Genetic epidemiological analysis of hypouricaemia from 4993 Japanese on non-functional variants of URAT1/SLC22A12 gene.

OBJECTIVES: Up to 0.3% of Japanese have hypouricaemia. Most cases appear to result from a hereditary disease, renal hypouricaemia (RHUC), which causes exercise-induced acute kidney injury and urolithiasis. However, to what extent RHUC accounts for hypouricaemia is not known. We therefore investigated its frequency and evaluated its risks by genotyping a general Japanese population. METHODS: A cohort of 4993 Japanese was examined by genotyping the non-functional variants R90H (rs121907896) and W258X (rs121907892) of URAT1/SLC22A12, the two most common causative variants of RHUC in Japanese. RESULTS: Participants' fractional excretion of uric acid and risk allele frequencies markedly increased at lower serum uric acid (SUA) levels. Ten participants (0.200%) had an SUA level ≤2.0 mg/dl and nine had R90H or W258X and were likely to have RHUC. Logistic regression analysis revealed these URAT1 variants to be significantly and independently associated with the risk of hypouricaemia and mild hypouricaemia (SUA ≤3.0 mg/dl) as well as sex, age and BMI, but these URAT1 variants were the only risks in the hypouricaemia population (SUA ≤2.0 mg/dl). W258X was only a risk in males with SUA ≤3.0 mg/dl. CONCLUSION: Our study accurately reveals the prevalence of RHUC and provides genetic evidence for its definition (SUA ≤2.0 mg/dl). We also show that individuals with SUA ≤3.0 mg/dl, especially males, are prone to RHUC. Our findings will help to promote a better epidemiological understanding of RHUC as well as more accurate diagnosis, especially in males with mild hypouricaemia.

Subtype-specific gout susceptibility loci and enrichment of selection pressure on ABCG2 and ALDH2 identified by subtype genome-wide meta-analyses of clinically defined gout patients.

OBJECTIVES: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years. METHODS: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. RESULTS: In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10-8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. CONCLUSIONS: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.

PEPITEM/Cadherin 15 Axis Inhibits T Lymphocyte Infiltration and Glomerulonephritis in a Mouse Model of Systemic Lupus Erythematosus.

Control of lymphocyte infiltration in kidney is a potential therapeutic strategy for lupus nephritis, considering that control of lymphocyte migration by sphingosine 1 phosphate has been implicated in inflammation-related pathology. The peptide inhibitor of the transendothelial migration (PEPITEM)/cadherin (CDH) 15 axis was recently reported to promote sphingosine 1 phosphate secretion. In this study, we investigated whether CDH15 is expressed in the kidney of MRL/lpr mice and whether lymphocyte infiltration is suppressed by exogenously administered PEPITEM. Mice (18 wk old) were randomized into 4-wk treatment groups that received PEPITEM or PBS encapsulated in dipalmitoylphosphatidylcholine liposomes. Enlargement of the kidney, spleen, and axillary lymph nodes was suppressed by PEPITEM treatment, which also blocked infiltration of double-negative T lymphocytes into the kidney and glomerular IgG/C3 deposition, reduced proteinuria, and increased podocyte density. Immunohistochemical analysis revealed that the PEPITEM receptor CDH15 was expressed on vascular endothelial cells of glomeruli and kidney arterioles, skin, and peritoneum in lupus mice at 22 wk of age but not in 4-wk-old mice. These results suggest that PEPITEM inhibits lymphocyte migration and infiltration into the kidney, thereby preserving the kidney structure and reducing proteinuria. Thus, PEPITEM administration may be considered as a potential therapeutic tool for systemic lupus erythematosus.

Fluoride potentiates tubulointerstitial nephropathy caused by unilateral ureteral obstruction.

The contamination of ground water by fluoride has been reported worldwide. Most fluoride (approximately 70%) is filtered by the kidneys; humans or experimental animals with renal damage therefore may be more affected by fluoride exposure than those with normal kidney function. Tubulointerstitial fibrosis, which involves macrophage-promoted extracellular matrix production and myofibroblast migration, can be induced in rats by unilateral ureteral obstruction (UUO). We examined the effects of fluoride exposure on tubulointerstitial fibrosis in the obstructed kidney of UUO rats. The left ureters of 6-week-old male rats were ligated using silk sutures. Fluoride was then administered for 2 weeks at doses of 0, 75, and 150ppm in the drinking water. Real-time polymerase chain reaction was performed to analyze transforming growth factor beta 1 (TGF-ß1) transcription; histological and immunohistochemical staining were used to identify positive areas within the renal cortex and staining-positive cells by image analysis. Significant increases were observed in the obstructed kidneys of UUO rats exposed to 150ppm fluoride (compared to 0ppm) for areas or number of cells that stained with Masson trichrome or with antibodies against collagen type I, alpha-smooth muscle actin (α-SMA, a myofibroblast marker), ED1, ED2, and ED3 (macrophage markers), and TGF-ß1. Taken together, these observations suggested that fluoride exacerbates tuburointerstitial nephropathy resulting from UUO, and that this effect occurs via activation of the M2 macrophage-TGF-ß1-fibroblast/myofibroblast-collagen synthesis pathway.

Safety Evaluation of Self-assembling Peptide Gel after Intracranial Administration to Rats Using the Open Field Test.

Self-assembling peptides have been developed as clinical materials, which could scaffold to regenerate nerve cells and hemostatic materials in vivo. However, there has not been enough information for their in vivo application. The safety of self-assembling peptides for the application on the brain was examined using behavioral tests for each rat in this study. Self-assembling peptide gel was administered to the surface of the brain at a volume of 20 µL at 1.5%. After 2 months, the open field test and the prepulse inhibition (PPI) test were performed. There were no significant differences between the peptide gel and the control groups in locomotor distances and in %PPIs in the PPI test. The mean values of the percentage of time the rats stayed in the central area of the open field during the first 5 min and instances of center rearing or face washing in the peptide gel group were significantly higher than those in the control. There were amorphous substance in the subarachnoid region, and infiltrations of mononuclear cells were also observed in the self-assembling peptide gel group. Although the meaning of the effects observed in this study was not fully elucidated, the self-assembling gel produced marginal but significant behavioral and histological effects.

The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks.

BACKGROUND: The toxicity of multi-walled carbon nanotubes (MWCNT) may be related to the immune system. The objective of this study was to obtain information for immunotoxic mechanisms of MWCNT in situ. METHODS: Using whole-body inhalation, male and female rats were exposed to 0, 0.2, 1 or 5 mg MWCNT/m³ for 13 weeks. Thereafter, spleens were recovered from the rats. Real-time PCR was done to assess expression of TNFα, IL-1ß, IL-6, IL-10, MCP-1 and MIP-1α mRNA in the splenic macrophages; splenic T-lymphocytes were examined for IL-2 and TGF-ß1 mRNA expression. RESULTS: The relative expression of IL-1ß mRNA in the cells from female rats exposed to 5 mg MWCNT/m³ was significantly higher than that in control cells. For IL-6 and IL-10, cells from rats in the 0.2 and 5 mg MWCNT/m³ had significantly higher mRNA expressions than did cells from controls. Expression of IL-1ß, IL-6 and TNFα genes in cells from males in all exposure groups were higher than in control cells. Expression of MIP-1α in the cells from female 5-mg group was significantly higher than that in cells in the control. Only IL-2 was expression reduced, i.e. cells from male and female rats in all MWCNT groups had significantly lower mRNA expressions than control cells. CONCLUSIONS: Systemic inflammation would likely occur in rats (or other hosts) exposed to MWCNT via inhalation due to increases in the expression of inflammatory cytokines in splenic macrophages. Moreover, decreases in IL-2 expression in T-lymphocytes may be critical to the potential reductions in anti-tumor responses in MWCNT-exposed hosts.

The increases in mRNA expressions of inflammatory cytokines by adding cleaning solvent or tetrachloroethylene in the murine macrophage cell line J774.1 evaluated by real-time PCR.

The use of a petroleum-derived cleaning solvent for dry cleaning, instead of tetrachloroethylene (perchloroethylene, PCE), has increased. The cleaning solvent may induce immunological alteration. In this study, murine macrophage-lineage J774.1 cells were exposed to the cleaning solvent at 0, 25, 50, and 75 µg/ml or PCE at 0, 400, 600, 800, and 1,000 µg/ml by vigorous vortexing. Cell viability was determined. The mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), IL-6, IL-10, IL-12p40 (a dimer of IL-12), and IL-27p28 (a dimer of IL-27) were evaluated by real-time PCR. The mean viabilities in the 50 and 75 µg/ml groups of the cleaning solvent were significantly lower than that of the control. The mean mRNA expressions of TNF-α and IL-1ß in the 50 µg/ml group were significantly higher than those in the control. For PCE, the mean viabilities at 600 µg/ml and over were significantly lower than that of the control. The mean expressions of IL-6 and IL-10 in the 800 µg/ml group were significantly higher than that in the control. The productions of IL-1ß and TNF-α may be altered in human during intoxication of the cleaning solvent as well as those of IL-6 and IL-10 in human during that of PCE, and these may affect on immune cells.

A pilot study testing the dimensions of safety climate among Japanese nurses.

To investigate the dimensions of safety climate among Japanese nurses, an anonymous self-administered questionnaire survey was conducted. The subjects involved in the survey included 293 full-time nurses (registered nurses and licensed practical nurses) working in a public hospital, excluding directors of nursing. A total of 221 of the 293 nurses answered the questionnaires. Among 221 questionnaires, the questionnaires, which had missing values in the question items used in this study, were excluded from the analyses. Consequently, a total of 201 questionnaires were analyzed. The average age of the subjects was 34.7 yr. As a result of exploratory factor analysis, 5 factors were extracted as follows: intellectual development regarding medical safety among nurses, accumulated fatigue, nursing conditions, supervisors' attitudes, and communication with physicians. All the values of Cronback's coefficient alpha among these 5 factors were between 0.804 and 0.892. As a result of the confirmatory factor analysis of the 5 factors, the value of the GFI (Goodness of Fit Index) was 0.868. The value of the CFI (Comparative Fit Index) was 0.943. The value of the RMSEA (Root Mean Square Error of Approximation) was 0.062. The results of this study will contribute to the investigation of the dimensions of a nurses' safety climate scale in the future. The associations between the dimensions of the safety climate and the motivation to work toward improving patients' safety among Japanese nurses will need to be examined, as will those between the dimensions of the safety climate and actual clinical mistakes.

Magnetometric evaluation of the effects of man-made mineral fibers on the function of macrophages using the macrophage cell line RAW 264.7.

The toxic effects of man-made mineral fibers (MMMFs) have been evaluated by cell magnetometry using alveolar macrophages (AMs). Recently, on the other hand, the murine macrophage cell line, RAW 264.7, became available and has been used as an in vitro model of AMs. The objective of this study was to determine whether or not cell magnetometry using RAW 264.7 cells can be used to evaluate the toxic effects of MMMFs. RAW 264.7 cells were exposed to one of the MMMFs, potassium octatitanate (PT) or silicon carbide whisker (SiC) at 0, 20, 40 and 60 microg/ml, or chrysotile as a positive control at 0, 15, 20 and 25 microg/ml. The toxic effects of fibers were evaluated by cell magnetometry and LDH assay. For this comparison, AMs were also exposed to chrysotile fibers (CF). In the RAW 264.7 cells exposed to PT 20, 40, 60 or SiC 20, 40, 60, CF 15, 20 and 25 microg/ml, significant delayed relaxation were observed compared with the respective control. In the LDH assay, significant increases in LDH in the supernatant of the cells exposed to PT 20, 40, 60, SiC 20, 40, 60 and CF 15, 20, 25 microg/ml were observed. In AMs exposed to CF 20, 25 microg/ml significant delayed relaxation and significant increases in LDH compared with the control were observed. The levels of MMMFs that induced significant differences were similar for cell magnetometry and LDH. The levels of CF that induced significant differences in cell magnetometry and LDH were identical for RAW 264.7 cells and AMs. Our results suggest that cell magnetometry using RAW 264.7 cells is adequate to evaluate the cytotoxicity of exposure to MMMFs.

Associations of health behaviors on depressive symptoms among employed men in Japan.

The associations between health behaviors and depressive symptoms have been demonstrated in many studies. However, job strain has also been associated with health behaviors. The aim of this study was to analyze whether health behaviors such as physical activity, sleeping, smoking and alcohol intake are associated with depressive symptoms after adjusting for job strain. Workers were recruited from nine companies and factories located in east and central areas of Japan. The Center for Epidemiologic Studies Depression (CES-D) Scale was used to assess depressive symptoms. Psychological demand and control (decision-latitude) at work were measured with the Job Content Questionnaire. Multiple logistic regression analysis was used to determine the independent contribution of each health behavior to depressive symptoms. Among the total participants, 3,748 (22.7%) had depressive symptoms, which was defined as scoring 16 or higher on the CES-D scale. Using the multiple logistic regression analysis, depressive symptoms were significantly associated with physical activity less than once a week (adjusted relative risk [ARR] = 1.18, 95% confidence interval [CI], 1.14 to 1.25) and daily hours of sleep of 6 h or less (ARR, 1.25; 95% CI, 1.14 to 1.35). Smoking and frequency of alcohol intake were not significantly associated with depressive symptoms. This study suggests some health behaviors such as physical activity or daily hours of sleep are associated with depressive symptoms after adjusting for job strain.

Decreased membrane fluidity and hyperpolarization in aluminum-treated PC-12 cells correlates with increased production of cellular oxidants.

Effects of aluminum (Al) on membrane properties of excitable cells are not fully understood. Several reports have identified cellular membranes as sensitive targets for Al intoxication. In the present study, we tested the hypothesis that treatment with Al would alter membrane fluidity and potential and these changes would correlate with aberrant generation of cellular oxidants. The effects of in vitro Al exposure in resting rat pheochromocytoma (PC-12) cells, a model that exhibits neuron-like properties, were investigated. Treatment of PC-12 cells with Al (>0.01mM) resulted in a concentration-dependent decrease in membrane fluidity. Similar concentrations of Al increased the rate of extracellular acidification, measured by a cytosensor microphysiometer, indicating stimulation of proton extrusion from cells. This change in proton extrusion was accompanied by a rapid and concentration-dependent hyperpolarizion of the cell membrane as determined by decreased fluorescence of a potential-sensitive dye, bis-[1,3-dibutylbarbituric acid]trimethine oxonol [Dibac(4)(3)]. Al-induced perturbations of membrane properties correlated with an increased level of cellular oxidants, indicated by increasing dihydrorhodamine 123 oxidation. Results suggest that acute exposure to Al modifies membrane properties of neuron-like cells and therefore cellular membranes represent a plausible target for Al neurotoxicity. Alterations in membrane potential can have a dramatic impact on cellular communication especially in neurons and may be an important mechanism in Al neurotoxicity.

Tributyltin (TBT) increases TNFα mRNA expression and induces apoptosis in the murine macrophage cell line in vitro.

OBJECTIVE: Tributyltin (TBT) compounds have been widely used as antifouling agents for shipbottom paint. The immune system is a target of TBT intoxication. We evaluated the effects of TBT chloride in macrophages, which have critical roles in the immune system, using a murine macrophage lineage cell line, J774.1,in vitro. METHODS: We examined tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß) andc-jun mRNA expression in J774.1 cells. The effects of TBT on the apoptosis of J774.1 cells were examined by determining the percentage of TUNEL-positive cells and caspase-3 activity. RESULTS: The mean values of the viabilities of J774.1 cells exposed to TBT decreased dose-dependently. The relative mRNA expression of TNFα increased dose-dependently, however, that of IL-1ß was not significantly different among the groups. The mean percentage of TUNEL-positive cells increased dose-dependently. Increases in the caspase-3 activities of J774.1 cells were observed in the groups exposed to higher concentrations of TBT. The mean value of relative mRNA expression of c-Jun transcription factor increased dose-dependently. CONCLUSIONS: The increases in the percentage of TUNEL-positive cells and in caspase-3 activity suggested that exposure to TBT induces apoptosis of J774.1 cells. The increases in the mRNA expression of TNFα andc-jun by TBT may be related to apoptosis in macrophages.

Serum cytokine levels, cigarette smoking and airway responsiveness among pregnant women.

BACKGROUND: Five to twenty percent of healthy, nonasthmatic individuals exhibit airway hyperreactivity. Because cytokines are important intermediates in airway responses, we investigated the relationship between serum cytokines and airway responsiveness in a well-characterized population of pregnant women. METHODS: We analyzed serum levels of interleukin-4 (IL-4), IL-5, IL-6, interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in frozen sera from 240 pregnant nonasthmatic subjects from a previous study of airway responsiveness and preterm labor for their relationship to methacholine challenge test results. RESULTS: Serum IFN-gamma and GM-CSF levels were significantly related to levels of methacholine airway responsiveness among nonasthmatics in this population. Nonasthmatics with PD(20) <8 micromol (the highest level of airway responsiveness) had the highest mean serum levels of IFN-gamma and GM-CSF, those with 8 50 micromol had intermediate levels, and those with PD(20) >>50 mol had the lowest levels. Both serum IFN-gamma and GM-CSF levels were also significantly related to the log dose-response slope for methacholine responsiveness. These relationships were confirmed in a multiple linear regression analysis controlling for age, ethnic background, and cigarette smoking. Current smoking was uniformly associated with decreased cytokine levels. CONCLUSION: IFN-gamma, GM-CSF, and other cytokines may be useful serum markers in population studies of airway responsiveness.

The relation between serum cytokine levels and common laboratory tests in healthy Japanese males.

OBJECTIVES: Cytokines are critical regulatory molecules that circulate in blood at measurable levels. However, the epidemiology of serum cytokine levels in healthy populations is still limited, particularly for Japanese populations. The objective of this study was to examine the relation between serum cytokine levels and common laboratory tests in a healthy Japanese population. METHODS: We studied serum levels of six cytokines (interleukin-4 (IL-4), IL-5, IL-6, IL-10, interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF)) in a healthy Japanese population using a sensitive chemiluminescence ELISA (CL-ELISA). We examined the relations between serum cytokine levels and common laboratory tests using multiple regression analysis. We were particularly interested in potential relations between serum cytokine levels and C-reactive protein (CRP) in light of its relation to the risk of cardiovascular events. We also investigated the relation between cytokine levels, alcohol consumption and smoking. RESULTS: After adjusting for the influence of other cytokines and laboratory tests, the serum level of IL-4 was associated with IL-5 and GM-CSF. In addition to IL-4, IL-5 was also associated with IL-10 and IFN-γ. IL-6 was associated with IL-10 and IFN-γ, and IL-10 and IFN-γ were associated with GM-CSF. CRP was significantly negatively associated with serum IL-4 level. IL-6 was weakly correlated with alcohol consumption level in this population. IL-5 among smokers tended to be lower than that among nonsmokers. CONCLUSIONS: Close relations among serum cytokine levels were observed in a healthy population. It is interesting that the level of IL-4, an anti-inflammatory cytokine, was inversely related to CRP level, suggested to be a marker of inflammation.

Long-term survivors with adult acute leukemia in complete remission: complications and return to work.

For addressing, and eventually being able to predict and prevent, both disease-related complications and changes in social status in long-term acute leukemia survivors, the follow-up is the most important factor after treatment. To this end, we assessed the complications following the attainment of complete remission in adult acute leukemia patients and the changes in social status of patients surviving more than 5 years after disease onset. In our study population of 42 survivors, 24 (57.1%) suffered from various combinations of 18 types of identified complications including posttransfusion hepatitis, diabetes mellitus, and idiopathic osteonecrosis. Regarding fertility, 9 live births were recorded in this cohort, from 2 female patients and the partner of a male patient. Of these 42 long-term survivors, at the time of this report 48.5% were working full- or part-time, 9.0% were unemployed, 30.3% were homemakers, and 12.2% were retired.