SSCP screening of the dihydropyrimidine dehydrogenase gene polymorphisms of the Japanese population using a semi-automated electrophoresis unit.

The single-strand conformation polymorphism (SSCP) procedure has been applied in routine testing for hereditary diseases. Temperature, running buffer, gel composition, and fragment length can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes, such as the dihydropyrimidine dehydrogenase (DPYD) gene, which codes the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). The authors have optimized the condition of SSCP with an automated system (GenePhor system, GE Healthcare UK Ltd.) to screen genetic polymorphisms in the DPYD gene. The efficiency of the method was evaluated using 21 positive controls (DNA samples with polymorphisms in the DPYD gene, previously characterized) and DNA samples from 35 Japanese. Results showed that the use of three different running buffers (pH 7.4, 8.3, and 9.0) in combination with other optimized conditions (10% polyacrylamide gel, 60-90 min at constant 900 V at 5 degrees C) resulted in a high polymorphism detection rate (95.3%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for pharmacogenetic studies on 5FU.

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors.

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).

Dual-color ELISPOT assay for analyzing cytokine balance.

A dual-color enzyme-linked immunospot (ELISPOT) assay enabled us to analyze three kinds of cytokine-secreting cells simultaneously. T helper (Th) cells can be subdivided into at least two distinct functional subsets based on their cytokine secretion profiles. The first type of clones (Th1) produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 or IL-5. The second type of clones (Th2) produces IL-4 and IL-5 but not IL-2 or IFN-gamma. Furthermore, the presence of the third type (Th0) cell, which is a precursor of Th1 or Th2 cells, has been demonstrated to produce both Th1- and Th2-type cytokines. The dual-color ELISPOT assay is developed to differentiate these three subtypes of Th cells in an identical well. In the system, the red spots corresponding to IL-2-secreting cells (Th1) were developed with horseradish peroxidase and amino-ethyl-carbazole/H2O2. The light blue spots corresponding to IL-4-secreting cells (Th2) were developed with alkaline phosphatase and Vector blue (chromogenic substrate for alkaline phosphatase). The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (Th0 cells) were developed with both chromogenic substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in a murine spleen cell or human peripheral mononuclear cell preparation.

Age-dependent decrease in serum transforming growth factor (TGF)-beta 1 in healthy Japanese individuals; population study of serum TGF-beta 1 level in Japanese.

Transforming growth factor-beta1 (TGF-beta1), a multi-functional cytokine, is involved in regulating a variety of cellular activities and the serum/plasma TGF-beta1 level is altered with various diseases. However, most published reports have described adult patients, and so we investigated the clinical significance of serum TGF-beta1 level in pediatric patients. The diagnostic application of the measurement of serum TGF-beta1 level depends critically on the control value, however, there is no information on the control value of serum TGF-beta1 for children. In the present study, we determined the serum TGF-beta1 level of healthy Japanese children as a control value with enzyme-linked immunosorbent assay (ELISA). The serum TGF-beta1 level of children (0-14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01). Thus, it is recommended that when the serum TGF-beta1 levels of patients are evaluated, they should be compared with those of age-matched controls.

17alpha-hydroxylase/C17-20 lyase cytochrome P450 mRNA expressions and enzyme activities during the development of arthritis in collagen-induced arthritis mice.

In our previous study, we have investigated the serum levels of dehydroepiandrosterone (DHEA) in type II collagen (CII)-induced arthritis (CIA) mice. During the study, we found that in normal control mice, serum levels of DHEA in the latter half of the experimental period (13-16 weeks old) were significantly lower than those at the beginning of the experiment (10 weeks old). However, in CIA mice, such decreases were not observed by CII treatment. To examine the cause of the retention of DHEA during the development of arthritis in CIA mice in this study, 17alpha-hydroxylase/C17-20 lyase P450 (CYP17) mRNA expressions were measured by real time RT-PCR and the CYP17 enzyme activities were investigated in the liver and testis on days 6, 13, 28 and 48 after CII treatment in DBA/1J mice. There were no significant differences of CYP17 expressions in the liver between control and CIA mice at each experimental day, while a significant increase of expression in the testis of CIA mice was observed on day 48. On the other hand, CYP17 enzyme activities on days 28 and 48 in testis microsome (Mc) from the CIA mice were significantly higher than those of the control on the same day, while no significant differences of activities in liver Mc were observed between the CIA and control mice. These findings suggested that the cause of the retention of DHEA on days 28 and 48 after CII treatment may be the increase of CYP17 expression and the enzyme activities in the testis.

Participation of endogenous dehydroepiandrosterone and its sulfate in the pathology of collagen-induced arthritis in mice.

We have investigated the serum levels of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) in type II collagen (CII)-induced arthritis (CIA) DBA/1J mice, an experimental model of human rheumatoid arthritis (RA). Serum levels of DHEA and DHEAS were measured by EIA and GC/MS, respectively. Sera were obtained from the mice on day 6, 13, 28 and 48 after the CII treatment. The disease onset of CIA was observed from day 28 (7%) to day 48 (80%) after CII immunization. The serum concentration of DHEA on day 13 did not differ from that on day 6 in CIA mice and untreated controls. Serum levels of DHEA on day 28 and 48 were significantly low compared with those on day 6 in controls. However, in CIA mice, DHEA levels on day 28 and 48 were not decreased from those on day 6. No difference in the serum DHEAS level on day 13 compared with day 6 was observed in either control or CIA mice. A significant decrease of DHEAS levels on day 28 and 48 compared with day 6 was found in both groups. The time point for the retention of DHEA in CIA mice, day 28 and day 48, coincided with the disease onset of CIA. In conclusion, endogenous DHEA may be produced as a result of physiological response for the protection against CIA.

A wide interindividual variability of urinary 6beta-hydroxycortisol to free cortisol in 487 healthy Japanese subjects in near basal condition.

The frequency distribution of CYP3A activity was investigated by measuring ratios of urinary 6beta-hydroxycortisol to free cortisol in 487 healthy subjects to determine whether a genetic polymorphism for this cytochrome enzyme exists in "native-born" Japanese persons. Spot urine samples (from 9:00 am to 12:00 pm) were collected for measurement of 6beta-hydroxycortisol and free cortisol by high-performance liquid chromatography with a CN column after extracting with a solid-phase column (Bond-Elut C18). The frequency distribution of the urinary 6beta-hydroxycortisol to free cortisol was widely distributed among subjects but with no clear bimodality by a probit plot. Furthermore, the frequency distribution assessed on a new normal test variable plot indicated the possible existence of a CYP3A sexual dimorphism. Mean 6beta-hydroxycortisol levels were higher in women (n = 249) than in men (n = 238) by 1.7-fold, and this difference was statistically significant (P < 0.01). These results show that a CYP3A genetic polymorphism in Japanese persons, based on 6beta-hydroxycortisol excretions, likely does not exist, but there appears to be a broad unimodal distribution of enzyme activity in the population.

Effect of dietary fiber on morphine-induced constipation in rats.

Morphine is used to alleviate chronic cancer pain. However, constipation is a major adverse effect that often detracts from the patient's quality of life. In this study, we investigated the effectiveness of dietary fiber on morphine-induced constipation. Rats were fed on a normal diet or one containing either 10% or 20% apple fiber for two weeks before morphine was administered. In the control diet group, the fecal number and dry weight were decreased by treating with morphine in a dose-dependent manner. Moreover, the motility of the small and large intestines was reduced. The fecal number and weight were increased and the colon motility was promoted by dietary fiber, regardless of whether morphine was being administered. The dietary fiber increased the concentration of short-chain fatty acids (SCFAs) in the cecum. These results suggest that dietary fiber has a preventative effect on morphine-induced constipation by increasing SCFAs in the cecum, and thereby promoting colon motility in rats.