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1.  Novel Positively Charged Nanoparticle Labeling for In Vivo Imaging of Adipose Tissue-Derived Stem Cells 
PLoS ONE  2014;9(11):e110142.
Stem cell transplantation has been expected to have various applications for regenerative medicine. However, in order to detect and trace the transplanted stem cells in the body, non-invasive and widely clinically available cell imaging technologies are required. In this paper, we focused on magnetic resonance (MR) imaging technology, and investigated whether the trimethylamino dextran-coated magnetic iron oxide nanoparticle -03 (TMADM-03), which was newly developed by our group, could be used for labeling adipose tissue-derived stem cells (ASCs) as a contrast agent. No cytotoxicity was observed in ASCs transduced with less than 100 µg-Fe/mL of TMADM-03 after a one hour transduction time. The transduction efficiency of TMADM-03 into ASCs was about four-fold more efficient than that of the alkali-treated dextran-coated magnetic iron oxide nanoparticle (ATDM), which is a major component of commercially available contrast agents such as ferucarbotran (Resovist), and the level of labeling was maintained for at least two weeks. In addition, the differentiation ability of ASCs labeled with TMADM-03 and their ability to produce cytokines such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), were confirmed to be maintained. The ASCs labeled with TMADM-03 were transplanted into the left kidney capsule of a mouse. The labeled ASCs could be imaged with good contrast using a 1T MR imaging system. These data suggest that TMADM-03 can therefore be utilized as a contrast agent for the MR imaging of stem cells.
PMCID: PMC4217721  PMID: 25365191
2.  Maintaining a Surgery Service for Local Hospitals Under the Situation of a Decreasing Number of Surgeons in a Region of Japan 
World Journal of Surgery  2014;38(12):3063-3066.
The number of surgeons is decreasing in Japan, leading to the problem of how to maintain a surgery service in local hospitals. We introduce our strategy for supporting ongoing surgical services in regional hospitals by dispatching surgeons temporarily to assist in operations.
We conducted a questionnaire-based survey at three local hospitals in Tottori and a neighboring prefecture to which surgeons from our department were temporarily dispatched over 5 years from January 2008 to March 2013.
We supported 686 operations at three hospitals over 5 years. The average age of the patients was 72.4 years. Of the diseases treated, 45.1 % were malignant, and 54.9 % were benign. The emergency operation rate was 17.3 %.
Our strategy has produced a continuous surgical service at local hospitals in the face of diminishing numbers of surgeons. We recommend that such a strategy be adopted in other regions in which there are a decreasing number of surgeons and where it is not easy to move patients elsewhere for care.
PMCID: PMC4232756  PMID: 25217111
3.  Gastric Adenocarcinoma with Thymic Metastasis after Curative Resection: A Case Report 
Journal of Gastric Cancer  2014;14(3):207-210.
The peritoneum is the most frequent site of recurrence for gastric cancer after gastrectomy, followed by the liver and lymph nodes. In contrast, metastasis to the thymus is rare. Annual surveillance with computed tomography was performed on a 67-year-old man who previously underwent a distal gastrectomy and D2 lymph node dissection for gastric cancer at Tottori University. Five years after the initial operation, an anterior mediastinal tumor was detected by computed tomography. The patient underwent video-assisted thoracic surgery to remove the tumor. Histopathology revealed adenocarcinoma cells similar to those of the gastric cancer resected 5 years previously. Thymic metastasis was considered likely based on the location of the tumor. The recognition that gastric cancer can metastasize to unusual anatomic locations, such as the thymus, can facilitate an accurate, prompt diagnosis and appropriate treatment.
PMCID: PMC4199889  PMID: 25328767
Stomach neoplasms; Neoplasm metastasis
4.  A solid pseudopapillary neoplasm without cysts that occurred in a patient diagnosed by endoscopic ultrasound-guided fine-needle aspiration: a case report 
Solid pseudopapillary neoplasm of the pancreas is a rare neoplasm that has been reported to account for between 0.17% and 2.7% of all non-endocrine tumors of the pancreas. It is usually seen in young women. Because solid pseudopapillary neoplasms are rarely aggressive and have low-grade malignant potential and an excellent prognosis after complete resection, it is an ideal pancreatic tumor for treatment by minimally invasive surgery. Therefore, making an accurate pre-operative diagnosis is very important.
Case presentation
A 24-year-old Japanese man who had been found to have mild transaminase elevations at a medical check-up visited our hospital for further examination. Abdominal computed tomography showed a 40mm-diameter tumor in the pancreatic tail and mild fatty liver. He was admitted to our hospital for additional examination. The abdominal contrast-enhanced computed tomography scan taken at our institution showed an increasingly enhanced mass of 40mm diameter in the pancreatic tail. Ultrasonography showed a low-level echoic mass of 35mm diameter in the pancreatic tail. T1-weighted magnetic resonance imaging showed low signal intensity in the tail of the pancreas. T2-weighted magnetic resonance imaging showed high signal intensity there. Diffusion magnetic resonance imaging showed high signal intensity. An endoscopic ultrasound yielded the same results as the abdominal ultrasonogram. In addition, [18F]-fluorodeoxyglucose positron emission tomography/computed tomography showed abnormal accumulation (maximum standardized uptake value, 6.53). This finding raised our suspicion of a pancreatic malignant tumor. However, the patient could not be confidently diagnosed solely on the basis of imaging. Endoscopic ultrasound-guided fine-needle aspiration was performed, which led us to a diagnosis of solid pseudopapillary neoplasm. On that basis, we performed minimally invasive surgery (spleen-preserving laparoscopic distal pancreatectomy).
Atypical solid pseudopapillary neoplasm without cysts should be considered when diagnosing pancreatic tumors. A definitive pre-operative diagnosis of solid pseudopapillary neoplasm made on the basis of endoscopic ultrasound-guided fine-needle aspiration can guide the surgical approach used.
PMCID: PMC4090630  PMID: 24993459
Atypical; Endoscopic ultrasound-guided fine-needle aspiration; Minimally invasive surgery; Positron emission tomography/computed tomography; Solid pseudopapillary neoplasm
5.  Seasonal variations in the onset of ulcerative colitis in Japan 
AIM: To investigate seasonal variations in the onset and relapse of ulcerative colitis (UC) in Japanese patients.
METHODS: Between 1994 and 2006, 198 Japanese patients diagnosed with UC according to conventional criteria in an academic hospital were enrolled for onset evaluation. Among 265 Japanese patients with UC who were observed for more than 12 mo, 165 patients relapsed (239 times) and were enrolled for relapse evaluation. The patients’ symptoms were recorded each month for 12 consecutive years.
RESULTS: There was monthly seasonality in symptom onset during October and March for UC. The onset of symptoms in UC patients frequently occurred during the winter. Variation in UC onset was observed according to both month (P = 0.015) and season (P = 0.048). Relapse commonly occurred in October, and variations in relapse were not significant either in month (P = 0.52) or season (P = 0.12). Upper respiratory inflammation was the main factor responsible for relapse.
CONCLUSION: Our results suggest that environmental factors associated with winter and spring seasonality may be responsible for triggering the clinical onset of UC in Japan.
PMCID: PMC3870560  PMID: 24379632
Japanese population; Onset; Relapse; Seasonal variations; Ulcerative colitis
6.  Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages 
Absence of the transcriptional regulator Zfp521 results in decreased bone formation by osteoblasts and increased osteoclast differentiation, largely via Zfp521’s regulation of the transcription factor Ebf1.
Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521+/− mice was rescued in Zfp521+/−:Ebf1+/− mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis.
PMCID: PMC3646489  PMID: 23569325
7.  Clinicopathologic Characteristics and Prognosis of Advanced Gastric Cancer Simulating Early Gastric Cancer 
Yonago Acta medica  2013;56(3):73-78.
Although the clinicopathologic features and prognosis of Borrmann type advanced gastric cancer has been well characterized, those of advanced gastric cancer simulating early gastric cancer (AGC simulating EGC) still remains unclear.
We reviewed 1985 gastric cancer patients who had undergone gastrectomy at our hospital to determine the clinicopathologic characteristics, susceptible sites for lymph node metastasis, and prognosis of AGC simulating EGC in comparison with Borrmann type advanced gastric cancer.
Among 102 patients with AGC simulating EGC, 100 patients (98%) had tumors with depressed type appearance. The frequencies of serosal invasion, lymph node metastasis, lymphatic vessel invasion, blood vessel invasion, and liver metastasis were significantly lower in AGC simulating EGC than in Borrmann type tumors. The prognosis of AGC simulating EGC was significantly better than that of the Borrmann type tumors. Multivariate analysis indicated that the gross appearance was an independent prognostic factor. In patients with AGC simulating EGC which invaded to the the muscularis propria (MP), most lymph node metastasis was restricted with the perigastric lymph nodes (1st-titer lymph nodes) and lymph node metastasis to 2nd-titer lymph nodes was only observed at station 8a.
AGC simulating EGC is less advanced in comparison with Borrmann type advanced gastric cancer. Based on the results of susceptible sites for lymph node metastasis in the current study, limited lymph node dissection could be indicated for AGC simulating EGC whose depth of invasion is MP.
PMCID: PMC3809722  PMID: 24170961
gastric cancer; gross appearance; lymph node dissection; prognosis
8.  Scoring of Prognostic Parameters in Patients with Unresectable Advanced or Recurrent Colorectal Cancer Undergoing Chemotherapy 
Yonago Acta medica  2013;56(3):69-72.
Suitable chemotherapy is needed to prolong the survival of patients with unresectable advanced or recurrent colorectal cancer. We scored the periodical changes of several prognostic markers during chemotherapy in patients with this type of cancer to discern the effectiveness of chemotherapy.
Twenty consecutive patients with unresectable advanced or recurrent colorectal cancer were enrolled. All patients underwent combination chemotherapy with oxaliplatin or irinotecan plus 5-fluorouracil/leucovorin. Neutrophil/lymphocyte ratio (NLR), serum C-reactive protein (CRP), serum carcinoembryonic antigen (CEA) and serum albumin (ALB) were compared between the two periods (before chemotherapy and 3 months after it was started) in each patient. The scoring system was as follows: points are added when a patient shows a decrease of NLR, CRP and CEA and an increase of ALB at 3 months after the start of chemotherapy with a possible final score of +4. On the other hand, points are reduced if a patient shows an elevation of NLR, CRP and CEA and a decrease of ALB at 3 months after the start of chemotherapy with a possible final score of −4.
At 3 months after the start of first line chemotherapy, 13 patients showed positive scores but 7 patients showed zero or minus scores. According to our scoring system, we found the mean survival time (MST) of the 13 patients with plus scores was 34 months and this was significantly better than that of the 7 patients who showed zero or minus scores (P = 0.0008).
Our new scoring system is useful but when we find that first line chemotherapy is ineffective, we need to change it to second line chemotherapy as soon as possible. That may be the best treatment for patients with unresectable advanced or recurrent colorectal cancer.
PMCID: PMC3812795  PMID: 24179314
chemotherapy; colorectal cancer; inflammation; immunity; nutrition
9.  Long-term biopersistence of tangled oxidized carbon nanotubes inside and outside macrophages in rat subcutaneous tissue 
Scientific Reports  2013;3:2516.
Because of their mechanical strength, chemical stability, and low molecular weight, carbon nanotubes (CNTs) are attractive biological implant materials. Biomaterials are typically implanted into subcutaneous tissue or bone; however, the long-term biopersistence of CNTs in these tissues is unknown. Here, tangled oxidized multi-walled CNTs (t-ox-MWCNTs) were implanted into rat subcutaneous tissues and structural changes in the t-ox-MWCNTs located inside and outside of macrophages were studied for 2 years post-implantation. The majority of the large agglomerates were present in the intercellular space, maintained a layered structure, and did not undergo degradation. By contrast, small agglomerates were found inside macrophages, where they were gradually degraded in lysosomes. None of the rats displayed symptoms of cancer or severe inflammatory reactions such as necrosis. These results indicate that t-ox-MWCNTs have high biopersistence and do not evoke adverse events in rat subcutaneous tissue in vivo, demonstrating their potential utility as implantable biomaterials.
PMCID: PMC3755288  PMID: 23981952
10.  Energy Expenditure and Bone Formation Share a Common Sensitivity to AP-1 Transcription in the Hypothalamus 
Journal of Bone and Mineral Research  2012;27(8):1649-1658.
The regulation of bone and fat homeostasis and its relationship to energy expenditure has recently been the focus of increased attention due to its potential relevance to osteoporosis, obesity and diabetes. Although central effectors within the hypothalamus have been shown to contribute to the regulation of both energy balance and bone homeostasis, little is known of the underlying mechanisms, including the possible involvement of transcriptional factors within the hypothalamus. Transgenic mice overexpressing ΔFosB, a splice variant of the AP1 transcription factor FosB with mixed agonist-antagonistic properties, have increased energy expenditure and bone mass. Since these mice express ΔFosB in bone, fat and hypothalamus, we sought to determine 1) whether overexpression of ΔFosB within the hypothalamus was sufficient to regulate energy expenditure and whether it would also regulate bone mass, and 2) whether these effects were due to antagonism to AP1. Our results show that stereotactic injection of an adeno-associated virus vector to restrict overexpression of ΔFosB to the ventral hypothalamus of wildtype mice induced a profound increase in both energy expenditure and bone formation and bone mass. This effect was phenocopied, at an even stronger level, by overexpressiong of a dominant-negative DNJunD, a pure AP1 antagonist. Taken together these results suggest that downregulation of AP1 activity in the hypothalamus profoundly increases energy expenditure and bone formation, leading to both a decrease in adipose mass and an increase in bone mass. These findings may have physiological implications since ΔFosB is expressed and regulated in the hypothalamus.
PMCID: PMC3399943  PMID: 22461201
AP-1; hypothalamus; energy expenditure; bone formation
11.  Prognosis of Patients With Gastric Cancer Who Underwent Proximal Gastrectomy 
International Surgery  2012;97(3):275-279.
Proximal gastrectomy (PG) has been introduced for patients who are preoperatively diagnosed with early gastric cancer located in the upper third of the stomach. In the present study, we compared the prognosis of patients who underwent PG with that of patients who underwent total gastrectomy (TG). Between 1997 and 2006, 51 patients were diagnosed with early gastric cancer located in the upper third of the stomach and underwent PG. In the same period, 35 patients were diagnosed with early gastric cancer and underwent TG. Of these, in 24 patients, the cancer was localized in the middle to upper part of the stomach, and 11 patients had multiple cancers. We compared the clinicopathologic differences and prognoses between the two groups. Significantly fewer lymph nodes were dissected in the PG group (mean, 18.2) than in the TG group (mean, 36.6;P < 0.001). Complications were detected in 17.6% of patients in the PG group and in 14.3% of patients in the TG group, which was not significant (P = 0.678). The overall and disease-specific 5-year survival rates in the 51 patients who underwent PG (88.7% and 97.1%, respectively) were not different from those in the 35 patients who underwent TG (87.6% and 93.4%; P = 0.971 and P = 0.553; respectively). These findings indicate that PG can be performed safely and may have various advantages compared with TG in terms of patients' daily lives.
PMCID: PMC3723220  PMID: 23113860
Early gastric cancer; Proximal gastrectomy; Prognosis; Total gastrectomy
12.  Ex vivo induction of IFN-λ3 by a TLR7 agonist determines response to Peg-IFN/Ribavirin therapy in chronic hepatitis C patients 
Journal of Gastroenterology  2013;49(1):126-137.
Genetic variation around interleukin-28B (IL28B), encoding IFN-λ3, predict non-responders to pegylated interferon-α/ribavirin (Peg-IFN/RBV) therapy in chronic hepatitis C (CHC). However, it remains unclear the expression and the role of IL28B itself. The aim of this study is to develop easy and useful methods for the prediction of treatment outcomes.
The mRNA and protein levels of IFN-λ3 induced by ex vivo stimulation of peripheral blood mononuclear cells (PBMC) or magnetically selected dendritic cells (DCs) with toll-like receptor agonists (TLR3; poly I:C, TLR7; R-837) were measured by the quantitative real-time polymerase chain reaction and our newly developed chemiluminescence enzyme immunoassays, respectively, and compared with the clinical data.
We found that BDCA-4+ plasmacytoid and BDCA-3+ myeloid DCs were the main producers of IFN-λs when stimulated with R-837 and poly I:C, respectively. Detectable levels of IFN-λs were inducible even in a small amount of PBMC, and IFN-λ3 was more robustly up-regulated by R-837 in PBMC of CHC patients with favorable genotype for the response to Peg-IFN/RBV (TT in rs8099917) than those with TG/GG. Importantly, the protein levels of IFN-λ3 induced by R-837 clearly differentiated the response to Peg-IFN/RBV treatment (p = 1.0 × 10−10), including cases that IL28B genotyping failed to predict the treatment response. The measurement of IFN-λ3 protein more accurately predicted treatment efficacies (95.7 %) than that of IL28B genotyping (65.2 %).
Genetic variations around IL28B basically affect IFN-λ3 production, but different amounts of IFN-λ3 protein determines the outcomes of Peg-IFN/RBV treatment. This study, for the first time, presents compelling evidence that IL28B confer a functional phenotype.
Electronic supplementary material
The online version of this article (doi:10.1007/s00535-013-0814-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3895210  PMID: 23591768
Chronic hepatitis C; IL28B; IFN-λ3; Peg-IFN/RBV
13.  In Vivo Imaging of Transplanted Islets Labeled with a Novel Cationic Nanoparticle 
PLoS ONE  2013;8(2):e57046.
To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a β-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients.
PMCID: PMC3579774  PMID: 23451139
14.  Usefulness of palliative prognostic score in the treatment of patients with non-resectable gastric cancer 
Molecular and Clinical Oncology  2013;1(2):253-256.
The aim of this study was to evaluate the clinical usefulness of the palliative prognostic (PaP) score in patients with non-resectable advanced gastric cancer. The PaP score was calculated prior to each course of chemotherapy in 44 consecutive patients with non-resectable advanced gastric cancer between 2003 and 2010 at the Tottori University Hospital, Yonago, Japan. The prognosis was evaluated according to the PaP score and the different chemotherapeutic agents. The median survival time (MST) was 10 months. The PaP score classified the heterogeneous patient sample into three isoprognostic groups with regard to the possibility of a 1-month survival period, with 28 patients in group A (>70% chance), 12 in group B (30–70% chance) and 4 in group C (<30% chance). The MST of the three groups was 11, 3 and 1 months for group A, B and C, respectively. In group A, chemotherapeutic regimens did not affect patient survival, although the docetaxel regimen prolonged survival of patients in group B. In conclusion, the PaP score may be useful in selecting the best chemotherapeutic regimen in patients with non-resectable gastric cancer.
PMCID: PMC3915674  PMID: 24649156
docetaxel; palliative prognostic score; non-resectable gastric cancer
15.  Clinicopathologic Characteristics and Prognosis of Gastric Cancer in Young Patients 
Yonago Acta Medica  2012;55(3):57-61.
To determine the clinicopathologic characteristics and prognosis of gastric cancer in young patients, a total of 1985 gastric cancer patients who had undergone gastrectomy at our hospital were reviewed. The male-to-female ratio was significantly lower in the young patients than in either the middle-aged (P < 0.0001) or elderly patients (P < 0.0001). Undifferentiated carcinoma was observed more frequently in the young patients compared with either the middle-aged (P < 0.0001) or elderly patients (P < 0.0001). Furthermore, peritoneal metastasis was observed more frequently in the young patients than in either the middle-aged (P < 0.005) or elderly patients (P < 0.005). Five-year survival rates were 61.0, 73.6 and 68.1% in the young, middle-aged and elderly patients, respectively. The prognosis of the middle-aged patients was significantly better than that of either the young or the elderly patients (P < 0.05). Multivariate analysis indicated that age was an independent prognostic factor. Peritoneal recurrence was more frequently observed in the young patients than either the middle-aged or the elderly patients (P < 0.05). Gastric cancer in young patients has unique characteristics, namely, a predominance of female patients and a high frequency of undifferentiated cancer and peritoneal metastasis and recurrence.
PMCID: PMC3727677  PMID: 24031140
age; gastric cancer; prognosis
16.  Heterotrophic bacterial production and extracellular enzymatic activity in sinking particulate matter in the western North Pacific Ocean 
Heterotrophic activities on sinking particulate matter (SPM) play an important role in SPM fluxes in the ocean. To demonstrate regional differences in heterotrophic activities on SPM, we measured heterotrophic bacterial production (HBP) in seawater (HBPSW) and SPM (HBPSPM) as well as potential extracellular enzyme activity (EEA) in SPM on a transect along 155°E in the western North Pacific Ocean in the subarctic (44°N), the Kuroshio Extension area (35°N), and the subtropical gyre (20°N). Depth-integrated HBPSW from the surface to 500 m was comparable between the locations, whereas HBPSPM at 44°N was substantially lower than at the other sites. We found the highest particulate organic carbon (POC) export flux and export efficiency to bathypelagic depths, and the lowest water temperatures, at 44°N. We found significant correlations between leucine aminopeptidase (LAPase) activity, β-glucosidase (BGase) activity, POC flux and particulate organic nitrogen flux. LAPase activity was two orders of magnitude higher than BGase activity, with a BGase:LAPase activity ratio of 0.027. There were no significant correlations between HBP and EEA in SPM except for lipase, and lipase activity was significantly correlated with temperature. We propose that hydrographic conditions are an important factor controlling heterotrophic bacterial activity and export efficiency of organic carbon to the deep ocean, as are the sources and abundance of SPM produced in the euphotic zone via primary production.
PMCID: PMC3478788  PMID: 23109933
sinking particulate matter; sediment trap; heterotrophic bacterial activity; extracellular enzyme activity; western North Pacific
17.  Deletion of PTH Rescues Skeletal Abnormalities and High Osteopontin Levels in Klotho−/− Mice 
PLoS Genetics  2012;8(5):e1002726.
Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. Parathyroid hormone (PTH), Klotho, and FGF23 have been shown to act as key regulators of serum calcium and phosphate homeostasis through a complex feedback mechanism. The phenotypes of Fgf23−/− and Klotho−/− (Kl−/−) mice are very similar and include hypercalcemia, hyperphosphatemia, hypervitaminosis D, suppressed PTH levels, and severe osteomalacia/osteoidosis. We recently reported that complete ablation of PTH from Fgf23−/− mice ameliorated the phenotype in Fgf23−/−/PTH−/− mice by suppressing serum vitamin D and calcium levels. The severe osteomalacia in Fgf23−/− mice, however, persisted, suggesting that a different mechanism is responsible for this mineralization defect. In the current study, we demonstrate that deletion of PTH from Kl−/− (Kl−/−/PTH−/− or DKO) mice corrects the abnormal skeletal phenotype. Bone turnover markers are restored to wild-type levels; and, more importantly, the skeletal mineralization defect is completely rescued in Kl−/−/PTH−/− mice. Interestingly, the correction of the osteomalacia is accompanied by a reduction in the high levels of osteopontin (Opn) in bone and serum. Such a reduction in Opn levels could not be observed in Fgf23−/−/PTH−/− mice, and these mice showed sustained osteomalacia. This significant in vivo finding is corroborated by in vitro studies using calvarial osteoblast cultures that show normalized Opn expression and rescued mineralization in Kl−/−/PTH−/− mice. Moreover, continuous PTH infusion of Kl−/− mice significantly increased Opn levels and osteoid volume, and decreased trabecular bone volume. In summary, our results demonstrate for the first time that PTH directly impacts the mineralization disorders and skeletal deformities of Kl−/−, but not of Fgf23−/− mice, possibly by regulating Opn expression. These are significant new perceptions into the role of PTH in skeletal and disease processes and suggest FGF23-independent interactions of PTH with Klotho.
Author Summary
Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. PTH, Klotho, and FGF23 are the key regulators of blood mineral ion homeostasis. Klotho is a type-I membrane protein and has been identified as cofactor required for FGF23 to bind and activate its receptor. Loss of either Klotho or Fgf23 activity results in a similar abnormal phenotype, including severe defects in skeletal mineralization and alterations in mineral ion balance. Here we describe a new mouse model in which we eliminated PTH from Kl−/− mice, and we can show that the skeletal mineralization defect was completely rescued in Kl−/−/PTH−/− mice and that this phenomenon was accompanied by a reduction in the high levels of osteopontin in bone and serum. We also present additional data showing that continuous infusion of Kl−/− mice with PTH results in an elevation in Opn levels and subsequently increased osteoid volume. Interestingly, this result differs from our previous report in which we describe that the osteomalacia and the high Opn levels in Fgf23−/−/PTH−/− mice persisted. Our finding suggests that PTH, possibly by regulating osteopontin, is responsible for the skeletal mineralization defect in Kl−/− mice, but not in Fgf23−/− mice.
PMCID: PMC3355080  PMID: 22615584
18.  Upregulation of TIM-3 and PD-1 on CD4+ and CD8+ T Cells Associated with Dysfunction of Cell-Mediated Immunity after Colorectal Cancer Operation 
Yonago Acta medica  2012;55(1):1-9.
Surgery has been reported to suppress cell-mediated immunity; however, the detailed mechanisms responsible for this remain unclear. We obtained information on expressions of T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and programmed cell death 1 (PD-1) on both CD4+ and CD8+ T cells postoperatively from colorectal cancer patients and evaluated by multicolor flow cytometry. The functions of CD4+ and CD8+ T cells based on TIM-3 and PD-1 expressions were determined by quantitating the concentration of interferon-γ (IFN-γ) by enzyme-linked immunosorbent assay. The study demonstrated a rapid and significant decrease in number of total lymphocytes, CD4+ T cells and CD8+ T cells. These immune cells reached the minimum on day 1 and then increased, but still significantly decreased on days 3 and 7. The frequency of PD-1+CD4+ T cells and TIM-3+CD4+ T cells significantly increased after colorectal surgery. IFN-γ secretion by PD-1+TIM-3+CD4+ T cells was significantly decreased compared to secretion by either PD-1+TIM3-CD4+ T cells or PD-1–TIM-3–CD4+ T cells. Furthermore, IFN-γ secretion by PD-1+TIM-3+CD8+ T cells was significantly decreased compared to secretion by either PD-1–TIM-3+CD8+ T cells or PD-1–TIM-3–CD8+ T cells. The expression of PD-1 and TIM-3 was closely related to impaired cell-mediated immunity that was observed after surgery for colorectal cancer. New treatment targeting TIM-3 and PD-1 on CD4+ and CD8+ T cells during the perioperative period may provide a breakthrough in the treatment of colorectal cancer patients.
PMCID: PMC3727487  PMID: 24031134
colorectal cancer; programmed cell death 1; surgical trauma; T-cell immunoglobulin domain and mucin domain 3
19.  Operative Procedure for Laparoscopy-Assisted Vagus Nerve and Pylorus-Preserving Gastrectomy (LAVNPPG) for Early Gastric Cancer 
Yonago Acta Medica  2011;54(4):59-63.
Laparosocpy-assisted pylorus-preserving gastrectomy (LAPPG) is a widely accepted surgical procedure for the treatment of early gastric cancer in the middle third of the stomach. We have been performing this operation since 2007. Compared with traditional distal gastrectomy, LAPPG has postoperative nutritional benefits for patients. However, this procedure preserves only the pyloric branch of the vagus nerve and not the celiac branch. We found that patients retain a large amount of residual food in the gastric remnant, which interferes with the detection of secondary cancer on endoscopic follow-up. To improve the pyloric function and postoperative gastrointestinal motility, we changed our procedure during 2009 to preserve both the pyloric and celiac branches of the vagus nerve, and we named this new procedure laparoscopy-assisted vagus nerve and pylorus-preserving gastrectomy (LAVNPPG). From 2009 to 2011, 11 patients underwent LAVNPPG at our hospital. Retrospective comparison of the safety of operation, postoperative complications, and condition of the gastric remnant between LAPPG (n = 13) and LAVNPPG (n = 11) found that the occurrence of postprandial stasis and food residue in the gastric remnant tended to be lower following LAVNPPG, though the differences were not significant. These findings indicate that LAVNPPG may be an operative procedure that could replace LAPPG.
PMCID: PMC3763795  PMID: 24031130
gastrectomy; gastric cancer; laparoscopy-assisted; pylorus; vagus nerve
20.  Nutritional Assessment of Patients After Pylorus-Preserving Gastrectomy for Early Gastric Cancer 
The Indian Journal of Surgery  2010;72(6):453-457.
The aim of this study was to evaluate the nutritional advantages of pylorus-preserving gastrectomy (PPG) in comparison with distal gastrectomy with Billroth I anastomosis (DG) in early gastric cancer (EGC). Between 2005 and 2007, 24 patients underwent PPG and 30 underwent DG. Subjective global assessment, objective data assessment, and endoscopic findings of the remnant stomach were compared between the two groups. Two years after surgery, the patients’ body weights recovered to 97% in PPG, but they continued to decrease in DG. Postoperative blood lymphocyte counts remained low in DG, but recovered to preoperative levels 6 months after surgery in PPG. Food residue in the gastric remnant was frequently observed in PPG (71.4%) than in DG (15.8%, P = 0.001). In nutritional aspect, PPG may be a more ideal operation than DG. However, food residue in the gastric remnant should be considered in PPG.
Electronic supplementary material
The online version of this article (doi:10.1007/s12262-010-0167-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3077201  PMID: 22131654
Gastrectomy; Gastric cancer; Pylorus; Surgical procedures; Treatment outcome
21.  The rs8099917 Polymorphism, When Determined by a Suitable Genotyping Method, Is a Better Predictor for Response to Pegylated Alpha Interferon/Ribavirin Therapy in Japanese Patients than Other Single Nucleotide Polymorphisms Associated with Interleukin-28B▿† 
Journal of Clinical Microbiology  2011;49(5):1853-1860.
We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.
PMCID: PMC3122695  PMID: 21389156
22.  Zfp521 Is a Target Gene and Key Effector of Parathyroid Hormone-Related Peptide Signaling in Growth Plate Chondrocytes 
Developmental cell  2010;19(4):533-546.
In the growth plate, the interplay between Parathyroid Hormone-Related Peptide (PTHrP) and Indian Hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional co-regulator, in pre-hypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP-/- and chondrocyte-specific PTHR1-/- mice, with decreased chondrocyte proliferation, early hypertrophic transition and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased cyclin D1 and Bcl-2 expression while increasing caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to up-regulate cyclin D1 and to antagonize Runx2, Ihh and Collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.
PMCID: PMC2958174  PMID: 20951345
23.  Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity 
The Journal of Cell Biology  2010;191(7):1271-1283.
Zfp521 regulates osteoblast development during lineage commitment and osteoblast maturation by suppressing Runx2 transcriptional activity.
Runx2 is indispensable for osteoblast lineage commitment and early differentiation but also blocks osteoblast maturation, thereby causing bone loss in Runx2 transgenic mice. Zinc finger protein 521 (Zfp521) antagonizes Runx2 in vivo. Eliminating one Zfp521 allele mitigates the cleidocranial dysplasia–like phenotype of newborn Runx2+/− mice, whereas overexpressing Zfp521 exacerbates it. Overexpressing Zfp521 also reverses the severe osteopenia of adult Runx2 transgenic mice. Zfp521 binds to both Runx2 and histone deacetylase 3 (HDAC3), promotes their association, and antagonizes Runx2 transcriptional activity in an HDAC3-dependent manner. Mutating the Zfp521 zinc finger domains 6 and 26 reduces the binding of Zfp521 to Runx2 and inhibition of Runx2 activity. These data provide evidence that Zfp521 antagonizes Runx2 in vivo and thereby regulates two stages of osteoblast development, early during mesenchymal cell lineage commitment and later during osteoblast maturation. Thus, the balance and molecular interplay between Zfp521 and Runx2 contribute to the control of osteoblast differentiation, skeletal development, and bone homeostasis.
PMCID: PMC3010073  PMID: 21173110
24.  Increased B7-H1 and B7-H4 Expressions on Circulating Monocytes and Tumor-Associated Macrophages are Involved in Immune Evasion in Patients with Gastric Cancer 
Yonago Acta Medica  2011;54(1):1-10.
Expression of the costimulatory molecule B7-H4, a member of the inhibitory B7 family, has been reported to be upregulated on tumor-associated macrophages, and this overexpression may be involved in immune evasion in cancer patients. The present study was designed to investigate B7-H4 expression on monocytes/macrophages and its relationship with immune evasion in gastric cancer patients. B7-H4 expression on circulating monocytes and tumor-associated macrophages was evaluated by multicolor flow cytometry. Carboxyfluorescein succinimidyl ester proliferation assays and quantitative interferon-gamma enzyme-linked immunosorbent assays were carried out to determine the inhibitory effect of B7-H4+ monocytes on CD4+ T cells. B7-H4 expression on circulating monocytes was upregulated and these B7-H4+ monocytes showed immunosuppressive properties. B7-H4 expression was closely related to the depth of invasion, as well as the presence of lymphatic and venous invasion. There were significant correlations between B7-H4 expression and B7-H1 or HLA-DR expression on monocytes/macrophages in gastric cancer patients. B7-H4 expression was remarkably upregulated in gastric cancer tissues compared with peripheral blood samples. Complete surgical removal of the tumor decreased B7-H4 expression on circulating monocytes from gastric cancer patients. Cocultures of gastric cancer cell lines and monocytes led to upregulation of B7-H4 expression on monocytes. Increased B7-H4 expression on circulating monocytes and tumor-associated macrophages may be one of the key mechanisms responsible for immune evasion by tumors in gastric cancer.
PMCID: PMC3763800  PMID: 24031123
B7-H1; B7-H4; gastric cancer; macrophage; monocyte
25.  Suppression of Wnt signaling by Dkk1 attenuates PTH-mediated stromal cell response and new bone formation 
Cell metabolism  2010;11(2):161.
Parathyroid hormone (PTH) suppresses Dickkopf 1 (Dkk1) expression in osteoblasts. To determine whether this suppression is essential for PTH-mediated Wnt signaling and bone formation, we examined mice that overexpress Dkk1 in osteoblasts (Dkk1 mice). Dkk1 mice were osteopenic due to abnormal osteoblast and osteoclast activity. When fed a low calcium diet, and in two other models of hyperparathyroidism, these mice failed to develop the peritrabecular stromal cell response (“osteitis fibrosis”) and new bone formation seen in wild type mice. Despite these effects of Dkk1 overexpression, PTH still activated Wnt signaling in Dkk1 mice and in osteoblastic cells cultured from these mice. In cultured MC3T3E1 preosteoblastic cells, PTH dramatically suppressed Dkk1 expression, induced PKA-mediated phosphorylation of β-catenin and significantly enhanced Lef1 expression. Our findings indicate that the full actions of PTH require intact Wnt signaling but that PTH can activate the Wnt pathway despite overexpression of Dkk1.
PMCID: PMC2819982  PMID: 20142103
PTH; PTH/PTHrP receptor; Dkk1; Wnt signaling

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