Preparation and Characterization of a Polyclonal Antibody against Brominated Protein

(Di)bromotyrosine is formed by the specific reaction of eosinophil peroxidase and can be used as an eosinophil activation marker. In the present study, an antibody for (di)bromotyrosine in proteins was prepared to investigate the pathogenesis of eosinophil-related diseases such as allergic responses. A rabbit polyclonal antibody was raised against brominated keyhole limpet hemocyanin. The specificity of the antiserum was investigated with an enzyme-linked immunosorbent assay (ELISA). The antiserum recognized brominated bovine serum albumin (BSA) and dibromotyrosine-conjugated BSA. The antiserum also reacted with chlorinated BSA and di-iodotyrosine-conjugated BSA. Moreover, the specificity of the antiserum was investigated using competitive ELISA. Dibromotyrosine and di-iodotyrosine inhibited the recognition of brominated BSA by the antiserum. However, the recognition of brominated BSA by the antiserum was not inhibited by bromotyrosine, chlorotyrosine, iodotyrosine, nitrotyrosine, aminotyrosine, phosphotyrosine, or tyrosine. These results suggested that the epitope of the antiserum is dihalogenated tyrosine. Immunohistochemically, the antiserum stained brominated rat eosinophils but not chlorinated or nitrated eosinophils. In conclusion, an antiserum for dihalogenated protein was prepared. It is expected that the antiserum will be useful for the analysis of the pathogenesis of allergic diseases such as asthma and atopic dermatitis

Association of Dietary Fat and Carbohydrate Consumption and Predicted Ten-year Risk for Developing Coronary Heart Disease in a General Japanese Population

We examined the relationships between dietary carbohydrate, protein, fat, and the ratio of n6/n3 fatty acid intakes with the predicted 10-year coronary heart disease (CHD) risk in a general Japanese population. We used the Framingham risk score to determine the 10-year CHD risk of the subjects, who were employees of 6 companies in a single prefecture in Japan. After excluding the subjects who reported any history of angina pectoris, myocardial infarction, diabetes, or cancer, and those with missing data resulting in the inability of estimation of 10-year CHD risk and food intakes, the final data analysis was carried out for 809 subjects. The logistic regression models revealed a significantly increased odds ratio of 10-year CHD risk in the subjects with the highest tertile of carbohydrate intake (% energy) (odds ratio 3.64, 95% CI, 2.07-6.40);after adjustment for other variables, the odds ratio for the 10-year CHD risk was also higher in the subjects with the highest tertile of carbohydrate intake (odds ratio 1.72, 95% CI, 0.70-4.25). We also found that fat intake and the ratio of n6/n3 fatty acids were inversely associated with the predicted 10-year CHD risk (p for trend＜0.01). The present findings added evidence of a positive association of dietary carbohydrate and inverse associations of total fat and n6/n3 fatty acid ratio with the predicted 10-year CHD risk in a general Japanese population

Biochemical characterization of reactive nitrogen species by eosinophil peroxidase in tyrosine nitration

It is well known that eosinophils are involved in tyrosine nitration. In this study, we evaluated tyrosine nitration by rat eosinophils isolated from peritoneal fl uid and constituent eosinophils in the stomach. Rat peritoneal eosinophils activated with 1 &#956;M phorbol myristate acetate (PMA) and 50 &#956;M NO2 &#65437; showed immunostaining for nitrotyrosine only in smaller cells, despite the fact that eosinophils are capable of producing superoxide (O2·&#65437;). Free tyrosine nitrating capacity after incubation with PMA and NO2 &#65437; was 4-fold higher in eosinophils than in neutrophils. Catalase and &#65400;- and &#65402; -tocopherol inhibited free tyrosine nitration by reactive nitrogen species from eosinophils but not that by peroxynitrite. Superoxide dismutase augmented free tyrosine nitration by activated eosinophils and peroxynitrite. The concentration of nitric oxide released from eosinophils was relatively low (0.32 &#956;M/106 cells/h) and did not contribute to the formation of nitrotyrosine. On the other hand, most constituent eosinophils constituent in the rat stomach stimulated by PMA and NO2 &#65437; showed tyrosine nitration capacity. These results suggest that intact cells other than apoptotic-like eosinophils eluted in the intraperitoneal cavity could not generate reactive species responsible for nitration by a peroxidase-dependent mechanism. In contrast, normal eosinophils in the stomach were capable of nitration, suggesting that the characteristics of eosinophils in gastric mucosa are diff erent from those eluted in the peritoneal cavity.</p

Comprehensive molecular diagnosis of a large cohort of Japanese retinitis pigmentosa and Usher syndrome patients by next-generation sequencing. Invest Ophthalmol Vis Sci

PURPOSE. Retinitis pigmentosa (RP), a major cause of blindness in developed countries, has multiple causative genes; its prevalence differs by ethnicity. Usher syndrome is the most common form of syndromic RP and is accompanied by hearing impairment. Although molecular diagnosis is challenging, recent technological advances such as targeted highthroughput resequencing are efficient screening tools. METHODS. We performed comprehensive molecular testing in 329 Japanese RP and Usher syndrome patients by using a custom capture panel that covered the coding exons and exon/ intron boundaries of all 193 known inherited eye disease genes combined with Illumina HiSequation 2500. Candidate variants were screened using systematic data analyses, and their potential pathogenicity was assessed according to the frequency of the variants in normal populations, in silico prediction tools, and compatibility with known phenotypes or inheritance patterns. RESULTS. Molecular diagnoses were made in 115/317 RP patients (36.3%) and 6/12 Usher syndrome patients (50%). We identified 104 distinct mutations, including 66 novel mutations. EYS, USH2A, and RHO were common causative genes. In particular, mutations in EYS accounted for 15.0% of the autosomal recessive/simplex RP patients or 10.7% of the entire RP cohort. Among the 189 previously reported mutations detected in the current study, 55 (29.1%) were found commonly in Japanese or other public databases and were excluded from molecular diagnoses. CONCLUSIONS. By screening a large cohort of patients, this study catalogued the genetic variations involved in RP and Usher syndrome in a Japanese population and highlighted the different distribution of causative genes among populations

Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD

金沢大学医薬保健研究域医学系3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 ± 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 ± 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 ± 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo. © 2007 The Japanese Biochemical Society

Associations of Serum Retinol, alpha-Tocopherol, and gamma-Tocopherol with Biomarkers among Healthy Japanese Men

Retinol, alpha-tocopherol, and gamma-tocopherol are fat-soluble vitamins acting as antioxidants via the prevention of lipid oxidation. Little is known about circulatory levels in healthy individuals. The present cross-sectional study aimed at elucidating the relationship between these antioxidants and clinical biomarkers in 206 male (median age 41 years, range 23-67) employees from companies located in the Okayama Prefecture, Japan. Subjects younger than 40 years (n = 94) showed a positive association of the frequency of alcohol consumption with the circulating retinol (beta = 0.344, p = 0.001) and gamma-tocopherol levels (beta = 0.219, p = 0.041), and an inverse association of fast insulin with serum retinol (beta = -0.301, p = 0.009). In participants older than 40 years (n = 112) we found that an inverse association of HOMA-R with serum retinol (beta = -0.262, p = 0.021), a-tocopherol (beta = -0.236, p = 0.035), and gamma-tocopherol levels (beta = -0.224, p = 0.052); and cigarette smoking was inversely associated with the levels of serum a-tocopherol (beta = -0.286, p = 0.008) and gamma-tocopherol (beta = -0.229, p = 0.040). We further found negative relationships between serum ferritin and the retinol (beta = -0.211, p = 0.032) and a-tocopherol levels (beta = -0.223, p = 0.022) in men over 40 years of age. The present study suggests that the circulatory levels of antioxidant vitamins may modulate the action of insulin and that higher levels of iron might decrease the levels of antioxidant vitamins in the blood

Proton beam therapy for intrahepatic cholangiocarcinoma: A multicenter prospective registry study in Japan

Introduction: Intrahepatic cholangiocarcinoma (ICC) can be treated with chemotherapy in unresectable cases, but outcomes are poor. Proton beam therapy (PBT) may provide an alternative treatment and has good dose concentration that may improve local control. Methods: Fifty-nine patients who received initial PBT for ICC from May 2016 to June 2018 at nine centers were included in the study. The treatment protocol was based on the policy of the Japanese Society for Radiation Oncology. Forty patients received 72.6-76 Gy (RBE) in 20-22 fr, 13 received 74.0-76.0 Gy (RBE) in 37-38 fr, and 6 received 60-70.2 Gy (RBE) in 20-30 fr. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier analysis. Results: The 59 patients (35 men, 24 women; median age 71 years; range 41-91 years) had PS of 0 (n=47), 1 (n=10) and 2 (n=2). Nine patients had hepatitis and all 59 cases were considered inoperable. The Child-Pugh class was A (n=46), B (n=7), and unknown (n=6); the median maximum tumor diameter was 5.0 cm (range 2.0-15.2 cm); and the clinical stage was I (n=12), II (n=19), III (n=10), and IV (n=18). At the last follow-up, 17 patients were alive (median follow-up 36.7 months; range 24.1-49.9 months) and 42 had died. The median OS was 21.7 months (95% CI 14.8-34.4 months). At the last follow-up, 37 cases had recurrence, including 10 with local recurrence. The median PFS was 7.5 months (95% CI 6.1-11.3 months). In multivariable analyses, Child-Pugh class was significantly associated with OS and PFS, and Child-Pugh class and hepatitis were significantly associated with local recurrence. Four patients (6.8%) had late adverse events of Grade 3 or higher. Discussion/Conclusion. PBT gives favorable treatment outcomes for unresectable ICC without distant metastasis and may be particularly effective in cases with large tumors

インフルエンザ カンセンショウ ニ ガッペイ シタ キュウセイ チュウスイエン ノ 2 ショウニ レイ

症例1は6歳女児.インフルエンザ感染症初日に発熱しオセルタミビルを開始.第3病日,右下腹部に限局した圧痛が出現.腹部造影CTで糞石を認め急性虫垂炎と診断.保存的に加療し炎症反応と腹痛は改善した.症例2は5歳女児.第1病日に発熱と腹痛を認め,第3病日に鼻咽腔迅速検査でインフルエンザB型と診断しザナミビル吸入を開始.触診で右下腹部に反跳痛を認め,腹部単純CTで虫垂壁の肥厚と糞石を確認.急性虫垂炎の併発と診断し,第4病日に虫垂切除術を施行.切除虫垂に膿瘍を認め腹腔ドレーンを留置.第5病日に解熱し経過は順調であった.インフルエンザに伴う腹痛では感染に付随する腹痛と断定せず急性虫垂炎の可能性も考慮し腹部CTなどの画像検査を行うことが肝要である.We report herein two pediatric cases of influenza virus infection associated with acute appendicitis. Patient 1 was a 6-year-old girl. She presented to hospital with a high fever and received oral administration of oseltamivir phosphate. On the third day from the onset, tenderness developed in the right lower area at McBurney\u27s point. Abdominal enhanced computed tomography( CT) showed intestinal fecalith, and acute appendicitis was subsequently diagnosed. Antibiotic therapy was performed, and abdominal pain and inflammation data improved. Patient 2 was a 5-year-old girl. On the first day, abdominal pain developed. On the third day from the onset, influenza type B was diagnosed using rapid antigen testing of the nasal mucosa, and inhalation therapy was started with zanamivir. Rebound tenderness was seen in the lower right abdomen. Abdominal CT showed both hypertrophy of the appendix wall and intestinal fecalith, and influenza infection associated with acute appendicitis was subsequently diagnosed. On the fourth day, appendectomy was performed. Drainage treatment was required to prevent peri-appendicitis abscess. On the fifth day, the patient defervesced and, as for the progress, was favorable. When encountering influenza infection associated with abdominal pain, performing abdominal CT is important

Machine learning analysis to identify the association between risk factors and onset of nosocomial diarrhea: a retrospective cohort study

Background Although several risk factors for nosocomial diarrhea have been identified, the detail of association between these factors and onset of nosocomial diarrhea, such as degree of importance or temporal pattern of influence, remains unclear. We aimed to determine the association between risk factors and onset of nosocomial diarrhea using machine learning algorithms. Methods We retrospectively collected data of patients with acute cerebral infarction. Seven variables, including age, sex, modified Rankin Scale (mRS) score, and number of days of antibiotics, tube feeding, proton pump inhibitors, and histamine 2-receptor antagonist use, were used in the analysis. We split the data into a training dataset and independant test dataset. Based on the training dataset, we developed a random forest, support vector machine (SVM), and radial basis function (RBF) network model. By calculating an area under the curve (AUC) of the receiver operating characteristic curve using 5-fold cross-validation, we performed feature selection and hyperparameter optimization in each model. According to their final performances, we selected the optimal model and also validated it in the independent test dataset. Based on the selected model, we visualized the variable importance and the association between each variable and the outcome using partial dependence plots. Results Two-hundred and eighteen patients were included. In the cross-validation within the training dataset, the random forest model achieved an AUC of 0.944, which was higher than in the SVM and RBF network models. The random forest model also achieved an AUC of 0.832 in the independent test dataset. Tube feeding use days, mRS score, antibiotic use days, age and sex were strongly associated with the onset of nosocomial diarrhea, in this order. Tube feeding use had an inverse U-shaped association with the outcome. The mRS score and age had a convex downward and increasing association, while antibiotic use had a convex upward association with the outcome. Conclusion We revealed the degree of importance and temporal pattern of the influence of several risk factors for nosocomial diarrhea, which could help clinicians manage nosocomial diarrhea