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Dental caries is the most prevalent of chronic oral conditions. We investigated child and parental factors associated with early childhood caries (ECC) in 17- to 23-month-old children in Aichi Prefecture, Japan. Of the 61,714 children who underwent a health examination at 18 months of age, 54,206 (27,860 males, 26,346 females) were included in the analysis. The parents of the children completed a self-administered questionnaire comprising items related to the child’s eating habits and lifestyle and the parent’s lifestyle and childcare factors. Logistic regression analyses were performed using ECC as the dependent variable, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The prevalence of ECC was 0.8%. In the multivariate analysis, children who consumed sweetened beverages daily and snacked frequently had significantly higher ORs for ECC: 1.29 (95% CI: 1.05–1.59) and 1.49 (95% CI: 1.15–1.91), respectively. Bedtime breastfeeding and bottle-feeding were significantly associated with higher ORs for ECC: 4.88 (95% CI: 4.01–5.94) and 2.31 (95% CI: 1.72–3.10), respectively. Not eating breakfast daily and late bedtime were associated with high ORs for ECC: 1.41 (95% CI: 1.02–1.96) and 1.31 (95% CI: 1.05–1.64), respectively. The OR for ECC was significantly higher in children whose father was a smoker than in those whose father was not: 1.44 (95% CI: 1.18–1.76). The OR for ECC was significantly higher in children with no childcare adviser than in those with a childcare adviser: 1.67 (95% CI: 1.06–2.65). Children who had not been vaccinated had a significantly higher OR for ECC compared to children who had: 1.49 (95% CI: 1.14–1.94). These results imply the importance of enhancing parental guidance on the lifestyle habits of children and creating an environment in which parents can consult with others regarding parenting.

Supplemental Tables 1-4. Supplemental Table 1: Characteristics of primary adenocarcinoma from which CAFs were obtained. Supplemental Table 2: Primary tumor characteristics(N=106). Supplemental Table 3: Impact of predictive factors on progression free survival of EGFR-TKI in patients with EGFR activating mutation by univariate and multivariate analysis (n=106). Supplemental Table 4: The correlation between PDPN expression status and clinical response to EGFR-TKIs in patients with EGFR common mutations, exon 19 deletion and L858R (N=102).

Supplemental Figures 1-6. Supplemental Figure 1: Fluorescence microscopy showing mRFP-labeled PC-9 (red) and Venus-labeled CAFs (green) with or without gefitinib treatment. Supplemental Figure 2: mRNA levels of podoplanin (PDPN) in CAF-Ctrl and CAF PDPN-WT. Supplemental Figure 3: Co-culture with other CAFs induces gefitinib resistance. mRFP-labeled PC-9 and HCC-827 cells were co-cultured with CAFs (CAF-Ctrl, PDPN-WT, or CAF-PDPN-Del.IC) in the presence or absence of gefitinib treatment. The total cell numbers of PC-9 or HCC-827 cells treated for 72 hours with gefitinib in the presence or absence of Venus-labeled CAF-Ctrl, PDPN-WT, or CAF-PDPN-Del.IC are shown. Supplemental Figure 4: The number of the cases according to the PDPN-positive CAF area/stromal area (%). Supplemental Figure 5: PFS according to podoplanin expression in patients with EGFR common mutations (exon 19 deletion and L858R). Supplemental Figure 6: mRNA levels of HGF in CAF-Ctrl and CAF PDPN-WT.

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Purpose: The biologic characteristics of microenvironmental constituents, especially cancer-associated fibroblasts (CAF), can be key regulators of the cellular sensitivity to molecular-targeted therapy. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) have marked therapeutic effects against non–small cell lung cancer (NSCLC) with EGFR mutations, but some patients have exhibited primary resistance to EGFR-TKIs. We recently reported that podoplanin-positive fibroblasts are associated with a tumor-promoting phenotype of CAFs in lung adenocarcinoma. The aim of this study was to evaluate whether the susceptibility of NSCLC to EGFR-TKIs could be affected by podoplanin-expressing CAFs.Experimental Design: We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell lines cocultured with podoplanin-expressing CAFs. We also examined the association between the expression of podoplanin in CAFs in surgical specimens and EGFR-TKI response of postoperative recurrent patients with EGFR mutations (N = 106).Results: Lung adenocarcinoma cell lines became more resistant to EGFR-TKI when cocultured with podoplanin-expressing CAFs, compared with control CAFs in vitro. The knockdown of podoplanin expression on CAFs cancelled the resistance to EGFR-TKIs in cancer cells. Compared with control CAFs, the cancer cells that were cocultured with podoplanin-positive CAFs continued to exhibit significantly higher p-ERK levels after treatment with gefitinib. Furthermore, postoperative recurrent patients with podoplanin-positive CAFs had a significantly lower overall response rate to EGFR-TKIs compared with those with podoplanin-negative CAFs (53% vs. 83%; P < 0.01).Conclusions: Podoplanin-positive CAFs play an important role in primary resistance to EGFR-TKIs and may be an ideal therapeutic target for use in combination therapy with EGFR-TKIs. Clin Cancer Res; 21(3); 642–51. ©2014 AACR.

Introduction: This study aimed to clarify the correlation between the number of AMs and prognosis and to examine the gene expression of AMs in lung squamous cell carcinoma (SqCC). Methods: We reviewed 124 stage I lung SqCC cases in our hospital and 139 stage I lung SqCC cases in The Cancer Genome Atlas (TCGA) cohort in this study. We counted the number of AMs in the peritumoral lung field (P-AMs), and in the lung field distant from the tumor (D-AMs). Moreover, we performed a novel ex vivo bronchoalveolar lavage fluid (BALF) analysis to select AMs from surgically resected lung SqCC cases and examined the expression of IL10, CCL2, IL6, TGFβ, and TNFα (n=3). Results: Patients with high P-AMs had significantly shorter overall survival (OS) (pEx vivoBALF analysis revealed that AMs collected from the tumor vicinity showed higher expression of IL10 and CCL2 than AMs from distant lung fields in all 3 cases (IL-10: 2.2, 3.0, and 10.0-fold; CCL-2: 3.0, 3.1, and 3.2-fold). Moreover, addition of recombinant CCL2 significantly increased the proliferation of RERF-LC-AI, a lung SqCC cell line. Conclusion: The current results indicated the prognostic impact of the number of peritumoral AMs and suggested the importance of peritumoral tumor microenvironment in lung SqCC progression.

Purpose Cancer cells are known to exhibit a cancer-associated fibroblast (CAF)-dependent invasive mode in the presence of CAFs. The purpose of this study was to investigate whether intrinsic factors of cancer cells influence the CAF-dependent invasive mode of cancer cells. Methods We observed the dynamic movement of CAFs and cancer cells by time-lapse imaging of 2-D and 3-D collagen invasion models and evaluated the invasion modes of gastric cancer cell lines (MKN-7, MKN-45, and HSC44PE). We further examined whether modification of the invasive capacity of CAFs can alter the invasive mode of MKN-7 and HSC44PE cells. Results When MKN-7 and MKN-45 were co-cultured with CAFs, CAFs first invade collagen matrix followed by cancer cells (CAF-dependent invasion), whereas HSC44PE invaded collagen matrix independently of CAFs invasion. Overexpression or suppression of podoplanin in CAFs, respectively increased or decreased the invasive capacity of CAFs themselves and significantly increased or decreased the number of invading MKN-7, respectively. CAFs overexpressing a podoplanin mutant lacking the cytoplasmic domain had a significantly reduced invasive capacity compared to CAFs overexpressing wild-type podoplanin, and it also reduced the number of invading MKN-7 cells significantly. When HSC44PE and CAFs were co-cultured, changes in the podoplanin expression in CAFs similarly altered the invasive capacity of CAFs themselves, but it did not affect the number of invading HSC44PE cells. Conclusion These results indicate that in the presence of CAFs, there are CAF-dependent and -independent modes of cancer cell invasion, the determinants of which may depend on the intrinsic properties of cancer cells.

Cancer cells are known to exhibit a cancer-associated fibroblast (CAF)-dependent invasive mode in the presence of CAFs. The purpose of this study was to investigate whether intrinsic factors of gastric cancer cells influence the CAF-dependent invasive mode of cancer cells.We observed dynamic movement of CAFs, and cancer cells, by time-lapse imaging of 2-D and 3-D collagen invasion models, and evaluated invasion modes of gastric cancer cell lines (MKN-7, MKN-45, and HSC44PE). We further examined whether modification of invasive capacity of CAFs can alter invasive mode of MKN-7, and HSC44PE cells.When MKN-7 and MKN-45 cells were co-cultured with CAFs, CAFs first invade collagen matrix followed by cancer cells (CAF-dependent invasion), whereas HSC44PE cells invaded collagen matrix independent of CAFs' invasion. Overexpression or suppression of podoplanin in CAFs, respectively, increased or decreased the invasive capacity of CAFs, and significantly increased or decreased the number of invading MKN-7 cells, respectively. CAFs overexpressing a podoplanin mutant, lacking the cytoplasmic domain, had significantly reduced invasive capacity, compared to CAFs overexpressing wild-type podoplanin, and it also reduced the number of invading MKN-7 cells significantly. When HSC44PE cells, and CAFs were co-cultured, changes in the podoplanin expression in CAFs similarly altered the invasive capacity of CAFs, but it did not affect the number of invading HSC44PE cells.These results indicate that in presence of CAFs, gastric cancer cells exhibit both CAF-dependent and -independent modes of invasion, the determinants of which may depend on the intrinsic properties of the gastric cancer cells.

The intake ofRats with 2K1C or sham operation were fed a normal-/high-salt diet with some kinds of seaweeds (5.0%, w/w) or SJ extract with different Alg contents for 6 weeks. We measured systolic BP every week and mean arterial pressure at the end, and measured the total and molecular weights of Alg in each seaweed. Then, we evaluated the relationship of the Alg amount in each seaweed with the suppression of BP increase in 2K1C rats. Finally, urinary and fecal SE for 24 h was measured.The intake of SJ, SJ extract,The sodium excretion in feces by Alg in SJ may not be one of the major mechanisms by which SJ intake attenuates hypertension in 2K1C rats.

In a previous study, we observed that Saccharina japonica (SJ) intake reduced blood pressure (BP) in 2-clip, 1-kidney renovascular hypertension model (2K1C) rats. However, the mechanism is not clear. Dietary fibers such as alginate, fucoidan and laminaran are components of SJ and have been reported to change gut microbiota and increase short-chain fatty acids (SCFAs) in normotensive rats. SCFAs have been reported to have an antihypertensive effect when intraperitoneally or intracolonically administered, although it was reported that 2K1C rats reduce SCFAs and bacteria which produce SCFAs compared to sham operated control model (SHAM) rats. To investigate whether acetic acid (AA), one of SCFAs, is involved in the mechanism of BP reduction by an ingestion of SJ, we observed AA, in 2K1C rats fed SJ. Under anesthesia, 2K1C and SHAM were induced to Sprague Dawley rats. Both models were allowed to ingest a standard diet (C group) or a 5% (5 of 100) SJ mixed diet (SJ group) for 7 weeks, to a drinking water freely. During the breeding period, the amount of intake, body weight, and systolic BP (SBP) of each group were observed. After that, rats were euthanized, the contents of cecum were collected, and the pH was measured. The amounts of AA were measured using a commercial kit. The SBP of 2K1C-C was significantly higher than that of SHAM-C throughout the feeding (146 ± 3 vs 131 ± 1 mmHg, pvs 146 ± 3 mmHg, pvs 92 ± 9 mmol, pvs 87 ± 5 mmol, p

Objectives: One of foods used for Japanese cuisine “Washoku” is brown algae, including Saccharina japonica (SJ). The intake of SJ is reported to decrease blood pressure (BP) in spontaneously hypertensive rats in some studies, and in 2-kidney, 1-clip hypertensive (2K1C) rats in our studies. Several studies indicated that dietary fibers contained in SJ, such as alginate and fucoidan, may play an important role in reducing BP. We previously demonstrated that SJ intake did not decrease BP in normotensive rats fed a normal-salt (NS) diet. We also observed that the suppression of BP rise by SJ intake was stronger in 2K1C rats fed a high-salt (HS) diet than in 2K1C rats fed an NS diet. An HS diet may lead to the development of hypertension in both humans and rats. However, to our knowledge, it has not been reported that the effect of SJ intake on BP in normotensive rats fed an HS diet. In this study, we compared the effects of SJ intake on BP between in normotensive and hypertensive rats when fed an HS diet. Methods: Male Sprague Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, rats were received a control diet (CTL) or a diet contained 5.0% (5 of 100, w/w) SJ, with an NS (0.7% NaCl) or HS (6.0% NaCl) in diets for 6 weeks. Systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the protocol, mean arterial pressure (MAP) was measured in each rat under anesthesia. Results and Discussion: Analysis of variance showed that SBP was significantly higher in 2K1C-HS-CTL than in SHAM-HS-CTL (P

Background Associations between tooth loss and mortality have been reported in older individuals. However, limited information is available regarding the association between occlusion and mortality in nursing home residents. Objective We investigated the association between posterior occluding pairs (POPs) of teeth or dentures and 1-year mortality in nursing home residents. Methods The subjects of this study were residents from four nursing homes in Aichi prefecture, Japan, who were capable of eating and were enrolled in baseline examinations. We examined the number of present teeth and POPs (0-8 pairs), defined as pairs of occluding natural, restored or fixed prosthetic post-canine teeth. Then, we defined prosthetic POPs (0-8 pairs) as pairs of occluding natural, restored or fixed prosthetic post-canine teeth and removable dentures. Nutritional status, activities of daily living, cognitive function and comorbid conditions were assessed. One year later, we followed up the subjects. Ultimately, we analysed 173 elderly people (mean age ± SD: 87.1 ± 8.6 years; the survival group: N = 145, the deceased group: N = 28). Results In univariate analyses, mortality was significantly associated with age, peripheral vascular disease, nutritional status, diet texture and POPs. In multivariate logistic regression analysis, subjects who had 0 prosthetic POPs had significantly higher odds of a high mortality, compared with 8 prosthetic POPs. Conclusion Lack of properly occluding posterior teeth, whether remaining teeth or dentures, is associated with 1-year mortality. (228 words/250).

Podoplanin-positive cancer-associated fibroblasts (CAFs) play an important role in tumor progression. The aim of this study was to evaluate the effect of podoplanin (+) CAFs on the proliferation of cancer cells using a three-dimensional (3D) organoid model.We examined the success rate of organoid culture containing PC-9 cancer cells and CAFs. Thereafter, we compared the proliferating index (MIB-1 index) of PC-9 cells co-cultured with podoplanin-overexpressing CAFs and control CAFs using organoid specimens. Furthermore, we compared the MIB-1 labeling index of cancer cells in podoplanin (+) CAFs cases (n = 13) and podoplanin (-) CAFs cases (n = 14) using surgically resected adenocarcinoma specimens.Without CAFs, PC-9 cells did not form any organoid (success rate: 0%). When PC-9 cells were mixed with CAFs (1:10), the mixed cells generated round and steric aggregates (hybrid cancer organoids, success rate: 100%). In three independent experiments, the MIB-1 index of PC-9 cells in hybrid cancer organoids containing podoplanin-overexpressing CAFs was significantly higher than that of PC-9 cells in organoids containing control CAFs (Exp. 1: 40.4% vs. 24.4%; Exp. 2: 40.0% vs. 24.5%; Exp. 3: 40.3% vs. 25.2%; p 0.001). Surgically resected human tumors revealed that the MIB-1 index of adenocarcinoma cells was significantly higher in the case of podoplanin (+) CAFs than in the case of podoplanin (-) CAFs (34.8% vs. 16.2%; p 0.01).Our data suggested that the hybrid cancer organoid model might reflect the growth-promoting effect of podoplanin (+) CAFs in cancer cells, and this new system can be a useful tool for evaluating the tumor microenvironment.

Humoral factors from cancer-associated fibroblasts (CAFs) reportedly affect epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance in cancer cells with EGFR mutations. The aim of this study was to identify the robust humoral factors secreted from CAFs that induce the primary resistance to EGFR-TKI. We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell line (PC-9) treated with condition media (CM) from 18 cases of CAFs and matched non-cancerous-tissue-associated fibroblasts (NCAFs). We measured the expression levels of hepatocyte growth factor (HGF), interleukin-6, fibroblast growth factor-2, insulin-like growth factor-1, and vascular endothelial growth factor-A in CAFs and NCAFs. We examined whether HGF neutralizing antibody could annul the EGFR-TKI resistance induced by CM from CAFs. Compared to CM from NCAFs, CM from CAFs increased the resistance of PC-9 cells to EGFR-TKI in five out of 18 cases. Relative expression ratio of HGF messenger RNA was significantly higher in these five CAFs compared to others (P = 0.0013), whereas other cytokines were not. In four of these five cases, the addition of HGF neutralizing antibody significantly decreased the survival ratio of PC-9 cells. This study suggests that the secretion of higher amounts of HGF is the robust feature of EGFR-TKI resistance-promoting CAFs.

Nano-immunotherapy has high potential to improve cancer patient outcomes, as already demonstrated in triple negative breast cancer with the combination of nanoparticle albumin-bound paclitaxel and the immune checkpoint blocker antibody, atezolizumab. This regimen, however, does not lead to complete cure with median survival lasting less than two years. To increase patient survival, research focuses on repurposing common drugs to modulate the tumor microenvironment (TME), aiming to increase delivery of nanoparticles, antibodies and other drug regimens and improve immunostimulation. These drugs, however, exhibit certain dose-limitations and are associated with adverse effects. Here, taking advantage of the TME reprogramming capabilities of the approved antihistamine drug tranilast, we developed polymeric tranilast-loaded nanoparticles and explored their potential to modulate the TME more efficiently than the free drug. The results were striking as the micellar formulation of tranilast administered in a dose 100 times lower than that of the free drug reported superior reprogramming effects in two syngeneic triple negative breast tumor models. These effects were due to the enhanced intratumoral accumulation and cancer-associated fibroblast uptake of the nanoparticles compared to the free drug. Tranilast micelles were also found to promote significantly the delivery and efficacy of epirubicin micelles, overcome immune checkpoint resistance and induce tumor immunogenicity. Overall, the proposed micelles improved the efficacy of nano-immunotherapy in both breast cancer tumor models leading to complete cure. Furthermore, we employed the non-invasive and clinically applied ultrasound shear wave elastography method to monitor changes in tumor stiffness, which is the first effect caused by the tranilast micelles, and thus to determine an optimal dose-scheduling. Importantly, we found that tumor stiffness before the initiation of nano-immunotherapy was able to predict the efficacy of the treatment providing strong evidence for the potential of shear wave elastography to be used as a biomarker predictive of response. Our findings are very encouraging indicating that a) encapsulation of TME modulating drugs improves their pharmacokinetic properties, while allows for drastic reduction in the dose of administration, b) modulation of the TME can lead to complete cures of nano-immunotherapy in resistant to this therapy breast cancer tumor models, and c) ultrasound shear wave elastography can be employed for optimizing the use of TME modulating agents and for the prediction of tumor response to nano-immunotherapy allowing for optimized treatment protocols. Citation Format: Myrofora Panagi, Fotios Mpekris, Pengwen Chen, Chrysovalantis Voutouri, Yasuhiro Nakagawa, John D. Martin, Tetsuro Hiroi, Hiroko Hashimoto, Genichiro Ishii, Motohiro Kojima, Kazunori Kataoka, Horacio Cabral, Triantafyllos Stylianopoulos. Superior effects of polymeric micelles in reprogramming tumor microenvironment and enhancing nano-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6381.

There is limited information regarding the association between tooth loss and the medications used for the treatment of rheumatoid arthritis (RA). Here, we examined the association between tooth loss, disease severity, and drug treatment regimens in RA patients. Method: This study recruited 94 Japanese patients with RA. The severity of RA was assessed using the Steinbrocker classification of class and stage. Data on RA medications were obtained from medical records. We examined the associations between tooth loss, RA severity, and drug treatment regimens using multinomial logistic regression analyses. Results: Patients with 1–19 teeth had significantly higher odds ratios (ORs) of taking methotrexate (MTX) (OR, 8.74, 95% confidence interval (CI), 1.11–68.8) and biologic disease-modifying antirheumatic drugs (bDMARDs) (OR, 21.0, 95% CI, 1.3–339.1) compared to those with 27–28 teeth when adjusted for RA severity (class). Furthermore, patients with 1–19 teeth had significantly higher ORs of taking MTX (OR, 9.71, 95% CI, 1.22–77.1) and bDMARDs (OR, 50.2, 95% CI, 2.55–990.6) compared to those with 27–28 teeth when adjusted for RA severity (stage). Conclusion: RA patients with fewer teeth were more likely to take stronger RA therapies, independent of RA severity and other factors.

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Aim Cancer-associated fibroblasts (CAFs) expressing podoplanin (PDPN) harbor a fibrous tumor microenvironment that promotes cancer progression in lung adenocarcinoma. In this study, we investigated whether tumor-promoting PDPN+ CAFs contribute to the immunosuppressive microenvironment in lung squamous cell carcinoma (SqCC). MM CD8+ and FOXP3+ tumor infiltrating lymphocytes (TILs), and CD204+ tumor-associated macrophages (TAMs) were evaluated in cases with PDPN+ and PDPN− CAFs. Results Analysis of TCGA database revealed that the PDPN-high group exhibited significantly higher expression of interleukin (IL)-1A, IL-1B, IL-6, IL-10, monocyte chemoattractant protein-1 (CCL2), colony stimulating factor 1 (CSF1), fibroblast growth factor 2 (FGF2), galectin 1 (LGALS1), platelet derived growth factor subunit A (PDGFA), PDGFB, and transforming growth factor-β1 (TGFB1) than those in the PDPN-low group. Among them, it was found that TGFB1 expression was higher in patient-derived PDPN+ CAFs. Immunohistochemical analyses revealed that more CD204+ TAMs infiltrated the tumor tissues in cases with PDPN+ CAFs than in cases with PDPN− CAFs (P Conclusion PDPN+ CAFs showed higher expression of TGFB1 and were associated with CD204+ TAM infiltration in stage-I lung SqCC, suggesting that PDPN+ CAFs were associated with the immunosuppressive tumor microenvironment.

Objective: Dried bonito is a processed marine product unique to Japan, and the hot water extracted product is widely used as soup stock. It has been reported that the dried bonito extract (DBE) has antioxidants. 2-kidney, 1-clip hypertensive (2K1C) rats is an experimental model of renovascular hypertension which is known to enhance the production of oxidative stress. Plasma thiobarbituric acid reactive substances (TBARS) level, a indicator for oxidative stress, was reported to increase in 2K1C rats when compared with sham operation (SHAM) rats. Moreover, previous studies have reported that intake of a few antioxidants ameliorate hypertension in 2K1C rats. Therefore, the intake of DBE may alleviate hypertension through antioxidant effect in 2K1C rats. To test the hypothesis, in the present study, we examined the effects of dietary intake of DBE intake on blood pressure (BP) of 2K1C rats. Additionally, to investigate the mechanism, we evaluated TBARS level in plasma in those rats. Methods: Male Sprague-Dawley rats (6 wks) were treated with SHAM or clipping the left renal artery (2K1C). After surgery, the rats started receiving a control diet (CTL), a diet with 5.0% (5 of 100, w/w) DBE for 6 consecutive weeks. Systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the protocol, mean arterial pressure (MAP) was measured in each rat under anesthesia. Plasma lipid peroxide levels were determined by measuring TBARS using a colorimetric method. The TBARS level was determined by the absorbance at 532 nm. Results: Analysis of variance shows that SBP was significantly higher in 2K1C-CTL (152±4 mmHg) than SHAM-CTL (131±3 mmHg) through the experiment period (P Conclusion: The reduction of TBARS level may contribute to a mechanism by which DBE intake decreases BP in 2K1C rats.

Introduction: Saccharina japonica (SJ) is one of brown algae. The extract of SJ (SJE) is often used for soup stock in Japanese cuisine. We recently observed the decreases in blood pressure (BP) by intake of SJ or SJE diet in 2-kidney, 1-clip renovascular hypertensive (2K1C) rats. SJ is rich in aspartate (Asp), which is the umami taste of amino acid. The intake of Asp was reported to reduce BP in Dahl salt sensitive rats. As well, the potassium Asp (AspK) intake was reported to improve BP in hypertensive patients. 70% (70 of 100) of amino acids such as Asp are eluted into the SJE from SJ. Therefore, we hypothesized that Asp in SJE contributes to the mechanism of BP reduced by dietary SJE in 2K1C rats. In this study, we observed BP in 2K1C rats fed a diet containing Asp or AspK and investigated whether those diets alleviate hypertension to the same extent of the diet containing SJE. Methods: Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats fed a control diet (C), a diet containing Asp, AspK or SJE for 6 weeks. The content of SJE was set to be the same as that in our previous study. The amount of Asp in diet was set to be the same as that in our previous study which showed the antihypertensive effect of SJ. The amount of AspK in diet was set to be equimolar to Asp. The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the procedure, mean arterial BP (MAP) was measured in each rat under anesthesia. Results: Six weeks after the surgery, 2K1C-C significantly increased in SBP compared with SHAM-C (126±2 vs 159±4 mmHg, p Conclusions: Asp and AspK may attenuate hypertension in 2K1C renovascular hypertension as much as SJE which contains similar quantities of Asp. It is possible that Asp and/or AspK eluted into SJE contributes to the mechanism of alleviating hypertension by SJE in 2K1C.

Loss of bone mineral density (BMD) is a substantial risk of mortality in addition to fracture in hemodialysis patients. However, the factors affecting BMD are not fully determined. We conducted a single-center, cross-sectional study on 321 maintenance hemodialysis patients who underwent evaluation of femoral neck BMD using dual-energy X-ray absorptiometry from August 1, 2018, to July 31, 2019. We examined factors associated with osteoporosis defined by T-score of ≤ − 2.5, using logistic regression models. Median age of patients was 66 years, and 131 patients (41%) were diagnosed with osteoporosis. Older age, female, lower body mass index, diabetes mellitus, and higher Kt/V ratios were associated with higher osteoporosis risk. The only medication associated with lower osteoporosis risk was calcium-based phosphate binders (CBPBs) [odds ratio (OR), 0.41; 95% confidence interval (CI), 0.21–0.81]. In particular, CBPB reduced the osteoporosis risk within subgroups with dialysis vintage of ≥ 10 years, albumin level of

Background: The number of child abuse cases continues to increase in Japan. Under these circumstances, public health nurses (PHNs) who provide continued support to children and their families play important roles in dealing with child abuse. Therefore, it is necessary to consider the support that PHNs require to perform their duties. The purpose of this study was to clarify the training needs and issues recognized by PHNs involved in prevention of child abuse. Methods: A questionnaire survey was administered to PHNs involved in prevention of child abuse in municipalities and public health centers in Japan. Descriptions regarding training needs and issues were analyzed qualitatively. Results: The contents of training that PHNs required were diverse. Among them, the most common training need was “ways to approach and support parents”, followed by “case study” and “role and response as a PHN”. Training needs differed according to the duration of experience as a PHN. The issues recognized by PHNs were “improvement of necessary system for support”, “development system to support supporters”, “enhancement of human resources” and “advancement of cooperation with relevant organizations”. Conclusion: The training needs of PHNs involved in prevention of child abuse varied, but there were characteristics according to the duration of experience as a PHN. It is important to reflect these needs in training contents in order to implement effective training for PHNs. The issues recognized by PHNs cannot be solved immediately through personal efforts alone; therefore, it is necessary for entire organizations to approach the issues.

Cancer cells can invade the surrounding stroma with the aid of fibroblasts (fibroblasts-dependent invasion). The aim of this study was to explore the spatiotemporal characteristics of fibroblast-dependent invasion of cancer cells. We performed an in vitro three-dimensional collagen invasion assay using Fluorescent Ubiquitination-based Cell Cycle indicator (Fucci)-labeled A431 carcinoma cells co-cultured with fibroblasts. We used time-lapse imaging to analyze the total cell number, frequencies of small cancer cell nests and S/G2/M phase of A431 cells in the invasion area. We compared the frequencies of small cancer cell nests and geminin (+) cancer cells within fibroblast-rich areas and fibroblast-poor areas in surgically resected human invasive squamous cell carcinoma tissue. The total invasion number of A431 cells was significantly higher when cultured with fibroblasts than without. The formation of small cancer cell nests was observed within the invasion area only in the presence of fibroblasts. The frequency of S/G2/M phase cells was significantly higher in A431 cells when cultured with fibroblasts than without. Immunohistochemical analysis of surgically resected human invasive squamous cell carcinoma tissue revealed that the frequencies of small cancer cell nests and geminin-positive cancer cells were significantly higher in fibroblast-rich areas compared to those in fibroblast-poor areas within the same tumor region. Our current study clearly showed that fibroblast-dependent cancer cell invasion was characterized by the progression in cell cycle and formation of small cancer cell nests.

Background Functional disability of the finger joints in rheumatoid arthritis (RA) patients could affect their oral hygiene and periodontal status because of poor plaque control. We examined the influence of plaque control on the relationship between the severity of RA and periodontal status in RA patients. Methods This study recruited 89 Japanese RA patients who reported no difficulty in performing oral hygiene. We assessed RA severity using four indices: the Steinbrocker stage and class, the Health Assessment Questionnaire (HAQ) and the Disease Activity Score (DAS). Probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were taken as parameters of periodontal status. Oral hygiene status was assessed using the plaque index (PlI). We examined the association between the severity of RA and periodontal parameters using multivariable linear regression analyses. Results In multivariable linear regression analyses not including PlI, Steinbrocker stage III-IV patients had significantly higher PlI scores and greater PD compared with those of stage I. The class III-IV patients had significantly higher PlI scores and greater PD. Patients with an HAQ score ≥0.5 had a significantly higher PlI score than those with an HAQ score of 0. However, when PlI was added to each multivariable model as an independent variable, no significant relationship between RA severity and periodontal status was observed. Conclusions This study suggests that it is important to consider the influence of plaque control when assessing the relationship between RA severity and periodontal health status in RA patients.

Objective Metformin is an antidiabetic drug that is widely used to treat patients with diabetes mellitus. Recent studies have reported that treatment with metformin not only improved blood glucose levels but also reduced blood pressure. However, it remains unclear how metformin reduces blood pressure. We hypothesized that metformin affects sodium reabsorption in the kidneys. Methods Urinary sodium excretion and expression of renal sodium transporters were examined in 8-week-old male C57BL/6 mice with acute and chronic treatment of metformin. In addition, we examined metformin effects using ex vivo preparations of mice kidney slices. Results In this study, we demonstrated that metformin increased urinary sodium excretion by reducing phosphorylation of the thiazide-sensitive Na-Cl cotransporter (NCC) in acute and chronic metformin administration. We also confirmed reduction of phosphorylated NCC in an ex vivo study. The activity of other renal sodium transporters, such as NKCC2, ENaC, and NHE3 did not show significant changes. WNK-OSR1/SPAK kinase signals were not involved in this inactivation effect of metformin on NCC. Conclusion Metformin increased urinary sodium excretion by reducing phosphorylation of NCC, suggesting its role in improving hypertension.

Background: Health promotion for the elderly mainly targets improvements in physical function, and a lack of strengthening of subjective well-being as well as the creation of interaction-providing opportunities for activity and participation has been reported. Evaluations of positive and negative aspects for subjective well-being are needed. Elderly subjects were intervened through a 6-month complex-type program combining amusement and exercise and its influences on subjective well-being and physical and physiological functions were investigated. Methods: Subjects in this survey were 37 healthy elderly females living in M city, Hiroshima Prefecture (age: 69.7 ± 5.9 years old), and the survey was performed between September 2016 and March 2017. The complex-type program comprised amusement and exercise programs. The survey was performed at 4 time points during the intervention period: before the intervention and 1, 3, and 6 months during the intervention. Subjective well-being (Japanese version of the Subjective Well-being Inventory (SUBI): Mental health scores, Mental fatigue scores), salivary amylase levels, and the locomotive syndrome risk test (two-step test, stand-up test, and 25-question risk assessment) were surveyed at each time point. Regarding the duration of the intervention as a factor, changes in salivary amylase levels were analyzed using repeated measures one-way ANOVA and SUBI was tested using the Friedman test (EZR Ver 1.32). In addition, the relationship between the locomotive syndrome risk level and duration of the intervention was examined using Fisher’s test. Results: SUBI and the locomotive syndrome risk level significantly differed with time during the intervention. Slight changes were observed in salivary amylase levels during the intervention. Conclusion: The present results suggest that the complex-type program promoted interactions among community-dwelling elderly subjects and positively influenced the maintenance of and improvements in physical and physiological functions. These changes may be associated with favorable influences on the positive and negative aspects of subjective well-being.

One of the characteristics of Autism Spectrum Disorder (ASD) is social disorder. The specificity of facial and expression recognition for people with ASD is gathering attention as a factor of this social disorder. The study examined the hemodynamic activities in the prefrontal cortex using near-infrared spectroscopy (NIRS) when a person with ASD performed an expression recognition task. The subjects were twenty males (18 - 22 years old) with ASD and without intellectual disabilities. Forty-five healthy males matched for age and sex were included as a control group. In both groups, the degree of autistic tendencies was evaluated using the Autism-Spectrum Quotient (AQ). Using eight standard emotional expressions of Japanese people, two expression recognition tasks were set. An NIRS was used to measure the prefrontal cortex blood mobilization during the expression-processing process. The AQ was significantly higher in the ASD group, while the rate of overall correct expression response was significantly lower (p < 0.001). A negative correlation was found when the relationship between the AQ and the rate of overall correct expression response was evaluated by combining the results of the control group with those of the ASD group (ρ = −0.40 p < 0.001). In the automatic expression-processing task, no activation in the prefrontal cortex was found in either the ASD or the control group. In the conscious expression-processing task, the activation of the left and right lateral prefrontal cortex was weaker in the ASD group compared to the control group. Unlike in the control group, a mild activation of posterior prefrontal cortex was found in the ASD group. The expression-processing process of the ASD group was found to be different from that of the control group. NIRS was effective in detecting a brain function disorder in people with ASD during an expression-processing process.

More than a few pediatric nurses experience difficulty in communicating with children's family members. However, effective means of providing communication support for pediatric nurses have not been examined sufficiently. This study aimed to develop and implement a communication support program for nurses to facilitate improved communication with families of hospitalized children, and to clarify changes in nurses' recognition and behavior toward communication with families in clinical settings. The program lasted 6 months and consisted of lectures, role-play, 4 communication models in which nurses experienced difficulty communicating with family members, and continued individual support. The effects of the program were evaluated qualitatively and descriptively using semi-structured interviews. A total of 7 nurses with less than 5 years of pediatric nursing experience completed the program. Subsequent to program completion, nurses' awareness of careful communication with families increased, and they began to approach families actively using thoughtful words. Furthermore, as nurses received favorable reactions from families, they realized that communication was interactive and recognized that their perception of their communication skills as poor had changed. This program could contribute in reducing nurses' difficulty in communicating with families and encourage them to improve their communication.

Background: Parents’ own histories of growth any experience of being abused or bullied) are regarded as one of the factors contributing to the difficulty in child-raising that those parents face. The objective of this research is to find the features of the difficulty in child-raising and resilience of mothers with past experience of adversity. Methods: A questionnaire survey was conducted targeting 4,013 mothers who were raising young children aged 1.5 years or older. The survey items were the history of growth, the child raising environment, difficulty in child-raising, and A chi squared test and the two sample t test were used for the analysis. This research was approved by the Ethics Committee of Tokushima University Hospital Results: The level of difficulty in child-raising was significantly high in the group “without stable family environments” during childhood in contrast with the group “with stable family environments” and in the group “with past experience of being bullied” in contrast with the group without such experience. The resilience of the group “without stable family environments” during childhood were significantly lower than the group “with stable family environments,” and so were the resilience of the group “with past experience of being bullied” than the group without such experience. The resilience of mothers with past experience of adversity was characterized by being low, especially in terms of sociability. Conclusion: This research has found that past experience of adversity has an impact on the difficulty in child-raising that currently nurturing mothers face. It is important for childcare support with a focus on resilience for mothers with experience of adversity to not only assist them in terms of environmental factors but also be based on the understanding of what perception they have.

Regrowth of cancer cells following chemotherapy is a significant problem for cancer patients. This study examined whether cancer-associated fibroblasts (CAFs), a major component of a tumor microenvironment, promote cancer cell regrowth after chemotherapy. First, we treated human lung adenocarcinoma cell line A549 and CAFs from four patients with cisplatin. Cisplatin treatment inhibited the viable cell number of A549 cells and induced epithelial–mesenchymal transition. After cisplatin was removed, A549 cells continued to manifest the mesenchymal phenotype and proliferated 2.2-fold in 4 days (regrowth of A549 cells). Cisplatin treatment inhibited the viable cell number of CAFs from four patients also. The CM (derived from cisplatin-pretreated CAFs from two patients) significantly enhanced the regrowth of cisplatin-pretreated A549 cells, and the CM derived from cisplatin-naive CAFs marginally enhanced A549 regrowth. By contrast, the CM derived from either cisplatin-pretreated CAFs or cisplatin-naive CAFs failed to enhance the growth of cisplatin-naive A549 cells. The CM derived from cisplatin-pretreated CAFs did not enhance the proliferation of A549 cells in which epithelial–mesenchymal transition was induced by TGFβ-1. Our findings indicate the possibility that humoral factors from cisplatin-pretreated CAFs promote the regrowth of cisplatin-pretreated A549 cells. These results suggest that interactions between cancer cells and CAFs may significantly enhance cancer cell regrowth within the tumor microenvironment after cisplatin treatment.

Objective : To improve primary prevention of child maltreatment, the association between at-risk parents identified by maternity hospitals and local health centers and intervention outcomes was investigated. Methods : A screening program for primary prevention of child maltreatment has been implemented for parents with children aged

Cancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified. CAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed. The nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential. Our studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.

The inappropriate over-activation of the with-no-lysine kinase (WNK)–STE20/SPS1–related proline/alanine-rich kinase (SPAK)–sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1–SPAK–NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1–SPAK–NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1– SPAK–NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.

Objective: Allicin contained in Allium Bakeri (AB) , a transient receptor potential (TRP) vanilloid type-1 (TRPV1) and TRP ankyrin-1 (TRPA1) agonist, increases phosphorylated (p-) endothelial nitric oxide synthase (eNOS). Another TRPV1 agonist, capsaicin (CAP), causes a vasodilatory effect through the increment of p-eNOS. We recently demonstrated the alleviation of blood pressure (BP) elevation by each intake of AB and CAP in 2-kidney, 1-clip (2K1C) hypertensive rats. AB is often used with red hot chili pepper containing CAP in the Japanese cuisine. We hypothesized that a combination diet of AB and CAP enhances the antihypertensive effect compared with each diet. In this study, to test the hypothesis, we investigated the interactive effects of dietary AB and CAP on BP, as well as the p-eNOS/eNOS in the protein expression of aorta, in 2K1C rats. Method: Male Sprague-Dawley rats (6wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, SHAM rats received a control diet (CTL), and 2K1C rats received CTL, a diet with 1% (10 of 1,000, w/w) freeze-dried AB powder, with 0.006% (60 of 1,000,000, w/w) CAP or with both (AB+CAP) for 6 weeks. The systolic BP (SBP) was measured by a tail-cuff method once per week. At the end of protocol, rats were euthanized and the thoracic aortas were collected for extracting the protein. The p-eNOS/eNOS in the protein expression in aorta was evaluated by Western blotting. Result: Analysis of variance showed that SBP was significantly higher in 2K1C-CTL than SHAM-CTL through the protocol (123±2.3 vs 165±3.2 mmHg, p < 0.05, at 6 weeks after the surgery). SBP was lower in 2K1C-AB and -CAP in 2K1C-CTL (151±2.4 and 139±3.7 vs 165±3.2, p < 0.05, each). Compared with 2K1C-AB and -CAP, a significant reduction in SBP was observed in 2K1C-AB+CAP (135±3.1, at 6 weeks) throughout the procedure ( p < 0.05). The p-eNOS/eNOS was tended to be enhanced in 2K1C-AB and -CAP, but not in 2K1C-AB+CAP, compared to 2K1C-CTL. Conclusion: A combination diet of AB and CAP may enhance antihypertensive effects compared with each diet of AB and CAP, in 2K1C rats. Although the increase in eNOS phosphorylation may participate in the mechanism of the antihypertensive effects by each diet of AB and CAP, it may not do in that of their combined effect.

Objective: One of foods to Japanese cuisine “Washoku” is algae, including Saccharina japonica (SJ). The intake of SJ is reported to decrease blood pressure (BP) in spontaneously hypertensive rats in some studies, and in 2-kidney, 1-clip hypertensive (2K1C) rats in our studies. Water soluble-alginate is rich in SJ. Since alginate has a physiological function to suppress sodium absorption in the gastrointestinal tract, SJ intake may alleviate hypertension to the extent according to the salt intake in 2K1C rats. In the present study, we observed the effects of SJ intake on BP in 2K1C rats fed a normal-sodium (NS) or high-sodium (HS) diet. Methods: Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats started receiving a NS (0.7% NaCl) or HS diet (6.0% NaCl), with or without 5.0% (w/w) SJ for 6 consecutive weeks. Systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the protocol, mean arterial pressure (MAP) was measured in each rat under anesthesia. Results and Discussion: Analysis of variance showed that SBP was significantly higher in 2K1C-NS-CTL than in SHAM-NS-CTL through the experiment period (P

As one form of tumor invasion, cancer cells can invade the extracellular matrix (ECM) through tracks that have been physically remodeled by cancer-associated fibroblasts (CAFs). However, CAFs are a heterogeneous population with diverse matrix-remodeling capacities. The purpose of this study was to investigate how CAFs with various matrix-remodeling capacities influence cancer cell invasion. We established single-cell-derived clones from three primary cultures of CAFs from lung adenocarcinoma patients (Case 1, 5 clones; Case 2, 5 clones; and Case 3, 7 clones). Using a co-culture model, we evaluated the correlations between the number of invaded cancer cells and the remodeling areas generated by CAF clones in each case. When A549 lung adenocarcinoma cells and CAF clones were co-cultured, both the numbers of invaded cancer cells and the remodeling areas generated by the CAF clones varied greatly. The number of invaded cancer cells was moderately and strongly correlated with the remodeling areas generated by each CAF clone originating from Cases 1 and 2 (R 2 value = 0.53 and 0.68, respectively), suggesting that the remodeling areas in the ECM may determine the number of invaded cancer cells. In contrast, the number of invaded cancer cells was not correlated with the remodeling areas generated by CAF clones originating from Case 3, suggesting that factors other than the remodeling areas might determine the number of invading cancer cells. These findings showed two types of fibroblast-dependent cancer cell invasion that are dependent on and independent of the remodeling areas generated by CAFs.

Cancer-associated fibroblasts (CAFs) expressing podoplanin (PDPN) are a favorable prognosticator in surgically resected small cell lung cancer (SCLC). Here we explore whether CAFs expressing PDPN influence proliferation of SCLC cells. Compared with control group (SCLC cells co-cultured with CAFs-Ctrl), numbers of SCLC cells co-cultured with CAFs overexpressing PDPN were decreased. Suppression of PDPN expression by shRNA in CAFs resulted in increased numbers of SCLC cells. In surgically resected human SCLC specimens, the frequency of Geminin-positive cancer cells was significantly higher in the cases with PDPN-positive CAFs than in the cases with PDPN-negative CAFs. Thus CAFs expressing PDPN inhibit growth of SCLC cells, suggesting that CAFs expressing PDPN represent a tumor inhibitory stromal cell component in SCLC.

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance.