Development of a simple screening method for analyzing cereulide toxin in fried rice using liquid chromatography-tandem mass spectrometry.

PURPOSE: The presence of cereulide, an emetic toxin produced by Bacillus cereus, in fried rice samples is critical evidence of food poisoning even in situations where B. cereus could not be detected. This study aims to develop a screening method for analyzing cereulide in fried rice using the QuEChERS procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Cereulide was identified and quantified in fried rice samples using the QuEChERS extraction method and LC-MS/MS. The accuracies of the methods were determined by analyzing fortified blank samples at two concentrations (10 and 50 µg/kg) conducted on three samples daily for five days. RESULTS: The QuEChERS procedure removed matrix compounds from fried rice. Characteristic MS/MS spectra enabled the identification of cereulide. As the matrix effects in seven fried rice samples were within ± 6%, an external solvent calibration curve could be used for quantification. This method exhibited good accuracy ranging from 88 to 89%. The relative standard deviations for both repeatability and intra-laboratory reproducibility were < 4%. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification was 2 µg/kg. The applicability of this method was confirmed using the analysis of cereulide in fried rice samples incubated with emetic Bacillus cereus. CONCLUSIONS: The QuEChERS extraction procedure described herein showed substantial promise as a reliable screening tool for cereulide in fried rice sample.

Simultaneous determination of five carbapenems, highly polar antibiotics, in milk by LC-MS/MS.

The simultaneous determination of five carbapenems (biapenem, doripenem, ertapenem, imipenem, and meropenem) in raw and pasteurised bovine milk samples using LC-MS/MS was achieved and validated. Chromatographic separation was conducted on an InertSustain® AQ-C18 column using 0.1% formic acid in water and acetonitrile as the mobile phase. Target compounds were extracted using acetonitrile/water (20:80, v/v). After the removal of lipids with acetonitrile-saturated hexane, the dissolved protein was denatured with acetic acid. A portion of the supernatant was passed through an Oasis® PRiME HLB cartridge to remove the matrix. This novel method was validated in accordance with the Japanese validation guidelines and exhibited good trueness, ranging from 86.3% to 96.2%, using matrix-matched calibration curves. The relative standard deviation of repeatability ranged from 1.0% to 6.3%, and that of within-laboratory reproducibility ranged from 1.6% to 7.1%. The limit of quantification was 1.0 µg kg-1 for all analytes. None of the 60 milk samples commercially available in Tokyo contained any analytes. This novel method exhibited high-quality performance and can easily be implemented for the routine monitoring of carbapenems, which are highly polar antibiotics in milk.

Stepwise Treatment for TAFRO Syndrome.

TAFRO syndrome, a rapidly progressive and fatal disease, is rare, and its etiology remains unknown. It is characterized by thrombocytopenia, anasarca (edema, pleural effusion, and ascites), fever, reticulin fibrosis (or renal insufficiency), and organomegaly with Castleman disease (CD)-like histological features in the lymph nodes. CD is a rare, indolent, lymphoproliferative disorder with no established curative strategies. Most idiopathic multicentric CD cases are controlled with anti-interleukin (IL)-6 therapy (tocilizumab and siltuximab) and/or rituximab. However, it is unclear whether these therapies can be directly applied to treat TAFRO syndrome. Here, we describe stepwise immunotherapy (rituximab induction therapy and cyclosporine maintenance therapy) for two cases of steroid-refractory TAFRO syndrome. A 32-year-old man visited a local hospital with sudden onset of fever and epigastralgia. The diagnosis of TAFRO syndrome was established based on the diagnostic criteria. After rituximab administration, C-reactive protein and IL-6 levels were normalized. However, the ascites persisted, with increased resistance to rituximab. Tocilizumab was also ineffective; therefore, cyclosporine was administered. After the initiation of cyclosporine treatment, the ascites decreased and ultimately disappeared. Twelve months after immunotherapy, the patient remained asymptomatic under cyclosporine maintenance therapy. Similar stepwise immunosuppressive therapy was administered to a 72-year-old man with TAFRO syndrome complicated by renal failure. After rituximab infusion, C-reactive protein was decreased. Although methylprednisolone, rituximab, tocilizumab, and cyclosporine were administered, other laboratory data and clinical symptoms remained unchanged. His level of consciousness subsequently deteriorated due to herpes zoster encephalitis, and he died. We consider the combination of rituximab induction therapy and cyclosporine maintenance therapy to be effective for TAFRO syndrome if initiated at an early stage.

[Simultaneous Determination of Pesticide Residues in Brown Rice Using GC-MS/MS and LC-MS/MS].

This study developed a method that simultaneously detected 283 pesticide residues in brown rice using GC-MS/MS and LC-MS/MS. In this method, we examined the desirable amount of sodium chloride required for salting out and the SPE cartridge required for clean-up. Pesticide residues from the sample were extracted with acetonitrile using a homogenizer and mixed with salts including anhydrous magnesium, two types of citrate, and sodium chloride. The sample solution of the acetonitrile layer was cleaned up using the GCB/NH2 (200 mg/200 mg, 6 mL) SPE cartridge. The determination method was validated using two concentrations (0.01 and 0.1 µg/g) of 283 pesticides based on the validation guideline of the Ministry of Health, Labor and Welfare in Japan. Of the 283 pesticides, 250 were detected satisfactorily. In addition, 59 brown rice samples sold in Tokyo were surveyed using the same method. Out of 44 samples, 12 pesticide residues below MRLs were detected. Therefore, this developed method is useful for the simultaneous determination of pesticide residues in brown rice.

Survey of Nitrate Ion Concentrations in Vegetables Cultivated in Plant Factories: Comparison with Open-Culture Vegetables.

A survey of nitrate-ion concentrations in plant-factory-cultured leafy vegetables was conducted. 344 samples of twenty-one varieties of raw leafy vegetables were examined using HPLC. The nitrate-ion concentrations in plant-factory-cultured leafy vegetables were found to be LOD-6,800 mg/kg. Furthermore, the average concentration values varied among different leafy vegetables. The average values for plant-factory-cultured leafy vegetables were higher than those of open-cultured leafy vegetables reported in previous studies, such as the values listed in the Standard Tables of Food Composition in Japan- 2015 - (Seventh revised edition). For some plant-factory-cultured leafy vegetables, such as salad spinach, the average values were above the maximum permissible levels of nitrate concentration in EC No 1258/2011; however, even when these plant-factory-cultured vegetables were routinely eaten, the intake of nitrate ions in humans did not exceed the ADI.

Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

BACKGROUND: Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. METHODS: The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. RESULTS: The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P <0.001), and serum levels of TNF-α (P <0.001) and interleukin-6 (P <0.001) after ADA medication, although plaque levels were comparable. Among a total of 495 protein spots obtained, nine spots were significantly decreased in abundance at reassessment, corresponding to complement factor H, phospholipase D, serum amyloid A, complement component 4, and α-1-acid glycoprotein (P <0.01). CONCLUSION: These results suggest a beneficial effect of ADA therapy on the periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

Comparative analysis of serum proteins in relation to rheumatoid arthritis and chronic periodontitis.

BACKGROUND: Rheumatoid arthritis (RA) and chronic periodontitis (CP) are chronic inflammatory conditions and share many pathologic features. The common molecular pathogenesis of the two inflammatory diseases is unclear. The aim of the present study is to evaluate serum protein profiles specific for patients with RA and CP by a comprehensive proteomic analysis. METHODS: The study participants were: 10 patients with RA, 10 patients with CP, 10 patients with RA and CP, and 10 healthy controls. All groups were balanced for age, sex, and smoking status. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant differences in abundance among the four groups were determined with computer image analysis and identified with mass spectrometry and protein databases. RESULTS: A total of 1,694 protein spots were obtained in sera of the four groups. Seven spots were significantly different in abundance among the four groups. Of these, three spots (complement component 3, complement factor H, and ceruloplasmin) were significantly different in the RA+CP group compared with the other three groups (P <0.05). The similar profiles of complement component 3, complement factor H, and ceruloplasmin were observed by enzyme-linked immunosorbent assay. CONCLUSION: These results suggest that patients with RA and CP may exhibit three serum proteins with different abundance compared with healthy controls and patients with RA only or CP only.

[Clinical course of retinopathy of prematurity and other neonatal complications in Kobe Children's Hospital].

PURPOSE: To report changes in retinopathy of prematurity (ROP) and other neonatal complications for the last 15 years CASES AND METHOD: 638 cases with a birth weight of less than 1000 g were categorized into two groups in oxygen management changes based on reduced oxygen protocol before and after change (Group A: 218 cases from 1997 to 2001, Group B: 420 cases from 2002 to 2011). Prematurity and the clinical course of ROP were reviewed. In addition, the incidence of 5 main neonatal complications before and after oxygen control criterion change was compared. RESULTS: The differences in birth weight and gestational age among the two groups were not significant (A: 765 138 g/26.5 +/- 2.2 weeks, B: 753 +/- 158 g/26.1 +/- 2.3 weeks). The incidence of ROP and the rate of treatment for ROP in Group B were significantly less than in Group A (A: 83.0%/32.6% VS B: 54.8%/ 10.0%; p < 0.01). The rate of forming a cicatricial phase severer than grade 3 was 4.1% in Group A and 1.7% in Group B (p = 0.06). The incidence of neonatal complications before and after oxygen management change was not significantly different. CONCLUSION: Incidence and treatment of ROP were improved after changing to the reduced oxygen protocol. There was no increase in neonatal complications after the management change.

Glaucoma in atomic bomb survivors.

Radiation has been associated with increases in noncancerous diseases. An effect of low-dose radiation on the prevalence of clinically detected glaucoma has not been previously reported. We therefore investigated the prevalence of glaucoma in A-bomb survivors and its possible association with radiation dose. A total of 1,589 people who participated in the clinical examination program for A-bomb survivors at the Radiation Effects Research Foundation (RERF) between October 2006 and September 2008 and who had reconstructed radiation doses, were recruited into this cross-sectional screening study. The prevalence of glaucoma and its dose-response relationship to A-bomb radiation were measured. Each subject underwent an initial screening consisting of an interview and ophthalmological examination. Questionable cases with any indication of ocular disease, including glaucoma, were referred to local hospitals for more comprehensive evaluation. A diagnosis of glaucoma was made based on specific optic disc appearance, perimetric results and other ocular findings. Of 1,589 eligible people, we detected 284 (17.9%) cases of glaucoma overall, including 36 (2.3%) cases of primary open-angle glaucoma with intraocular pressure levels greater than 21 mmHg, 226 (14.2%) cases of normal-tension glaucoma and 25 (1.6%) cases of primary angle-closure glaucoma. Seven glaucoma risk factors were examined as potential confounders but only two needed to be included in the final model. Binary regression using a generalized estimating equation method, with adjustment for gender, age, city, cataract surgery or diabetes mellitus, revealed an odds ratio at 1 Gy of 1.31 (95% confidence interval 1.11-1.53, P = 0.001) in the case of normal-tension glaucoma, but no association for other types of glaucoma. The prevalence of normal-tension glaucoma may increase with A-bomb radiation dose, but uncertainties associated with nonparticipation (59% participation) suggest caution in the interpretation of these results until they are confirmed by other studies.

Periodontal treatment decreases levels of antibodies to Porphyromonas gingivalis and citrulline in patients with rheumatoid arthritis and periodontitis.

BACKGROUND: Porphyromonas gingivalis has been implicated as an etiologic agent of rheumatoid arthritis (RA) because of the expression of peptidylarginine deiminase. The present study evaluates whether periodontal treatment may affect serum antibodies to P. gingivalis and citrulline levels in relation to disease activity of RA. METHODS: Fifty-five patients with RA were randomly assigned to receive oral hygiene instruction and supragingival scaling (treatment group, n = 26) or no periodontal treatment (control group, n = 29). Periodontal and rheumatologic parameters and serum levels of cytokine and inflammatory markers citrulline and immunoglobulin (Ig)G to P. gingivalis were examined at baseline and 8 weeks later. RESULTS: Both groups did not differ statistically in any parameters except percentage of sites with probing depth and clinical attachment level ≥ 4 mm at baseline. The treatment group exhibited a significantly greater decrease in disease activity score including 28 joints using C-reactive protein (DAS28-CRP) (P = 0.02), serum levels of IgG to P. gingivalis hemin binding protein (HBP)35 (P = 0.04), and citrulline (P = 0.02) than the control group. Serum levels of IgG to P. gingivalis HBP35 were significantly correlated positively with those of anti-cyclic citrullinated peptide antibodies (P = 0.0002). The same correlation was obtained between serum levels of IgG to P. gingivalis-sonicated extracts and those of rheumatoid factor (P = 0.02). CONCLUSIONS: These results suggest that supragingival scaling decreases DAS28-CRP and serum levels of IgG to P. gingivalis HBP35 and citrulline in patients with RA. These observations may reflect a role of P. gingivalis in the protein citrullination, which is related to the pathogenesis of RA.

Interleukin-6 gene promoter methylation in rheumatoid arthritis and chronic periodontitis.

BACKGROUND: Methylation status of the cytokine genes may play a role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA) and chronic periodontitis (CP). This study was undertaken to evaluate whether the DNA methylation profile of the interleukin-6 (IL-6) gene promoter was unique to individuals with RA and CP. METHODS: The study participants consisted of 30 patients with RA, 30 patients with CP, and 30 age-, sex-, and smoking status-balanced healthy controls. Genomic DNA isolated from peripheral blood was modified by sodium bisulfite and analyzed for DNA methylation levels of IL-6 gene with direct sequencing. Levels of IL-6 were determined by an enzyme-linked immunosorbent assay. RESULTS: The region of IL-6 gene promoter from -1200 to +27 bp was shown to contain 19 CpG motifs. The methylation levels of the CpG motif at -74 bp were significantly lower in patients with RA and CP than those in controls (P = 0.0001). Both levels of serum IL-6 and IL-6 production by mononuclear cells were significantly different between individuals with and without the methylation at -74 bp (P = 0.03). The +19 bp motif exhibited differential levels of the methylation among the groups, which was not associated with serum levels of IL-6. The other 17 CpG motifs exhibited comparable levels of the methylation between the groups. CONCLUSION: These results suggest that hypomethylated status of a single CpG in the IL-6 promoter region may lead to increased levels of serum IL-6, implicating a role in the pathogenesis of RA and CP.

The efficacy of ezetimibe on nonalcoholic fatty liver disease (NAFLD).

The usefulness of ezetimibe was examined in 297 patients with dyslipidemia who did not achieve LDL-C target levels set in JAS 2007 Guidelines by lifestyle modification. The mean period of administration was 178.2±295.4 days. Ezetimibe significantly improved serum lipid levels in the patients with and without non-alcoholic fatty liver disease (NAFLD) (p<0.01). Significant improvement of AST, ALT and γ GTP levels were also observed in the patients with NAFLD (p<0.01, p<0.05, and p<0.01, respectively). Seventy of the patients with NAFLD who underwent abdominal ultrasound before and after administration of ezetimibe were followed. Of those, 38.6% of the patients showed disappearance of steatosis, indicating that administration of ezetimibe is useful in patients with NAFLD.

Antibody responses to periodontopathic bacteria in relation to rheumatoid arthritis in Japanese adults.

BACKGROUND: Periodontopathic bacteria have been implicated as contributory to the etiology of rheumatoid arthritis (RA). Anticyclic citrullinated peptide (CCP) antibodies and rheumatoid factor (RF) were shown to be associated with RA. This study examines whether serum levels of antibodies to periodontopathic bacteria may affect clinical and laboratory profiles of RA. METHODS: The study participants consisted of 80 patients with RA, and 38 age-, sex-, smoking status-, and periodontal condition-balanced healthy controls. After periodontal and rheumatologic examination, serum levels of immunoglobulin G (IgG) antibodies to Porphyromonas gingivalis (Pg), Prevotella intermedia, Aggregatibacter actinomycetemcomitans (Aa) (previously Actinobacillus actinomycetemcomitans), and Eikenella corrodens (Ec) and those of anti-CCP antibodies and RF were determined by an enzyme-linked immunosorbent assay. RESULTS: Patients with RA showed significantly higher levels of anti-Pg and anti-CCP antibodies than controls (P = 0.04 and P <0.0001). In contrast, IgG responses to Aa and Ec in patients with RA were significantly lower than those in controls (P <0.0001 and P = 0.0001). Multiple logistic regression analysis revealed a significant association of anti-Pg and anti-Aa IgG responses with RA, after adjustment for age, sex, and smoking (P = 0.005 and P = 0.02). Anti-Pg titer displayed a significant correlation with RF levels, probing depth, and clinical attachment level (P = 0.03, P = 0.03, and P = 0.02). CONCLUSION: These results suggest that serum levels of anti-Pg IgG antibodies were associated with RA, and might affect serum levels of RF and periodontal condition in patients with RA.

Twenty-four-hour ocular hypotensive effects of 0.0015% tafluprost and 0.005% latanoprost in healthy subjects.

PURPOSE: To compare the intraocular pressure (IOP) reduction over 24 h achieved with tafluprost (0.0015%) with that achieved with latanoprost (0.005%). METHODS: Twenty-seven healthy volunteers were studied. After a 24-h IOP baseline measurement was taken, one ophthalmic solution was applied to the right eye daily for 7 days. The drug was then withdrawn for 2 weeks. The other agent was then applied to the left eye in the same manner. IOP was measured every 3 h for 24 h on the seventh day of treatment. RESULTS: The 24-h IOP after 7 days' treatment with latanoprost decreased from 11.5 mmHg at baseline to 9.7 mmHg (-1.8 mmHg) and that with tafluprost from 11.8 to 9.8 mmHg (-1.9 mmHg). Tafluprost was statistically more effective after 24 h (P = 0.007; paired t test). The number of subjects with a 24-h mean IOP reduction of <10% was 8/27 (29.6%) with latanoprost versus 4/27 (14.8%) with tafluprost. The incidence of conjunctival hyperemia with latanoprost was 4/27 (14.8%) and that with tafluprost was 8/27 (29.6%). CONCLUSION: The overall efficacies of the two agents were not different, but tafluprost was associated with a greater reduction in IOP at 24 h after administration. Tafluprost showed a higher rate of conjunctival hyperemia.

Anti-U1 RNP antibodies in cerebrospinal fluid are associated with central neuropsychiatric manifestations in systemic lupus erythematosus and mixed connective tissue disease.

OBJECTIVE: To determine the significance of anti-U1 RNP antibodies in the cerebrospinal fluid (CSF) of patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD) who have central neuropsychiatric SLE (NPSLE). METHODS: The frequency of antinuclear antibodies including anti-U1 RNP antibodies in the sera and CSF of 24 patients with SLE and 4 patients with MCTD, all of whom had neuropsychiatric syndromes, was determined using an RNA immunoprecipitation assay and an enzyme-linked immunosorbent assay. The frequency of anti-U1 RNP antibodies in the CSF of patients with central NPSLE was examined, and the anti-U1 RNP index ([CSF anti-U1 RNP antibodies/serum anti-U1 RNP antibodies]/[CSF IgG/serum IgG]) was compared with CSF interleukin-6 (IL-6) levels and the albumin quotient (Qalb, an indicator of blood-brain barrier damage). CSF and serum antibodies against U1-70K, U1-A, and U1-C, including autoantigenic regions, were examined, and the U1-70K, U1-A, and U1-C indices as well as the anti-U1 RNP index were calculated. RESULTS: CSF anti-U1 RNP antibodies with an increased anti-U1 RNP index showed 64.3% sensitivity and 92.9% specificity for central NPSLE. The anti-U1 RNP index did not correlate with CSF IL-6 levels or the Qalb. The anti-U1-70K index was higher than the anti-U1-A and anti-U1-C indices in the CSF of anti-U1 RNP antibody-positive patients with central NPSLE. The major autoantigenic region for CSF anti-U1-70K antibodies appeared to be localized in U1-70K amino acid 141-164 residue within the RNA-binding domain. CONCLUSION: The frequency of anti-U1 RNP antibodies in the CSF and the anti-U1 RNP index are useful indicators of central NPSLE in anti-U1 RNP antibody-positive patients. The predominance of anti-U1-70K antibodies in CSF suggests intrathecal anti-U1 RNP antibody production.

Serum cytokine and periodontal profiles in relation to disease activity of rheumatoid arthritis in Japanese adults.

BACKGROUND: Rheumatoid arthritis (RA) and periodontitis are common chronic inflammatory conditions and share many pathologic features. A similar profile of cytokines is involved in the pathogenesis of the two diseases. The relationship between the disease activity of RA and the periodontal condition remains unclear. This study examines whether the disease activity of RA affects serum cytokine and periodontal profiles. METHODS: The study subjects consisted of 84 Japanese adults with RA and 22 race-matched control individuals. After periodontal and rheumatologic examination, the disease activity of RA was determined with the Disease Activity Score including 28 joints using C-reactive protein (DAS28-CRP). Serum levels of cytokines including interleukin (IL)-1beta, IL-6, IL-12, IL-12 p40, IL-18, and tumor necrosis factor-alpha (TNF-alpha) were determined by an enzyme-linked immunosorbent assay. High-sensitive CRP was also measured with a latex particle-enhanced nephelometric method. RESULTS: Of 84 patients with RA, 28 and 56 patients exhibited low and moderate to high disease activity, respectively. Serum levels of IL-6, TNF-alpha, and CRP were significantly different between the two groups (P <0.05). Additionally, a significant correlation was observed between DAS28-CRP and percentage of sites with bleeding on probing (BOP) (P = 0.008) and between serum TNF-alpha levels and percentage of sites with BOP (P = 0.01) in 56 patients with RA with moderate to high activity. CONCLUSION: These results suggest that the disease activity of RA correlated with serum levels of IL-6, TNF-alpha, and CRP, and it might influence BOP in the patients with moderate to high disease activity.

Two cases of late-onset drug-induced lupus erythematosus caused by ticlopidine in elderly men.

We have experienced two cases of drug-induced lupus erythematosus caused by ticlopidine in the last three years. Both were late-onset cases (1 and 4 years) that occurred in elderly men (76 and 81 years old). The common features were fever, arthralgia, myalgia, serositis, and the presence of anti-histone autoantibodies. Because ticlopidine is widely used in elderly people with ischemic vascular disease, ticlopidine-induced lupus should be considered when patients taking ticlopidine present lupus-like symptoms.