE kit (Qiagen, Hilden, Germany). The isolated RNA was reverse-transcribed to the cDNA using the high-capacity cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) for the real-time PCR template, as described previously [34, 35]. The cDNA was subjected to PCR with the following primers: β-actin (NM_001101) sense primer, 5′-GCGAGAAGATGACCCAGA-3′ and anti-sense primer, 5′- CAGAGGCGTACAGGGATA-3′; REG Iα (NM_002909) sense primer, 5′-AGGAGAGTGGCACTGATGACTT-3′ and anti-sense primer 5′-TAGGAGACCAGGGACCCACTG-3′; REG Iβ (NM_006507) sense primer, 5′-GCTGATCTCCTCCCTGATGTTC-3′ and anti-sense primer, 5′-TGTCAGTGATCTTGGTTTGAA-3′; REG III (AB161037) sense primer, 5′-GAATATTCTCCCCAAACTG-3′ and anti-sense primer, 5′-GAGAAAAGCCTGAAATGAAG-3′; HIP/PAP (NM_138937) sense primer, 5′-AGAGAATATTCGCTTAATTCC-3′ and anti-sense primer, 5′-AATGAAGAGACTGAAATGACA-3′; REG IV (AY007243) sense primer, 5′-ATCCTGGTCTGGCAAGTC-3′ and anti-sense primer, 5′-CGTTGCTGCTCCAAGTTA-3′, IL-6 (NM_000600) sense primer, 5′-GGTACATCCTCGACGGCATC-3′ and anti-sense primer, 5′- GCCTCTTTGCTGCTTTCACAC-3′, IL-8 (NM_000584) sense primer, 5′-TAGCAAAATTGAGGCCAAGG-3′ and anti-sense primer, 5′-GGACTTGTGGATCCTGGCTA-3′ IL-22 (NM_020525) sense primer, 5′-GCAGGCTTGACAAGTCCAACT-3′ and anti-sense primer, 5′-GCCTCCTTAGCCAGCATGAA-3′, IL-22 receptor (IL-22R) (NM_021258) sense primer, 5′-CTACATGTGCCGAGTGAAGA-3′ and anti-sense primer, 5′-ACATATCTGTAGCTCAGGTA-3′, IFN-β (NM_002176) sense primer, 5′-CATTACCTGAAGGCCAAGGA-3′ and anti-sense primer, 5′-CAGCATCTGCTGGTTGAAGA-3′, IFN-γ (NM_000619) sense primer, 5′-ATTCGGTAACTGACTTGAATGTCC-3′ and anti-sense primer, 5′-CTCTTCGACCTCGAAACAGC-3′, IL-11 (NM_000641) sense primer, 5′-TCTCTCCTGGCGGACACG-3′ and anti-sense primer, 5′-AATCCAGGTTGTGGTCCCC-3′, CXCL1 (NM_001511) sense primer, 5′-GAAAGCTTGCCTCAATCCTG-3′ and anti-sense primer, 5′-TCCTAAGCGATGCTCAAACA-3′, IL-6 receptor (IL-6R) (X12830) sense primer, 5′-TGAGCTCAGATATCGGGCTGAAC-3′ and anti-sense primer, 5′-CGTCGTGGATGACACAGTGATG-3′, and gp130 (NM_002184) sense primer, 5′-AGGACCAAAGATGCCTCAACT-3′ and anti-sense primer, 5′-TTGGACAGTGAATGAAGATCG-3′. All the PCR primers were synthesized by NGRL (Sendai, Japan). Real-time PCR was performed using KAPA SYBR® Fast qPCR Master Mix (Kapa Biosystems, Boston, MA, USA) and the Thermal Cycler Dice Real Time System (Takara, Otsu, Japan), as described previously . PCR was performed with an initial step of 3 min at 95°C followed by 40 cycles of 3 s at 95°C and 20 s at 60°C for β-actin, REG III and HIP/PAP, 40 cycles of 3 s at 95°C and 20 s at 64°C for REG Iα, REG Iβ and REG IV, 45 cycles of 3 s at 95°C and 20 s at 62°C for IL-6 and IL-22, 45 cycles of 3 s at 95°C and 20 s at 60°C for IL-8, IL-22R, IL-6R and gp130 and 45 cycles of 3 s at 95°C and 20 s at 63°C for IFN-β, IFN-γ, IL-11 and CXCL1. The level of target mRNA was normalized to the mRNA level of β-actin as an internal standard.
Immunohistochemical staining for REG Iα protein
Sections were prepared on 7-μm thick glass slides processed by deparaffinization/rehydration, and fixed thereafter in cold acetone at 4°C for 10 min. The antigen retrieval step was performed using Bond Epitope Retrieval Solution 1 (citrate-based pH 6·0 solution). The tissue slices were incubated with an anti-human REG I protein monoclonal antibody [2, 36] (1:250) as a primary antibody overnight at 4°C in a humidified environment. After washing, the antibody was detected by the Bond polymer method (Autoimmunostainer Bond MAX; Mitsubishi Chemical Medience Co., Tokyo, Japan).
Screening of αREG
Recombinant human REG Iα protein (20 μg)  was electrophoresed on a 12·5% sodium dodecyl sulphate (SDS)-polyacrylamide gel (9 × 7 × 0·1 cm) with a constant current at 20 mA/gel for 100 min and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane using a semidry electroblotter, as described previously [16, 37]. After blocking with 5% non-fat dry milk, the membrane was incubated with patient or control serum, which had been diluted 1024-fold with 5% non-fat dry milk, using a screener blotter (Screener Blotter Mini 56; Sanplatec, Osaka, Japan) [16, 37, 38]. The membrane was then rinsed with PBS containing 0·10% Tween 20 and incubated with goat anti-human IgG labelled with horseradish peroxidase (American Qualex, San Clemente, CA, USA) at 1/1600 dilution. The signals were visualized using an enhanced chemiluminescent (ECL) detection system (GE Healthcare, Little Chalfont, UK), as described previously [16, 37, 38]. The band intensities from positive blots were analysed by Image J software (National Institute of Health, Bethesda, MD, USA). The density was standardized using the value of an internal control sample treated as a relative value, as described previously [16, 37].
All values are presented as means ± standard error. Differences between the two groups were analysed with the Mann–Whitney U-test. A P-value of <0·05 was considered statistically significant. The frequency distribution of positive values [mean ± 3 standard deviations (s.d.) for control] was compared using the χ2-test.
REG gene expression in the MSG
We extracted total RNA from formalin fixed paraffin-embedded specimens and analysed mRNA levels of all the REG family genes (REG Iα, REG Iβ, REG III, HIP/PAP and REG IV) using qRT–PCR (Fig. 1). No REG Iβ mRNA was detected either in the control or the SS MSG. The mRNA levels of REG III, HIP/PAP and REG IV were not different between the control and SS MSG. In contrast, the mRNA level of REG Iα in the MSG of SS patients was significantly higher than that of control (P = 0·036).
Immunohistochemical staining of the MSG with SS for REG Iα protein
We then analysed REG Iα protein expression in MSG of SS patients by immunohistochemistry. REG Iα protein was stained strongly in ductal epithelial cells in 28 of 53 samples (53%), whereas acinar cells were immunostained only in two samples for REG Iα (Fig. 2a,b). In REG Iα-positive samples, the intensity of staining was not associated with the degree of inflammation, fibrosis or acinic atrophy (data not shown).
Cytokine/chemokine(s) gene expression in MSG
IL-6 and IL-8 are reported to induce REG Iα mRNA in vitro [18, 21, 22] and in vivo , and IL-11 , IL-22 , IFN-γ , IFN-β  and CINC-2β  are also reported to induce REG Iα mRNA. IL-6R and gp130 are known as signal transducers of IL-6. We measured IL-6, IL-8 IL-11, IL-22, IL-22R, IFN-γ, IFN-β, CXCL1 (human homologue of CINC-2β), IL-6R and gp130 mRNAs in the MSG by qRT–PCR. As shown in Fig. 3a, the IL-6 mRNA level in the SS MSG was significantly higher than that in normal MSG. The IL-8 mRNA level in SS MSG was also higher than that in normal MSG (Fig. 3b). The mRNA levels of IL-11, IL-22, IL-22R, IFN-γ, CXCL1, IL-6R and gp130 were not significantly different between the two groups (Fig. 3c–j). The mRNA of IFN-β was not detected in SS MSG (Fig. 3g). We performed correlation analyses of expression of cytokine mRNAs and REG Iα mRNA and found that IL-6 mRNA expression was correlated significantly with REG Iα mRNA expression (data not shown).
Detection of αREG in SS patient sera
The sera from 117 patients with primary SS and 271 controls were screened for αREG. A percentile graph of the relative αReg values is shown in Fig. 4. The relative αREG values for all controls, with the exception of six individuals, were within the mean ± 3 s.d. value. Henceforth, we treated this mean ± 3 s.d. value (3·9) of the controls as the cut-off value for all related data analyses. Eleven per cent (13 of 117) of patients with primary SS subjects tested positive for αREG, whereas only 2·2% (six of 271) were positive in controls (P = 0·00019, χ2 test).
Relationships among clinicopathological factors
The αREG-positive group showed significantly lower saliva secretion (0·64 ± 0·29 g/2 min) than the negative group (1·54 ± 0·17 g/2 min) using Saxon's test (P = 0·0073) (Table 1). The ratio of a destructive stage (stage 4 based on Rubin and Holt's criteria) in sialography in the αREG-positive group was significantly higher than that in the αREG-negative group (Table 2). In the patients with primary SS, no correlation was found in age, sex ratio, serum levels of SS-A/SS-B autoantibody, anti-nuclear antibody titre, rheumatoid factor, amylase, IgG and HbA1c between the αREG-positive group and the αREG-negative group (Table 1). Kerato-conjunctivitis sicca, Schirmer's test, also showed no significant difference between the two groups. In the histological features of labial salivary gland biopsy according to Greenspan's grade, there was no significant difference in the αREG-positive and the αREG-negative groups (Table 2). We also examined extraglandular disease, systemic or severe disease (skin rash, Raynaud's phenomenon, arthralgia, thyroid gland disease, interstitial pneumonia, primary biliary cirrhosis, renal tubular acidosis, peripheral neuropathy and lymphoma); there was no significant difference in the two groups (data not shown).
Table 1. General characteristics of patients with primary SS.
(+) n = 13
(−) n = 104
Not significant (n.s.): P ≥ 0·05. Ig: immunoglobulin; SS: Sjögren's syndrome; ANA: anti-nuclear antibody.
62 ± 4·0
55 ± 1·5
Sex ratio (male/female)
84/16 (n = 100)
44/56 (n = 100)
ANA titre ≥ 1:320 (+/−)
6/7 (n = 13)
60/33 (n = 93)
Rheumatoid factor (+/−)
7/3 (n = 10)
34/32 (n = 66)
70 ± 9·3 (n=12)
88 ± 4·1 (n = 97)
2041 ± 125
1790 ± 57 (n = 99)
5·3 ± 0·24 (n = 5)
5·2 ± 0·1 (n = 16)
Kerato-conjunctivitis sicca (KCS) (+/−)
9/2 (n = 11)
62/19 (n = 81)
Schirmer's test (≤5 mm/5 min/>5 mm/5 min)
8/4 (n = 12)
54/43 (n = 97)
Saxon's test (g/2 min)
0·64 ± 0·29 (n = 12)
1·54 ± 0·17 (n = 84)
Table 2. Relationship among sialography (Rubin–Holt stage), labial salivary gland biopsy (Greenspan's grade) and presence of αREG.
Not significant (n.s.): P ≥ 0·05. †In the grade 4 group, we classified ‘grade 5’ in which germinal centres were found in the histopathology.
Relationship between REG Iα protein expression and αREG
Twenty-one per cent of patients (11 of 53) who were examined for REG Iα protein expression in MSG by immunohistochemistry tested positive for αREG. All the 11 samples showed REG Iα expression in the αREG-positive group, whereas only 40% (17 of 42) showed REG Iα expression in MSG in the αREG-negative group (P = 0·00043) (Table 3).
Table 3. Relation between REG Iα expression in the ductal epithelial cells and presence of αREG.
REG Iα expression in the ductal epithelial cells
P = 0·00043.
αREG-positive (n = 11)
αREG-negative (n = 42)
In the present study, we found that REG Iα protein was expressed in the ductal cells of MSGs from patients with primary SS and that saliva secretion was reduced in primary SS patients with the αREG. Although the aetiology of SS is still unclear, it is thought to be an autoimmune disease characterized by marked ductal cell destruction with inflammatory cell infiltration in MSG . It was reported previously that REG Iα is expressed not only in various human inflammatory diseases such as gastritis , pancreatitis  and colitis, but also in various experimental models of inflammation in animal tissues [39, 43]. Thus, it is most likely that inflammation, regardless of whether or not it is autoimmune-associated, is a key event triggering REG Iα expression in many tissues. Therefore, whether or not REG Iα overexpression is associated directly with the immune disorder in patients with primary SS is an interesting question. We performed RT–PCR analyses of all the REG family genes in MSG and found that REG Iα mRNA was over-expressed specifically in MSG specimens from SS patients (Fig. 1). These results support the idea that REG Iα mRNA over-expression is associated with inflammation triggered by autoimmune disorders such as SS.
It was reported that Reg gene expression was regulated by several factors, such as nicotinamide [18, 19], glucocorticoids [18, 20], nutrient factors , IL-6 [12, 18], IL-8 , IL-11 , IL-22 , IFN-γ , IFN-β  and CINC-2β . Among the major inflammatory mediators involved in the induction of inflammation of the salivary glands with primary SS, IL-6 is an important proinflammatory cytokine in relation to lymphocyte infiltration [27, 28]. In addition, the presence of IL-8 was reported in the salivary glands of SS [29, 30].
We therefore examined the mRNA levels of IL-6, IL-8 and other cytokine/chemokine(s), which were reported to induce REG Iα mRNA in MSG specimens. The mRNA levels of IL-6 and IL-8 in the MSG of SS patients were significantly higher than those of the control. The mRNA levels of IL-11, IL-22, IL-22R, IFN-γ and CXCL1 were not significantly different between the two groups. The mRNA of IFN-β was not detected in SS MSG. These results suggest that the up-regulation of cytokines, especially IL-6 and IL-8, induces over-expression of the REG Iα gene in SS MSG. IL-6R and gp130 are known as signal transducers of IL-6. We examined the mRNA levels of IL-6R and gp130 in SS and normal MSG. The mRNA levels of IL-6R and gp130 were not significantly different between the two groups (Fig. 3i,j), suggesting that the increase of IL-6 in the salivary gland of SS patients (Fig. 3a) can function as a switch for the IL-6/gp130 signalling system to induce REG Iα gene expression.
Anti-REG autoantibodies that inhibited pancreatic β cell replication were detected in Japanese diabetes patients . In the present study, we detected autoantibodies against REG Iα in Japanese primary SS patients for the first time. We evaluated the correlation between αREG and the clinicopathological factors of primary SS patients.
The αREG-positive group showed significantly lower salivary secretion and a higher ratio of the destructive stage in sialography. REG Iα protein was expressed in MSG ductal epithelial cells from nearly half of SS patients, and some in MSG acinar cells. Interestingly, all the patients in the αREG-positive group showed REG Iα expression in MSG ductal cells, whereas only 40% in the αREG-negative group showed REG Iα expression in MSG (Table 3). These results suggest that autoimmunity to REG is associated with the regeneration of the ductal epithelial cells of MSG in primary SS patients.
When salivary glands are damaged by inflammation, REG Iα protein might be induced in progenitor cells for MSG ductal/acinar cells such as ductal cells for recovering damaged cell mass by regeneration. Accumulating evidence concerning development of the salivary gland suggests that the stem cell population of salivary glands is present in the intercalated duct [44, 45]. Additionally, the proliferation of pancreatic β cells was reported to be attenuated by the diabetic patient sera containing αREG in vitro . It is possible that αREG attenuates not only the growth-promoting affects of REG to fully differentiated acinar/ductal cells, but also regeneration of the stem cell population of salivary glands. As a result, salivary function, including saliva secretion, could deteriorate in SS patients with αREG.
Xerostomia is an important clinical concern in oral health and is known to induce various problems, including dental caries, periodontitis, denture problems, mastication and swallowing problems, burning sensations and dysgeusia . Muscarinic agonist medications such as pilocarpine and cevimeline induced salivary secretion from the residual functional tissue . However, these medications provided temporary relief of symptoms and had a limited effect on the recovery of damaged tissues. Accordingly, the development of a novel treatment to restore or regenerate damaged salivary gland tissues is eagerly awaited. It is unclear whether or not a specific signal is required for the regeneration of salivary gland. REG Iα could be a candidate growth factor for regeneration of the salivary gland cells, as hepatocyte growth factor is a well-known protein that promotes the regeneration of liver and even protects from tissue damage [48, 49]. Therefore, it is expected that the regenerative growth of ductal epithelial cells serves as a practical therapeutic approach for SS.
Detection of αREG is not so powerful in SS diagnosis. However, because of the correlation between the salivary function and existence of serum αREG (Tables 1 and 2), αREG detection can be a useful diagnostic marker for the prognosis of salivary function, such as saliva secretion. In addition, as described in Materials and methods, very small volumes of serum (less than 1 μl) were required to detect αREG.
In conclusion, we have shown that REG Iα mRNA was expressed in the SS patient salivary glands and that REG Iα protein was expressed in the ductal epithelial cells of MSGs from patients with primary SS. Saxon's test revealed clearly that saliva secretion was reduced in primary SS patients with αREG. Furthermore, there was a correlation between the presence of αREG and REG Iα protein expression on ductal cells of MSG. These data suggest strongly that autoimmunity to REG Iα could play a role in the regeneration of MSG ductal epithelial cells in primary SS.
This work was supported in part by Grants-in-Aid for Scientific Research (C) (22591096) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are grateful to Drs Shinobu Nakamura and Yoshiko Dohi for encouragement. This work is in partial fulfillment by K.Y. of the degree of Doctor of Medical Science at Nara Medical University.