An automated rapid detection system using the quenching probe method for detecting interleukin 28B and inosine triphosphatase single nucleotide polymorphisms in chronic hepatitis C.

Single nucleotide polymorphisms (SNPs) in the interleukin 28B gene (IL28B) are good pretreatment predictors of anti-hepatitis C virus (HCV) therapy with interferon. SNPs of the inosine triphosphatase (ITPA) gene are associated with reduced haemoglobin levels during treatment with ribavirin. The i-densy™ (Arkray, Inc.), which is based on the quenching probe (QP) method, automatically detects target genes in blood samples by fluorescence quenching within 100 min. Using a QP and primer set, a gene amplification response is generated that can quickly and easily detect a specific gene's arrangement by fluorometry. The present study was conducted to compare the utility of i-densy (QP method) with that of conventional direct sequencing (DS) for detecting SNPs in the IL28B and ITPA genes in chronic hepatitis C patients. Between June 2011 and January 2012, 73 consecutive patients underwent genotyping of IL28B, and 54 patients underwent genotyping of ITPA. All of the patients were seropositive for HCV-RNA. The IL28B and ITPA genotypes were tested for bi-allelic polymorphisms in rs8099917 (T/T, T/G and G/G; minor allele, G) and rs1127354 (C/C, C/A and A/A; minor allele, A), respectively. The results obtained with the QP method were identical to those obtained with the conventional DS method. The frequency of the IL28B genotypes TT, GT and GG were 74%, 24.7% and 1.4%, respectively, and those of the ITPA genotypes CC, AC and AA were 68.5%, 29.6% and 1.9%, respectively. These results indicate that the i-densy using the QP method can automatically, quickly and easily identify genotypes of IL28B and ITPA.

Changes in intraluminal pressure in rat large intestines with aging and effects of dietary fiber.

Changes in intraluminal pressure in rat colon with aging and with the effects of dietary fiber were measured. A pressure sensor was inserted into the rat large intestine under endoscopic guidance. The intraluminal pressure curve in the colon was recorded, and the motility index was calculated by this curve. The rats were divided into three groups with a fiber-free diet, a cellulose diet (10% w/w), or a pectin diet (10% w/w). Intraluminal pressure was measured in the proximal, middle, and distal colon at 2, 4, 8, 12, and 16 months after birth. Intraluminal pressure in three sites increased with age and decreased in the latter half of the study. The motility index was lower during the course in the fiber groups, especially the pectin group more than the nonfiber group. This result suggests that long-term ingestion of dietary fiber might have a prophylactic effect on the development of diverticula.

Long-term prognosis of patients with ulcerative colitis in Japan.

Even though it is important in improving the quality of life to evaluate the long-term prognosis of patients with ulcerative colitis in comparison with the general population, it is unknown in detail. One hundred and seventeen cases followed-up for 10 years or more were evaluated to define the long-term prognosis of ulcerative colitis by the person-years method. The estimated number of death (E) was 14.5, and the observed number of death (O) was 20. The O/E ratio was 1.38 and confidence interval was 0.84-2.13, showing no significant difference between E and O. Evaluation of change in the O/E ratio at 5-year intervals revealed a decrease in both the males and females, with a significant difference observed between the ratios in 1960 and 1965 (each p < 0.05). Generally, the death rate was significantly higher in the patients with ulcerative clitis than in the general population between 1953 and 1965 but did not significantly differ thereafter. On the other hand, the E from malignant tumors was 3.94, and the O was 4; the O/E ratio was 1.02 and 95% confidence interval was 0.27-2.60, showing no significant difference between E and O.

Trace element levels in drinking water and the incidence of colorectal cancer.

We determined the levels of 15 elements in drinking water from 34 water treatment plants in Aomori Prefecture and studied how element levels relate to colorectal cancer incidence by district. Colorectal cancer incidence was calculated from the data of Aomori Colorectal Cancer Registry. Multiple regression analysis was performed by using age-adjusted incidences of rectal cancer and colon cancer by gender as object variables and each element level as an explanatory variable. The standardized partial regression coefficient was significant in gold (p < 0.01), magnesium (p < 0.01), selenium (p < 0.01) and tin (p < 0.05) for age-adjusted rectal cancer incidence in men as objective variable; in gold (p < 0.05), calcium (p < 0.01) and phosphorus (p < 0.01) with age-adjusted colon cancer incidence in men as the objective variable; and in sodium (p < 0.05), phosphorus (p < 0.05), tin (p < 0.05) and strontium (p < 0.01) with age-adjusted colon cancer incidence in women as the objective variable. These results confirm the need to further study trace elements in drinking water and food, and relationship to colorectal carcinogenesis.

Inhibition of eosinophil activation in bronchoalveolar lavage fluid from atopic asthmatics by Y-24180, an antagonist to platelet-activating factor.

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptor, on the expression of adhesion molecules in peripheral blood and bronchoalveolar lavage fluid (BALF) eosinophils from atopic asthmatics. Y-24180 (20 mg/day) was administered to 4 atopic asthmatics for 8 weeks. The number of eosinophils, the level of eosinophil cationic protein (ECP), the bindings of soluble intercellular adhesion molecule-1 (sICAM-1) and fibronectin (FN), and the expressions of CD11b (alpha chain of Mac-1) and CD49d (alpha chain of VLA-4) on eosinophils were evaluated in peripheral blood (n=4) and BALF (n=3) before and after the administration of Y-24180. The infiltration of eosinophils into the bronchial wall was also examined by taking biopsies. Eosinophil count, sICAM-1 and FN binding to eosinophils in BALF significantly decreased after the administration of Y-24180 (p<0.05). The level of CD11b expression also decreased remarkably after the administration (n=2). In peripheral blood, eosinophil count and ECP level did not change. The binding of sICAM-1 and FN, and expression of CD11b on eosinophils in peripheral blood showed a tendency to decrease after the administration. The level of CD49d expression on eosinophils changed neither in BALF nor in blood. Eosinophil infiltration into the bronchial wall markedly decreased in one out of 3 cases after the administration. These results suggest that Y-24180 inhibits the activation of eosinophils by antagonizing the actions of PAF in atopic asthmatics.

Comparison of resistant starch with cellulose diet on 1,2-dimethylhydrazine-induced colonic carcinogenesis in rats.

BACKGROUND & AIMS: Epidemiological and experimental studies have shown that dietary fiber may prevent colon cancer. Resistant starch, like dietary fiber, is not subject to digestion in the small intestine. However, it is unknown whether resistant starch inhibits colonic carcinogenesis. In vitro studies have shown that butyrate slows the growth of cultured colon cancer cells. The effect of resistant starch diet on 1,2-dimethylhydrazine-induced colonic carcinogenesis in rats was evaluated, and the colonic butyrate concentration was measured. METHODS: Sprague-Dawley rats were randomly divided into five groups that were fed diets containing no fiber, 3% cellulose, 10% cellulose, 3% resistant starch, or 10% resistant starch. Colonic carcinogenesis and butyrate concentration of colonic contents and feces in each diet group were compared. RESULTS: Total cancer volume per rat in the 10% cellulose group was significantly lower than that in the basal group (109 +/- 54 mm3 and 247 +/- 83 mm3; P < 0.05), but the other groups showed no significant differences. The butyrate concentration in colonic content and in feces were higher in the resistant starch groups than in the cellulose groups. CONCLUSIONS: The resistant starch diet increased butyrate concentration but did not inhibit colonic carcinogenesis. It remains doubtful whether butyrate inhibits the proliferation of colon cancer cells.

Effects of dietary fiber on gastrointestinal transit time, fecal properties and fat absorption in rats.

The gastrointestinal transit time of food was determined by x-ray fluoroscopy using barium sulfate in rats fed with diets of various dietary fiber contents, and the effects of dietary fiber on the transit time, properties of feces, and fat absorption were examined. In 4- and 16-month-old rats fed with the diet for 3 and 15 month, respectively, the transit time of the cecum and colon in those receiving 20 and 40% wheat bran diets was shortened compared with that in the 0% group. The fecal pellet number and volume increased as the wheat bran content of the diet increased. In another experiments, the daily total fat excretion was found to be the greatest in rats receiving 15% pectin diet, followed by rats receiving 15% cellulose and non-fiber diets, respectively. These results suggest that shortening of the transit time through the cecum and colon with increase of fecal volume and suppression of fat absorption all participate in the mechanism of the inhibitory action of wheat bran on carcinogenesis and on the development of diverticulum of the large intestine.

[Dietary fiber intake among the general population in 25 prefectures].

The per capita daily intake of dietary fiber in 25 prefectures was calculated from measured values of dietary fiber in each food according to the modified Southgate method and the modified Prosky method. Results obtained were as follows; In the calculated values according to the modified Southgate method, mean per capita daily dietary fiber intake was highest in Gunma 20.5 g and lowest in Okinawa (13.5 g). On the other hand with Prosky method, dietary fiber intake was highest in Gunma (19.3 g) and lowest in Hyogo (12.4 g). Therefore, dietary fiber intake was higher in eastern Japan than in western Japan. In calculated values according to the modified Southgate method, the largest consumption was noted with rice at 30%. On the other hand, in Prosky method, vegetables were noted as the largest and rice ranked second. The mean per capita daily intake of hemicellulose was noted at 50% of the whole, cellulose at 30-35% and lignin at 15-20%.

Epidemiological evaluation of colonic diverticulosis and dietary fiber in Japan.

The incidence of colonic diverticulosis was examined in 5 hospitals geographically isolated from each other in Japan during the period between mid '70s and 1986. The incidence rapidly increased in all hospitals from 3.5-9.0% in mid '70s and 8.4-23.2% in 1986. The ratio of right-sided type colonic diverticulosis was approximately 70-80% in each hospital and the ratio did not fluctuate significantly during the period. Birth cohort analysis revealed that although the incidence of right-sided diverticula had increased with aging, in groups born in more recent decades right-sided diverticula appeared in younger age than other groups. It was suggested that environmental factors rather than congenital factors may cause the increase of the incidence of right-sided colon diverticula.

[Dietary fiber intake and intake patterns among the general population in Aomori, calculated using modified Southgate and Prosky methods].

Based on the result of the Aomori Nutrition Survey, we have calculated daily dietary fiber (DF) intake using modified Southgate and Prosky methods. Result obtained were as follows: DF intake in Aomori was 23.7 +/- 8.4g by the modified Southgate method, and 22.2 +/- 8.5g by the modified Prosky method. As for supply sources of DF, 32.3% was from grains; 22.7%, vegetables; 15.6%, beans; and 7.8%, fruits calculated by the modified Southgate method, and 31.3%, vegetables; 19.0%, grains; 18.0%, beans; and 11.6%, fruits by the modified Prosky method. As to types of DF, intake of hemicellulose was 11.5 +/- 4.2g, cellulose 7.8 +/- 3.0g and lignin 4.4 +/- 2.3g. In the group with DF intake below 10.0g, DF came mainly from grains and fruits, while the group with DF intake above 30.0g took DF from various foods.

Electron microscopic study on the homozygote (Ml/Ml) of the macular mutant mouse.

The macular mouse is a mutant mouse with a defect in copper transport and an X-linked recessive inheritance. Its hemizygote (Ml/y) is considered to be an appropriate model fo Menkes kinky hair disease (MKHD). In this study, homozygote (Ml/Ml) were bred by coupling CuCl2-treated Ml/y with heterozygote (Ml/+). Both Ml/Ml and Ml/y die around day 15 of age. However, treatment with CuCl2 enables them to live until adulthood. The brains of Ml/Ml were chronologically examined by light and electron microscopy. In the non-treated Ml/Ml, abnormal mitochondria increased in number in the cerebral cortical neurons and in the cerebellar Purkinje cells from day 7 to 14 of age. In the treated Ml/Ml, the administration of CuCl2 improved the abnormality of the mitochondria in the cerebrum by day 20, but those in the Purkinje cells remained until day 60. Flattened cisterns and intracytoplasmic inclusions were also observed in the Purkinje cells of treated Ml/Ml. These ultrastructural changes were quite similar to those observed in the Ml/y. Our mutant mice (treated Ml/Ml), when they are coupled with treated Ml/y, can give birth to offspring, all of which will be genetically Ml/y or Ml/Ml. These fetal mice will be very helpful for studying the pathological and biochemical condition of prenatal MKHD.

[Endoscopic study on the effect of dietary fiber against 1,2-dimethylhydrazine-induced colonic carcinogenesis in rats].

The effect of dietary fiber on colonic carcinogenesis was studied endoscopically in rats administered 1,2-dimethylhydrazine. Colonic tumors appeared about six weeks later in rats fed 15% cellulose diet or 40% wheat bran diet than in those fed basal diet. Furthermore, the incidence of colonic tumor was significantly lower from 23th week to 26th week in the cellulose diet group (p less than 0.01) and at 26th week in the wheat bran diet group (p less than 0.05) than in the basal diet group. However, the incidence of colonic tumors and the mean number of tumors per rat at sacrifice (30th week) were not significantly different between these two high-fiber diet groups and the basal diet group. These two diets significantly increased feces 3 to 4 times in weight and 5 times in volume over the base diet. However, 15% pectin diet could neither inhibit colonic carcinogenesis nor increase the fecal weight. These results suggested that cellulose and some dietary fibers of wheat bran were effective to retard colonic carcinogenesis, and that the increased fecal weight and volume by fibers may be involved in the inhibitory effect.

Golgi study on the homozygote (Ml/Ml) of macular mutant mouse.

The macular mutant mouse shows X-linked recessive inheritance and its hemizygote (Ml/y) is considered to be an appropriate model of Menkes kinky hair disease (MKHD). In this study the homozygote (Ml/Ml) was bred by coupling CuCl2-treated Ml/y with Ml/+ and was clinically and neuropathologically examined. The Ml/Ml had white fur color and curly whiskers from day 3, showed ataxia and tonic seizure on day 8 and gradually lost weight after day 10. It died with severe emaciation around day 15. These clinical features were improved by CuCl2 injection. Quantitative analysis showed that the dendritic arborization of the pyramidal cell in the treated Ml/Ml was delayed on days 14, 20, 30, 45 and 90 in comparison with that of the age-matched +/y. In the cerebellum of the Ml/Ml on day 14, some of the Purkinje cells showed abnormal changes such as somal sprouts, spine-like structures on the surface of the soma and stem dendrites, thick stem dendrites, multiple focal swellings of the stem and distal dendrites, reduction in the size of dendritic trees and axonal focal swellings. These changes were gradually improved in the Ml/Ml with CuCl2 treatment after day 20, with the exception of the multiple focal swellings of the stem and distal dendrites. The dendritic focal swelling gradually decreased after day 45. These clinical and neuropathological features of the Ml/Ml are almost same as those of the Ml/y. In our mutant mouse, when the treated Ml/Ml is coupled with the treated Ml/y all offspring from the Ml/Ml are genetically Ml/y or Ml/Ml. Our study indicates that these fetal mice may be useful for studying the pathological and biochemical condition of prenatal MKHD.

Electron microscopic study on brain of macular mutant mouse after copper therapy.

The hemizygote of the macular mutant mice, which is clinically and neuropathologically considered to be a model of Menkes kinky hair disease (MKHD), were injected intraperitoneally four times with 10, 20, 20 and 30 micrograms of cupric chloride on days 4, 6, 8 and 10 after birth, respectively. Their cerebral and cerebellar cortices were chronologically examined by electron microscopy. In the cerebral cortex, only a few abnormal mitochondria with electron-lucent matrix and short peripherally located cristae were scattered in the neurons on day 14, and these had almost entirely vanished after day 21. In the cerebellar cortex, abnormal mitochondria were frequently found on day 14 in the dendrites of the Purkinje cells, whereas they were only occasionally observed in their cytoplasm. Those in the dendrites had decreased in number on day 30, and only a few of them were seen in the cerebellum after day 45. These results show that the copper therapy reduced ultrastructural abnormalities in the hemizygote of this mutant mouse.

Golgi study on macular mutant mouse after copper therapy.

This study was undertaken to elucidate the clinical and neuropathological effects of copper administration on the macular mutant mouse. Its hemizygote, which is considered to be a model of Menkes kinky hair disease (MKHD), was injected intraperitoneally four times with 10, 20, 20 and 30 micrograms of cupric chloride on days 4, 6, 8 and 10, respectively. The hemizygote's curly whiskers gradually straightened and the frequent tonic seizures and ataxia disappeared after the injections. The body weight also gradually increased. In the cerebral cortex, the dendritic arborization of the pyramidal neurons in both the normal littermate and the treated hemizygote developed with time and reached the maximum around day 60. In the treated hemizygote, however, the arborization of the dendrites was significantly poor in comparison with that in the normal littermate from day 20 to 90. In the cerebellum of the treated hemizygote, the abnormal Purkinje cells with the few somal sprouts, thick stem dendrite and/or poor arborization, which were seen in the non-treated hemizygote, were improved by day 30, while their focal dendritic swellings remained even on day 60. These results indicate that the copper therapy improves not only the clinical manifestations but also the neuropathological changes, especially in the cerebellum.