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Although the early diagnosis of gastric cancer provides the opportunity for curative endoscopic resection, comprehensive screening endoscopy would be invasive and expensive. To date, there is a complete absence of clinically useful gastric cancer biomarkers. With the goal of discovering noninvasive biomarkers for the early diagnosis of gastric cancer, we have conducted a case–control study using urine samples from individuals with gastric cancer versus healthy control samples. Of the enrolled 106 patients from September, 2012 to April, 2013, a cohort of 70 patients composed of 35 patients with gastric cancer and 35 age- and sex-matched healthy controls was analyzed. The gastric cancer group was composed of stage IA of 62.9% (22/35). The urinary levels of MMP-9/NGAL complex (uMMP-9/NGAL) and ADAM12 (uADAM12) were significantly higher in the gastric cancer group compared with the healthy control group as determined by monospecific ELISAs (uMMP-9/NGAL: median, 85 pg/mL vs. 0 pg/mL; P = 0.020; uADAM12: median, 3.35 ng/mL vs. 1.44 ng/mL; P < 0.001). Multivariate analysis demonstrated that both uMMP-9/NGAL and uADAM12 were significant, independent diagnostic biomarkers for gastric cancer. Moreover, MMP-9/NGAL activity was significantly elevated as determined by gelatin zymography. The combination of uMMP-9/NGAL with uADAM12 distinguished between control samples and gastric cancer samples with an AUC of 0.825 (P < 0.001) in an ROC analysis. Significantly, immunohistochemical analyses demonstrated a high coexpression of MMP-9 and NGAL (P < 0.001) and high expression of ADAM12 (P < 0.001) in gastric cancer tissues compared with adjacent normal tissues (N = 35). In summary, uMMP-9/NGAL and uADAM12 are potential noninvasive biomarkers for gastric cancer, including early-stage disease. Cancer Prev Res; 8(3); 240–8. ©2015 AACR.

Supplementary Figure S2. Representative immunohistochemical images of MMP-9 and NGAL in early gastric cancer without urinary MMP-9/NGAL complex.

Supplementary Figure S1. Angiogenesis array analyses of urine sample.

Background Marital transitions are associated with adverse health events, such as mortality and cardiovascular disease. Since marital transitions (e.g., becoming widowed) are unavoidable life events, it is necessary to identify modifiable intermediate outcomes. Thus, we examined the association between marital transitions and vegetable intake among middle-aged and older Japanese adults. Methods This longitudinal study included Japanese adults aged 40-79 years who received an annual health checkup between 2007 and 2011 (baseline) and five years later (follow-up). Marital transitions were classified as whether and what type of transition occurred during the five-year period and comprised five groups: consistently married, married to widowed, married to divorced, not married to married, and remained not married. Changes in total vegetable, green and yellow vegetable, and light-colored vegetable intake from baseline to follow-up were calculated using the Food Frequency Questionnaire. Results Data from 4813 participants were analyzed (mean age: 59.4 years; 44.1% women). Regarding marital transitions, 3,960 participants were classified as "consistently married," 135 as "married to widowed," 40 as "married to divorced," 60 as "not married to married," and 529 as "remained not married." Multivariable linear regression analysis revealed that compared to consistently married, married to widowed was inversely associated with the change in total vegetable intake (β = -16.64, SE = 7.68, p = 0.030) and light-colored vegetable intake (β = -11.46, SE = 4.33, p = 0.008). Conclusion Our findings suggest that being widowed could result in a reduced intake of vegetables. Hence, dietary counseling according to marital situation is necessary.

This study aimed to examine if feeding lactating mice a calcium-insufficient diet while simultaneously administering alendronate (ALN) could potentially induce osteocytic osteolysis.Lactating mice were fed calcium (Ca)-insufficient diets with or without ALN administration, and then their femurs were examined for TRAP and ALP, and observed by Kossa staining and transmission electron microscopy (TEM). Mice that had been fed a Ca-insufficient diet were then fed aMice fed a Ca-insufficient diet had a reduced number of TRAP-positive osteoclasts after ALN administration. ALN-treated, lactating mice fed a Ca-insufficient diet had enlarged lacunae in their cortical bones, and TEM imaging demonstrated expanded regions between osteocytes and lacunar walls. In ALN-treated lactating mice fed a Ca-insufficient diet, huge areas of demineralized bone matrix occurred, centered around blood vessels in the cortical bone. Isotope microscopy showedBone demineralization likely takes place in the periphery of the osteocytic lacunae and in the broad regions around the blood vessels of lactating mice when they are exposed to severely reduced serum Ca through a Ca-insufficient diet coupled with ALN administration.

Simple Summary Early diagnosis is critically important to achieve life-saving therapy for colorectal cancer (CRC). Since colonoscopy is not suitable as a screening method for CRC due to its invasiveness and high-cost, reliable and non-invasive diagnostic biomarkers are hopeful for CRC. In this case-control study, we established completely non-invasive, novel urinary microRNA (miRNA) biomarker panel combining miR-129-1-3p and miR-566 for the diagnosis of CRC. In the independent age- and sex-matched three cohorts comprising 415 participants, urinary levels of these miRNAs were consistently elevated in the CRC group compared to the healthy controls. Notably, the panel of combining miR-129-1-3p and miR-566 revealed an AUC of 0.845 for stage 0/I CRC that can be treated with endoscopic resection. Abstract Since noninvasive biomarkers as an alternative to invasive colonoscopy to detect colorectal cancer (CRC) are desired, we conducted this study to determine the urinary biomarker consisting of microRNAs (miRNAs). In total, 415 age- and sex-matched participants, including 206 patients with CRC and 209 healthy controls (HCs), were randomly divided into three groups: (1) the discovery cohort (CRC, n = 3; HC, n = 6); (2) the training cohort (140 pairs); and (3) the validation cohort (63 pairs). Among 11 urinary miRNAs with aberrant expressions between the two groups, miR-129-1-3p and miR-566 were significantly independent biomarkers that detect CRC. The panel consisting of two miRNAs could distinguish patients with CRC from HC participants with an area under the curve (AUC) = 0.811 in the training cohort. This panel showed good efficacy with an AUC = 0.868 in the validation cohort. This urinary biomarker combining miR-129-1-3p and miR-566 could detect even stage 0/I CRC effectively with an AUC = 0.845. Moreover, the expression levels of both miR-129-1-3p and miR-566 were significantly higher in primary tumor tissues than in adjacent normal tissue. Our established novel biomarker consisting of urinary miR-129-1-3p and miR-566 enables noninvasive and early detection of CRC.

The clinical impact of direct-to-consumer genetic testing (DTC-GT) on health behavior change has remained controversial. The aim of this study is to clarify the short-term effects of DTC-GT on gynecological cancer screening uptake among middle-aged never-screened Japanese women in a randomized controlled trial (RCT). A total of 144 women aged 45–50 who had never undergone gynecological cancer screening were randomly selected to receive health education (control group), or health education and DTC-GT (intervention group), at a 1:1 ratio. We compared the gynecological screening uptake during the follow-up period. Furthermore, to estimate the impact of learning of an elevated genetic cancer risk in the intervention group, we conducted an analysis dichotomized by genetic risk category. A total of 139 women completed the one-year follow-up survey (69 in the control group and 70 in the intervention group). The follow-up period did not differ between control and intervention groups (the median follow-up period was 276 days and 279 days, respectively, p = 0.746). There were 7 (9.7%) women in the control group and 10 (13.9%) in the intervention group who attended breast cancer screening (p = 0.606), and 9 (12.5%) women from both groups attended cervical cancer screening (p = 1.000). Likewise, there were no significant differences in cancer screening uptake in the analysis stratified by risk category within the intervention group. In conclusion, there was no significant effect of DTC-GT on gynecological cancer screening uptake in this RCT setting. Increasing cancer screening attendance may require a combination of well-established intervention strategies and DTC-GT. Clinical Trial Registration: UMIN-CTR Identifier, UMIN000031709.

Background Helicobacter pylori (H.pylori) may be commonly transmitted among family members, especially from parents to their children. Therefore, we investigated the prevalence of H.pylori infection in parents and their children. Method The subjects (n = 607) were selected from participants of the Japan Multi-Institutional Collaborative Cohort Study in Okazaki, Japan, which was conducted from November 2018 to March 2019, and their families. Of the 607 subjects, 126 subjects (32 men=fathers, 94 women=mothers) participated in this study with at least one of their children (126 children in total). We defined ‘H.pylori infection+’ as 1) H.pylori antibody titer≥3U/mL or 2) pepsinogen I ≤ 70ng/mL with the ratio of pepsinogen I/II≤3.0 or 3) past eradication of H.pylori or 4) past history of gastric cancer. The prevalence of ‘H.pylori infection+’ was compared with between mothers and fathers who has infected children. Finally, the 94 mothers were divided into two groups according to their birth-year (1936-1963 and 1964-1973), and we compared the prevalence of ‘H.pylori infection+’ in mothers and their children by group. Result The prevalence of ‘H.pylori infection+’ in mothers and fathers was 44 (46.8%) and 9 (28.1%), respectively. 13 (29.5%) of the 44 infected mothers had at least one infected child, but the infected fathers did not have infected children. The prevalence of ‘H.pylori infection+’ in 50 mothers born in 1936-1963 was 28 (56.0%), and 9 (32.1%) of the 28 infected mothers had at least one infected child. The prevalence of ‘H.pylori infection+’ in 44 mothers born in 1964-1973 was 16 (36.4%), and 4 (25.0%) of the 16 infected mothers had at least one infected child. Conclusion In family members, H.pylori may be transmitted mainly from mothers, rather than from fathers, to their children. Key messages Among Japanese, the prevalence of H.pylori infection will decrease with decline of the infection in women.

Background We aimed to examine the association between educational history and second-hand smoke (SHS) exposure in Japanese adults. Methods We conducted a repeated cross-sectional study using data from the 2007-2011 baseline and the 2013-2016 follow-up survey in a Japanese suburban area. Overall, 5,562 male and 5,381 female non-smokers participated in the study, which evaluated the SHS exposure and educational history of the participants through self-reported questionnaires. We performed a multilevel ordered logistic regression analysis with survey year as the first level and the individual as the second level, SHS exposure as the dependent variable, educational history as the explanatory variable, and age, smoking history, marital status as covariates, stratified by gender and working status. Results The prevalence of SHS exposure was 45.4% in males and 41.9% in females in the 2007-2011 survey, and 31.6% in males and 28.7% in females in the 2013-2016 survey. Multilevel analysis indicated that less educational history was associated with higher SHS exposure in both genders (among males, the odds ratio compared with ≥13 years was 1.75 for 10-12 years and 1.64 for ≤9 years; among females, it was 1.79 for 10-12 years and 2.43 for ≤9 years). Stratified analysis by working status showed that for females, education-based inequalities in SHS exposure more strongly impacted for workers than non-workers (p for interaction Conclusions Education-based inequalities in SHS exposure exist in Japan and might be severe among female workers. Key messages Measures against SHS in Japan should be promoted to reduce social and gender inequalities.

Gastric cancer (GC) is one of the most common causes of cancer deaths worldwide; however, reliable and non-invasive screening methods for GC are not established. Therefore, we conducted this study to develop a biomarker for GC detection, consisting of urinary microRNAs (miRNAs). We matched 306 participants by age and sex [153 pairs consisting of patients with GC and healthy controls (HCs)], then randomly divided them across three groups: (1) the discovery cohort (4 pairs); (2) the training cohort (95 pairs); and (3) the validation cohort (54 pairs). There were 22 urinary miRNAs with significantly aberrant expressions between the two groups in the discovery cohort. Upon multivariate analysis of the training cohort, urinary expression levels of miR-6807-5p and miR-6856-5p were significantly independent biomarkers for diagnosis of GC, in addition to Helicobacter pylori (H. pylori) status. A diagnostic panel that combined these 2 miRNAs and H. pylori status distinguished between HC and GC samples with an area under the curve (AUC) = 0.736. In the validation cohort, urinary miR-6807-5p and miR-6856-5p showed significantly higher expression levels in the GC group, and the combination biomarker panel of miR-6807-5p, miR-6856-5p, and H. pylori status also showed excellent performance (AUC = 0.885). In addition, this biomarker panel could distinguish between HC and stage I GC patients with an AUC = 0.748. Urinary expression levels of miR-6807-5p and miR-6856-5p significantly decreased to undetectable level after curative resection of GC. This novel biomarker panel enables early and non-invasive detection of GC.

The purpose of this paper is to describe the treatment procedures for a solid/multicystic ameloblastoma which was treated with the “Dredging Method” modified by fractionated enucleation adopted to avoid the pathologic fracture because of the complicated expansive nature in the mandible. Ameloblastoma is defined as a benign epithelial odontogenic tumor with progressive growth potential. Currently employed surgical treatment as curative treatment is resection with adequate margins because of the characteristics of high recurrence rates with conservative treatment. In this article, a 38-year-old male with swelling on the right mandible was referred to our hospital. Image analysis showed an expansile partially honeycombed multilocular radiolucent lesion from the body to the ascending ramus of the mandible. A follicular ameloblastoma was diagnosed by the biopsy. The paper also details the management of the ameloblastoma with the “Dredging Method” to remove the tumor completely and maintain the form and function of the mandible and the 13-year follow-up post treatment.

Background It is known that physical activity affects glucose metabolism. However, there have been no reports on the influence of physical activity earlier in life on subsequent glucose metabolism. Therefore, we analyzed the influence of physical activity in earlier decades of life on insulin resistance in middle aged and older residents in Japan. Methods The subjects were 6,883 residents of Okazaki City between the ages of 40 and 79 years who underwent physical examinations at the Okazaki City Medical Association Public Health Center from April 2007 through August 2011. They gave informed consent for participation in the study. Data on individual characteristics were collected via a questionnaire and from the health examination records. Fasting blood glucose and insulin levels were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). HOMA-IR >1.6 was considered to indicate insulin resistance for the purpose of logistic regression models. Results The study sample included 3,683 men and 3,200 women for whom complete information was available. For those who exercised regularly throughout their teens to their 30s-40s, the odds ratio for having insulin resistance was 0.75 (95% confidence interval [CI], 0.58-0.96) for men and 0.76 (95% CI, 0.58-0.99) for women after adjusting for other variables, including age, body mass index, and present physical activity. A linear trend was also observed in both men and women. Conclusions Subjects who have exercised regularly in the early decades of life are less likely to have insulin resistance later in life.

Background: Esophageal cancer (EC) including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) generally exhibits poor prognosis; hence, a noninvasive biomarker enabling early detection is necessary. Thus, this study aimed to establish novel urinary miRNA biomarkers for diagnosing EC.Methods: Out of 343 patients (299 healthy controls [HCs] and 44 ESCCs), 150 HCs and 43 patients with ESCC, which were both age- and sex-matched, were analyzed and randomly divided into two groups: 9 patients (6 HCs and 3 ESCCs) in the discovery cohort for microarray analysis and 184 patients (144 HCs and 40 ESCCs) in the training/test cohort with cross-validation for qRT-PCR analysis. Using 152 urine samples (144 HCs and 8 EACs), we validated the urinary miRNA biomarkers for EAC diagnosis.Results: Eight miRNAs were selected as biomarker candidates in the discovery cohort. In the training/test cohort, five of the eight miRNAs (miR-1273f, miR-619-5p, miR-150-3p, miR-4327, and miR-3135b) in the ESCC group had significantly higher urinary levels than those in the HC group. Consistently, these five urinary miRNAs were significantly different between HC and ESCC in both training and test sets. Especially, urinary miR-1273f and miR-619-5p showed excellent values of area under the curve (AUC) ≥ 0.80 for diagnosing stage I ESCC. Consistent with the ESCC group, the EAC group had significantly higher urinary levels of these five miRNAs than the HC group, and it also exhibited AUC values of approximately 0.80.Conclusion: The present study established novel urinary miRNA biomarkers that can early detect ESCC and EAC.

Introduction After the onset of bone metastasis, tumor cells appear to modify surrounding microenvironments for their benefit, and particularly, the levels of circulating fibroblast growth factor (FGF) 23 in patients with tumors have been highlighted. Materials and methods We have attempted to verify if human breast carcinoma MDA-MB-231 cells metastasized in the long bone of nu/nu mice would synthesize FGF23. Serum concentrations of calcium, phosphate (Pi) and FGF23 were measured in control nu/nu mice, bone-metastasized mice, and mice with mammary gland injected with MDA-MB-231 cells mimicking primary mammary tumors. Results and conclusions MDA-MB-231 cells revealed intense FGF23 reactivity in metastasized lesions, whereas MDA-MB-231 cells cultured in vitro or when injected into the mammary glands (without bone metastasis) showed weak FGF23 immunoreactivity. Although the bone-metastasized MDA-MB-231 cells abundantly synthesized FGF23, osteocytes adjacent to the FGF23-immunopositive tumors, unlike intact osteocytes, showed no FGF23. Despite significantly elevated serum FGF23 levels in bone-metastasized mice, there was no significant decrease in the serum Pi concentration when compared with the intact mice and mice with a mass of MDA-MB-231 cells in mammary glands. The metastasized femora showed increased expression and FGFR1 immunoreactivity in fibroblastic stromal cells, whereas femora of control mice showed no obvious FGFR1 immunoreactivity. Taken together, it seems likely that MDA-MB-231 cells synthesize FGF23 when metastasized to a bone, and thus affect FGFR1-positive stromal cells in the metastasized tumor nest in a paracrine manner.

Background With the goal of discovering non-invasive biomarkers for early diagnosis of GC, we conducted a case-control study utilising urine samples from individuals with predominantly early GC vs. healthy control (HC). Methods Among urine samples from 372 patients, age- and sex-matched 282 patients were randomly divided into three groups: 18 patients in a discovery cohort; 176 patients in a training cohort and 88 patients in a validation cohort. Results Among urinary proteins identified in the comprehensive quantitative proteomics analysis, urinary levels of TFF1 (uTFF1) and ADAM12 (uADAM12) were significantly independent diagnostic biomarkers for GC, in addition to Helicobacter pylori status. A urinary biomarker panel combining uTFF1, uADAM12 and H. pylori significantly distinguished between HC and GC patients in both training and validation cohorts. On the analysis for sex-specific biomarkers, this combination panel demonstrated a good AUC of 0.858 for male GC, whereas another combination panel of uTFF1, uBARD1 and H. pylori also provided a good AUC of 0.893 for female GC. Notably, each panel could distinguish even stage I GC patients from HC patients (AUC = 0.850 for males; AUC = 0.845 for females). Conclusions Novel urinary protein biomarker panels represent promising non-invasive biomarkers for GC, including early-stage disease.

Glial choristoma is considered a developmental malformation characterized by brain heterotopias without connection to the central nervous system. In this article, we report a rare case of glial choristoma occurring on the dorsum of the tongue with cleft palate in a 3-month-old male infant. Complete surgical excision was performed prior to palatoplasty. There were no postoperative complications or evidence of recurrence after 5 years of follow-up.

The number of older adults who continue working after retirement is increasing in Japan. Little is known about how job conditions affect older adults' health. We examined the association between job conditions and health-related quality of life (HRQOL) during a five-year follow-up study.This study included participants aged 65 years or older from the Japan Multi-Institutional Collaborative Cohort Study in the Okazaki area recruited at baseline between 2007 and 2011 and followed up five years later. Participants completed a self-reported questionnaire on the physical and mental health aspects of HRQOL (SF-8™), employment status, and job conditions (job satisfaction, skill use, and job suitability).Data of 1,146 men and 522 women were analyzed (mean age: 69.1 and 68.6 years, respectively). Generalized mixed linear regression analysis revealed that, compared to the not-working group, skill use was positively associated with mental health aspects among men (skill use × time: β = 0.16, SE = 0.08, p0.05), while poor job satisfaction and job suitability were negatively associated with mental health aspects among women (job satisfaction, not satisfied × time: β = -0.93, SE = 0.47, p0.05; job suitability, not suitable × time: β = -1.06, SE = 0.50, p0.05).Regarding job conditions among older adults, skill use in men was marginally associated with mental health, and poor job satisfaction and suitability in women were negatively associated with mental health. Considering the job conditions of older workers is necessary to protect their mental health.

INTRODUCTION AND OBJECTIVE:Development of kidney stones has been linked to metabolic syndrome, which is characterized by insulin resistance (IR). In this prospective study, we followed the baseline...

Background: Second-hand smoke exposure has been associated with poor mental health. However, among Japanese adults, little is known about the association between second-hand smoking and depressive symptoms. We examined this association in a cross-sectional study among a Japanese general adult population sample. Methods: Japanese adults were recruited from the Japan Multi-Institutional Collaborative Cohort Study in the Okazaki area between 2012 and 2017. Second-hand smoke exposure and smoking status were assessed using a self-administered questionnaire. Based on their frequency of exposure to second-hand smoke, non-smokers and smokers were categorized as “almost never,” “sometimes,” and “almost every day”. Depressive symptoms were defined by a Kessler 6 score ≥5 points. We performed a multivariable Poisson regression analysis to obtain adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs) for depressive symptoms. Results: Overall, 5,121 participants (4,547 non-smokers and 574 smokers) were included whose mean age was 63.6 (standard deviation [SD], 10.3) years for non-smokers and 59.33 (SD, 10.2) years for smokers. The association between second-hand smoking and depressive symptoms was significant among non-smokers, but not among smokers. Among non-smokers, PRs compared with “almost never” were 1.25 (95% CI, 1.09–1.42) for “sometimes” and 1.41 (95% CI, 1.09–1.84) for “almost every day” (P for trend

INTRODUCTION AND OBJECTIVES:Kidney stone development has been linked to metabolic syndrome (MetS), which is characterized by insulin resistance. In a previous cross-sectional analysis, we reported ...

ObjectiveWe aimed to examine missing data in FFQ and to assess the effects on estimating dietary intake by comparing between multiple imputation and zero imputation.DesignWe used data from the Okazaki Japan Multi-Institutional Collaborative Cohort (J-MICC) study. A self-administered questionnaire including an FFQ was implemented at baseline (FFQ1) and 5-year follow-up (FFQ2). Missing values in FFQ2 were replaced by corresponding FFQ1 values, multiple imputation and zero imputation.SettingA methodological sub-study of the Okazaki J-MICC study.ParticipantsOf a total of 7585 men and women aged 35–79 years at baseline, we analysed data for 5120 participants who answered all items in FFQ1 and at least 50% of items in FFQ2.ResultsAmong 5120 participants, the proportion of missing data was 3·7%. The increasing number of missing food items in FFQ2 varied with personal characteristics. Missing food items not eaten often in FFQ2 were likely to represent zero intake in FFQ1. Most food items showed that the observed proportion of zero intake was likely to be similar to the probability that the missing value is zero intake. Compared with FFQ1 values, multiple imputation had smaller differences of total energy and nutrient estimates, except for alcohol, than zero imputation.ConclusionsOur results indicate that missing values due to zero intake, namely missing not at random, in FFQ can be predicted reasonably well from observed data. Multiple imputation performed better than zero imputation for most nutrients and may be applied to FFQ data when missing is low.

Usual sleep duration has substantial heritability and is associated with various physical and psychiatric conditions as well as mortality. However, for its genetic locus, only PAX8 and VRK2 have been replicated in previous genome-wide association studies (GWAS). We conducted a GWAS meta-analysis of self-reported usual sleep duration using three population-based cohorts totaling 31 230 Japanese individuals. A genome-wide significant locus was identified at 12q24 (p-value < 5.0 × 10-8). Subsequently, a functional variant in the ALDH2 locus, rs671, was replicated in an independent sample of 5140 Japanese individuals (p-value = 0.004). The association signal, however, disappeared after adjusting for alcohol consumption, indicating the possibility that the rs671 genotype modifies sleep duration via alcohol consumption. This hypothesis explained a modest genetic correlation observed between sleep duration and alcohol consumption (rG = 0.23). A Mendelian randomization analysis using rs671 and other variants as instrumental variables confirmed this by showing a causal effect of alcohol consumption, but not of coffee consumption on sleep duration. Another genome-wide significant locus was identified at 5q33 after adjusting for drinking frequency. However, this locus was not replicated, nor was the PAX8 and VRK2. Our study has confirmed that a functional ALDH2 variant, rs671, most strongly influences on usual sleep duration possibly via alcohol consumption in the Japanese population, and presumably in East Asian populations. This highlights the importance of considering the involvement of alcohol consumption in future GWAS of usual sleep duration, even in non-East Asian populations, where rs671 is monomorphic.

Oral lichen planus (OLP) is a chronic inflammatory disorder of the oral mucosa of unknown etiology. Clinically, the erosive type of OLP (erosive OLP) can show features similar to those of pemphigus vulgaris (PV), an autoimmune blistering disorder in which desmoglein (Dsg)3 is targeted. In addition to clinical and histopathological findings, immunological studies, including direct immunofluorescence (IF), indirect IF and enzyme-linked immunosorbent assay (ELISA) that detect autoantibodies to Dsg3, are helpful in differentiating erosive OLP from PV. Here, we show two cases of erosive OLP with autoantibodies to Dsg3. Patient 1 was a 68-year-old woman with chronic erosions of the oral mucosa, in which elevated levels of immunoglobulin (Ig)G autoantibodies to Dsg1 and Dsg3 were detected by ELISA. Patient 2 was an 85-year-old woman with white striae with erosions on the lateral sides of the buccal mucosa with elevated levels of IgG autoantibodies to Dsg3 detected by ELISA. Histopathological findings from both cases showed lichenoid dermatitis, and both direct and indirect IF showed no tissue-bound IgG autoantibodies. From these findings, the diagnosis of erosive OLP was made. Immunological assays revealed both cases to have IgG-directing calcium-independent linear epitopes on Dsg3, which are suggestive of non-pathogenic autoantibodies. In addition, autoantibodies to Dsg3 in patient 2 reacted with a prosequence-possessing precursor form of Dsg3 but not with the mature form of the molecule. The present study suggests that erosive OLP may develop anti-Dsg3 autoantibodies, which should be carefully assessed.

We examined the association between family caregiver burden and subjective well-being with social participation&rsquo, s moderating effect among Japanese adults. Data were obtained from a cross-sectional survey by the Japan Multi-Institutional Collaborative Cohort Study in the Okazaki area between 2013 and 2017. Study participants included 5321 adults who visited the Public Health Center for annual health check-ups and answered a questionnaire regarding health status and lifestyle. Subjective well-being was assessed by a single item, out of 10 points, and analyzed with multivariable linear regression analysis models by subjective family caregiver burden (&ldquo, none&rdquo, &ldquo, mild&rdquo, severe&rdquo, ), stratified by gender. Ultimately, 2857 men and 2223 women were included. Mean participant age (standard deviation) in years was 64.7 (10.4) for men and 61.3 (10.0) for women. Multivariable analysis revealed that, among women, higher caregiver burden was inversely associated with subjective well-being (p for trend &lt, 0.001), and the interaction of severe caregiver burden and social participation on subjective well-being was positive and significant (p for interaction &lt, 0.05). High family caregiver burden was inversely associated with subjective well-being among Japanese women, but moderated by the caregiver&rsquo, s social participation, suggesting the importance of community development that enables family caregivers&rsquo, social participation to protect their subjective well-being.

Since a fecal occult blood test for colorectal cancer (CRC) does not offer sufficient diagnostic power for CRC, novel non-invasive biomarkers are hopeful for CRC screening. We conducted the current study to discover non-invasive urinary biomarkers for diagnosing CRC. Among urine samples from 258 patients (CRC, n = 148; healthy controls, n = 110), a cohort of 176 patients composed of 88 patients with GC and 88 healthy controls was selected after age- and sex-matching using propensity score. This cohort was then randomly divided into 2 groups: 53 pairs (106 patients) in the training cohort, and 35 pairs (70 patients) in the validation cohort. No significant differences were found for baseline characteristics between the CRC and healthy control groups in both training and validation cohorts. On multivariate analysis in the training cohort, urinary levels of cysteine-rich protein 61 (uCyr61) and trefoil factor 3 (uTFF3) were identified as independent significant diagnostic markers for CRC. Moreover, uCyr61 alone and the combination of uCyr61 and uTFF3 allowed significant differentiation between healthy controls and CRC groups in the training set (uCyr61: area under the curve (AUC) = 0.745 [95% CI, 0.653–0.838]; uCyr61 + uTFF3: AUC = 0.753 [95% CI, 0.659–0.847]). In the validation cohort, uCyr61 and uTFF3 were significantly higher in the CRC group than in the healthy control group, and they also allowed significant differentiation between healthy control and CRC groups (uCyr61: AUC = 0.696 [95% CI, 0.571–0.822]; uTFF3: AUC = 0.639 [95% CI, 0.508–0.770]; uCyr61 + uTFF3: AUC = 0.720 [95% CI, 0.599–0.841]), as in the training cohort. A panel combining uCyr61 and uTFF3 offers a promising non-invasive biomarker for diagnosing CRC.

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.

Although the early diagnosis of gastric cancer provides the opportunity for curative endoscopic resection, comprehensive screening endoscopy would be invasive and expensive. To date, there is a complete absence of clinically useful gastric cancer biomarkers. With the goal of discovering noninvasive biomarkers for the early diagnosis of gastric cancer, we have conducted a case–control study using urine samples from individuals with gastric cancer versus healthy control samples. Of the enrolled 106 patients from September, 2012 to April, 2013, a cohort of 70 patients composed of 35 patients with gastric cancer and 35 age- and sex-matched healthy controls was analyzed. The gastric cancer group was composed of stage IA of 62.9% (22/35). The urinary levels of MMP-9/NGAL complex (uMMP-9/NGAL) and ADAM12 (uADAM12) were significantly higher in the gastric cancer group compared with the healthy control group as determined by monospecific ELISAs (uMMP-9/NGAL: median, 85 pg/mL vs. 0 pg/mL; P = 0.020; uADAM12: median, 3.35 ng/mL vs. 1.44 ng/mL; P < 0.001). Multivariate analysis demonstrated that both uMMP-9/NGAL and uADAM12 were significant, independent diagnostic biomarkers for gastric cancer. Moreover, MMP-9/NGAL activity was significantly elevated as determined by gelatin zymography. The combination of uMMP-9/NGAL with uADAM12 distinguished between control samples and gastric cancer samples with an AUC of 0.825 (P < 0.001) in an ROC analysis. Significantly, immunohistochemical analyses demonstrated a high coexpression of MMP-9 and NGAL (P < 0.001) and high expression of ADAM12 (P < 0.001) in gastric cancer tissues compared with adjacent normal tissues (N = 35). In summary, uMMP-9/NGAL and uADAM12 are potential noninvasive biomarkers for gastric cancer, including early-stage disease. Cancer Prev Res; 8(3); 240–8. ©2015 AACR.

This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig's epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial-mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle.

Examination of klotho-deficient (kl/kl) mice showed that they had developed vascular medial calcification. Von Kossa and van Gieson staining showed calcification of the straight elongated elastic lamellae. Interestingly, elastic fibers, but not the abundant collagen fibrils surrounding them, were preferentially calcified in the kl/kl aorta. Unlike normal vascular smooth muscle cells (VSMCs), tunica media cells of kl/kl mice had developed cisterns of rough endoplasmic reticulum and Golgi apparatus; thus, they resembled matrix-synthesizing cells rather than authentic VSMCs. Additionally, many matrix vesicle-like structures and calcifying nodules were observed in the vicinity of these VSMC-like cells in kl/kl tunica media. Several calcifying nodules made contact with the amorphous organic materials, allowing the extension of calcification into the amorphous materials from these contact points. In contrast, mineral deposition within the amorphous organic materials was also observed, indicating dystrophic calcification. These observations suggest 2 possible pathways of vascular calcification: one involving matrix vesicle-like structures that could include mineral crystals and the other involving the deposition of calcium phosphates within the amorphous materials. The former pathway suggests the possibility of trans-differentiation of VSMCs into the osteoblastic lineage that could induce biological calcification of vascular tissues, in addition to the classical hypothesis involving dystrophic calcification. Here, we review our current findings on the process of vascular medial calcification of the aorta in klotho-deficient mice.

We have histologically examined vascular invasion and calcification of the hypertrophic zone during endochondral ossification in matrix metalloproteinase (MMP)-9 deficient (MMP-9-/-) mice and in their littermates at 3 days, 3 weeks and 6 weeks after birth. Capillaries and osteoclasts at the chondro-osseous junction showed an intense MMP-9 immunopositivity, suggesting that they recognize chemical properties of cartilaginous matrices, and then release MMP-9 for cartilage degradation. CD31-positive capillaries and tartrate-resistant acid phosphatase-reactive osteoclasts could be found in the close proximity in the region of chondro-osseous junction in MMP-9-/- mice, while in wild-type mice, vascular invasion preceded osteoclastic migration into the epiphyseal cartilage. Although MMP-9-/- mice revealed larger hypertrophic zones, the index of calcified area was significantly smaller in MMP-9-/- mice. Interestingly, the lower layer of the MMP-9-/- hypertrophic zone showed intense MMP-13 staining, which could not be observed in wild-type mice. This indicates that MMP-13 may compensate for MMP-9 deficiency at that specific region, but not to a point at which the deficiency could be completely rescued. In conclusion, it seems that MMP-9 is the optimal enzyme for cartilage degradation during endochondral ossification by controlling vascular invasion and subsequent osteoclastic migration.

Sclerostin, an osteocyte-derived molecule, has been reported to serve as a negative regulator of osteoblastic activity as well as bone remodeling. However, there is no report that verified the regional difference for sclerostin synthesis, and in this study we have investigated immunolocalization of sclerostin by comparing dentin matrix protein (DMP) 1, an osteocyte-derived factor broadly expressed in tibial metaphyses and cortical bone. In metaphyseal primary trabecules, a site of bone modeling, strong DMP1-reactivity was observed in osteocytic lacunar-canalicular system (OLCS), while faint staining for sclerostin was visible only in a few osteocytes. In secondary trabecules, in which bone remodeling begins, some osteocytes showed intense sclerostin-immunopositivity, though there were many DMP1-positive osteocytes. In cortical bone, there were more osteocytes reactive for sclerostin, when compared with those in the secondary trabecules. Silver impregnation verified that immature, primary trabecules contained randomly-oriented OLCS, while mature, cortical bone showed geometrically well-arrangement of OLCS. Taken together, though DMP1 is broadly synthesized in bone, sclerostin appears to be abundantly synthesized in regular OLCS of cortical bone, but less produced in irregular OLCS as seen in primary trabecules, indicating the regional difference for sclerostin synthesis.

// Takaya Shimura 1 , Masahide Ebi 1, 2 , Tomonori Yamada 3 , Tamaki Yamada 4 , Takahito Katano 1 , Yu Nojiri 1, 3 , Hiroyasu Iwasaki 1, 3 , Satoshi Nomura 1 , Noriyuki Hayashi 1 , Yoshinori Mori 1 , Hiromi Kataoka 1 , Marsha A. Moses 5, 6 , Takashi Joh 1 1 Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan 2 Department of Gastroenterology, Aichi Medical University, Nagakute, Japan 3 Department of Gastroenterology, Japanese Red Cross Nagoya Daini Hospital, Nagoya, Japan 4 Okazaki Public Health Center, Okazaki, Japan 5 Vascular Biology Program, Boston Children’s Hospital, Boston, MA, USA 6 Department of Surgery, Harvard Medical School and Boston Children’s Hospital, Boston, MA, USA Correspondence to: Takaya Shimura, email: tshimura@med.nagoya-cu.ac.jp Keywords: biomarker, gastric cancer, inoperability, kallikrein 10, urine Received: November 08, 2016 Accepted: February 22, 2017 Published: March 22, 2017 ABSTRACT The current imaging modalities are not sufficient to identify inoperable tumor factors, including distant metastasis and local invasion. Hence, we conducted this study using urine samples to discover non-invasive biomarkers for the incurability of gastric cancer (GC). Urine samples from 111 GC patients were analyzed in this study. The GC cohort was categorized and analyzed according to disease stage and operability. In the discovery phase, protease protein array analysis identified 3 potential candidate proteins that were elevated in the urine of advanced GC patients compared to early GC patients. Among them, urinary kallikrein 10 (KLK10) was positively associated with tumor stage progression. Moreover, the urinary level of KLK10 (uKLK10) was significantly elevated in the urine of patients with inoperable GC compared to operable GC patients (median, 118 vs. 229; P =0.014). The combination of uKLK10, tumor location and tumor size distinguished operability of GC with an area under the curve of 0.859, 82.4% sensitivity and 86.2% specificity. Disease-free survival (DFS) was significantly shorter in GC patients with high uKLK10 compared to those with low uKLK10 (hazard ratio: 3.30 [95% confidence interval, 1.58-6.90] P

Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin β1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.

In order to provide a clue to understand the interplay between leptin and estrogen, we have examined femoral metaphyses of ovariectomized db/db mice carrying a mutated leptin receptor. We performed ovariectomy (OVX) or sham-operation (sham) on 12-week old female wild-type and db/db mice, and then, after 8 weeks, divided the animals into four groups: wild-type sham, wild-type OVX, db/db sham and db/db OVX. Samples from all groups were prepared for histochemical and ultrastructural examinations. As a result, db/db sham mice showed a reduced number and thickness of metaphyseal trabeculae and excessive adipose tissue when compared to wild-type sham mice. The wild-type OVX group exhibited markedly diminished trabecular number, as well as lower populations of osteoblasts and osteoclasts in comparison to wild-type sham group. On the other hand, trabecular numbers were similar for the two db/db groups, suggesting that the effect of the ovariectomy, i.e., estrogen deficiency may be lessened in this animal model. Leptin receptor was mainly found in osteoblasts and in bone marrow stromal cells including adipocytes. In addition, the expression of estrogen receptor did not seem to change after OVX in wild-type mice and in db/db mice. Both db/db sham and OVX mice featured many adipocytes close to the metaphyseal chondro-osseous junction, while osteoblasts accumulated glycogen granules and lipid droplets. Therefore, it seems likely that the disruption of leptin signaling in db/db mice shifts the cell differentiation cascade towards the adipocyte lineage, resulting in an osteoporotic bone independently of estrogen deficiency.

This study aimed at elucidating whether estrogen deficiency would affect the synthesis of an osteocyte-derived factor, sclerostin, in the mesial region of alveolar bone. Eight 9-week-old Wistar female rats were ovariectomized (OVX) and eight other rats were Sham-operated (Sham). After 4 weeks, the interradicular septa of mandibular first molar were embedded in paraffin and then histochemically examined. Sclerostin-positive osteocytes were located in the superficial layer of the mesial region of Sham bones, whereas the OVX mesial region showed less sclerostin-reactive osteocytes. There was no significant difference in the distribution of estrogen receptor α and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling -positive cells in the groups studied. The Sham mesial region featured many osteoclasts, and OVX specimens showed numerous osteoclasts in association with intense immunolabeling of the receptor activator of the nuclear factor kB ligand. Contrary to the observations in Sham specimens, a complex meshwork of cement lines was seen in the OVX mesial region, accompanied by an irregularly distributed osteocytic lacunar-canalicular system. In conclusion, estrogen deficiency appears to inhibit osteocyte-derived sclerostin synthesis in the mesial region of the interradicular septum, in a process that seems to be mediated by accelerated bone remodeling rather than by direct effects on osteocytes.

Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor κB ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment.

Objectives: It has been reported that kidney stones are linked to metabolic syndrome (MetS), which is characterized by insulin resistance. The aim of the present study was to examine the association of insulin resistance, insulin and adiponectin with kidney stones in a Japanese population. Methods: From February 2007 to March 2008, 1036 (529 men and 507 women) apparently healthy Japanese subjects, aged 35–79 years, were analyzed. Weight, height, waist circumference and blood pressure were measured. Overnight fasting blood was collected to measure insulin and adiponectin levels. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated to assess insulin resistance. Logistic regression analysis was used to estimate the odds ratio (OR) and 95% confidence intervals for a self-reported history of kidney stones across tertiles of HOMA-IR, insulin and adiponectin. Results: Of the participants, 84 men (15.6%) and 35 women (6.9%) had a history of kidney stones. Age, body mass index, waist circumference, systolic and diastolic blood pressures, HOMA-IR and insulin were significantly higher in women with than in women without kidney stones. There was no difference in adiponectin level between subjects with and without a history of kidney stones in either sex. Furthermore, a significant positive trend was observed in the age-adjusted OR for a history of kidney stones across insulin tertiles (P-value for trend = 0.04) in women. Conclusions: For Japanese women, HOMA-IR and insulin are associated with a history of kidney stones. The findings suggest that MetS components could increase the risk of kidney stones through subclinical hyperinsulinemia and insulin resistance.

Objectives The aim of the present study was to determine the potential for pancreatic calcification in autoimmune pancreatitis by investigating osteopontin and CD44 expression. Methods Human pancreatic tissues in normal pancreas, chronic pancreatitis, and autoimmune pancreatitis were obtained from the surgical specimens of 42 patients. Pancreatic tissues from male Wistar Bonn/Kobori rats were also used as an animal autoimmune pancreatitis model. Results The incidences of osteopontin expression in centroacinar cells in chronic pancreatitis with calcification and in autoimmune pancreatitis were significantly greater than that in normal pancreas (P < 0.05). Some cases of chronic pancreatitis and autoimmune pancreatitis expressed CD44 in centroacinar cells and ductal cells. In male Wistar Bonn/Kobori rats, the inflammatory area and percentage of osteopontin-CD44-positive cells increased with advancing age (P < 0.01 or 0.05). Conclusions These results suggest that autoimmune pancreatitis has the potential for pancreatic calcification over a long-term clinical course.

Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.