Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H.

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and ß2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

Obesity reduced the gene expressions of leptin receptors in hypothalamus and liver.

The expression of leptin receptor (OB-R) is downregulated by leptin in some cell lines. This study investigated the expressions of leptin receptors at central nerve system and peripheral site in a dietary model of obesity. Rats in the 8 week high-diet and control group were classified based on body weight gain into obese and control groups. Serum leptin and insulin concentrations were measured and gene expressions of short form of leptin receptor (OB-Ra) and long form (OB-Rb) in hypothalamus and liver were detected by RT-PCR. The levels of serum leptin in obese rats were increased compared with control rats (p<0.05). The levels of OB-Ra and OB-Rb gene expressions in both hypothalamus and liver in obese rats were reduced significantly (p<0.01). Serum leptin concentrations of obese rats had a significant negative relationship with both of OB-Ra or OB-Rb gene expression levels in hypothalamus and liver (p<0.01). On the other hand, serum insulin levels had no relationship with OB-Ra or OB-Rb gene expression levels in neither liver nor hypothalamus. Rats with diet-induced obesity have hyperleptinemia and reduced expressions of leptin receptors in hypothalamus and liver. The results suggest that a leptin downregulated OB-R expression is one of leptin resistant mechanisms for maintaining obesity.

Focal segmental glomerulosclerosis in a case of panhypopituitarism: a possible role of growth hormone treatment.

Panhypopituitarism manifests various symptoms including growth failure, hypothyroidism, adrenal insufficiency and hypogonadism. Dwarfism is an important problem in children with this condition, and long-term treatment with recombinant human growth hormone (GH) is usually required. We report a 24-year-old man with panhypopituitarism complicated by focal segmental glomerulosclerosis (FSGS). The patient had been treated with GH for hypopituitary dwarfism from 3 years of age. Proteinuria was initially noticed at 15 years of age and persisted despite cessation of GH supplementation at 18 years of age. A renal biopsy specimen showed glomerular hypertrophy and limited glomerulosclerosis, compatible with FSGS. To our knowledge, this is the first reported case of panhypopituitarism complicated by FSGS. Our case suggests that GH treatment for dwarfism may induce irreversible glomerular disease.

Comparison of computer-based information support to clinical research in Chinese and Japanese hospitals: a postal survey of clinicians' views.

OBJECTIVES: The objectives of this research are to examine the current situation of computer-based information support of clinical research in hospitals and to determine the expectations of clinicians toward clinical research support functions of hospital information systems (HISs) in both China and Japan. METHODS: 172 clinicians from 42 major hospitals in China (2 groups), and 568 clinicians from 79 university hospitals in Japan (2 groups), were surveyed by postal questionnaire during July and August, 1999. The Kruskal-Wallis test was performed to analyze the differences among the groups. RESULTS: The total response rate was 66.9%. The result shows that 94.8% of the Japanese clinicians, 3.5 times more than those in China, use computers almost every day. High significance was shown for the frequency of non-HIS based information resources used by clinicians between China and Japan (p < 0.001), whereas no significance for the frequency of HIS use by clinicians between the China I and Japan I groups (p = 0.725) was found. 33.3% clinicians in China thought they could obtain 30-50% of the necessary patient data for clinical research from the HIS, about 2 times more than in Japan (p = 0.009). CONCLUSIONS: Although the degree of computer involvement among clinicians in Japan is much higher than in China, the computer-based hospital information systems have not been developed well for supporting clinical research in both countries. The clinicians expect comprehensive computerized patient records (CPRs) and full use of patient related information in the existing HISs to support their clinical research.

Regulation of norepinephrine in the medial preoptic area of Siberian hamsters by gonadal steroids.

Photoperiod has profound effects upon the neuroendocrine axis underlying reproductive physiology in seasonally breeding mammals. For long-day (LD) breeders, such as the Siberian hamster, exposure to a short-day (SD) photoperiod results in declines in circulating levels of gonadal steroids, luteinizing hormone (LH), and prolactin (PRL). The current study sought to investigate the effects of photoperiod and steroid levels on norepinephrine (NE), one of the major neurochemical regulators of gonadotropin-releasing hormone (GnRH) function. Since NE release within the medial preoptic area (mPOA) has been shown to stimulate the activity of GnRH cells, it was hypothesized that exposure to a short photoperiod would decrease NE levels. Furthermore, since gonadal steroids show negative feedback on GnRH function, it was hypothesized that gonadectomy would result in increased levels of NE. Adult male and female Siberian hamsters were gonadectomized and implanted with silastic capsules containing either cholesterol (C) or a mixture of estradiol (E) or testosterone (T). Microdialysis sampling within the mPOA was conducted after 8 weeks of exposure to either an LD or an SD photoperiod. Blood samples were analyzed for LH and PRL, while dialysis samples were analyzed for NE and its major metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG). The results revealed significant suppression of LH and PRL by exposure to the SD photoperiod in both males and females. For LH, the steroid implants suppressed circulating hormone levels under both photoperiods, whereas for PRL, steroid treatment facilitated circulating levels. In contrast, there were no significant effects of photoperiod on NE or MHPG release for either males or females, but there was a significant decrease in extracellular levels of these neurochemicals in steroid-treated animals. These data suggest that photoperiodic modulation of GnRH neuronal function by NE is achieved largely through the indirect effects of photoperiod on circulating gonadal steroids.

What do radiographers think about teleradiology in Japan?

In order to understand radiographers' views of teleradiology, we sent questionnaires to all radiographers in Hokkaido, Japan. Questions concerned the understanding of, interest in, need for and problems related to teleradiology. A total of 1275 radiographers responded to the survey (a response rate of 65%). Almost all had heard about teleradiology and about 60% of them were interested in it. However, fewer radiographers working in the central region than in other regions expected to be involved in teleradiology. If teleradiology were to be introduced, 60% of the respondents thought that it should be used for interpreting difficult cases and 30% for emergency cases. Half thought that the system should be managed and operated by the radiographer. Thirty-seven per cent of radiographers expected that there would be problems concerning the management of the system within one facility and between facilities, and 34% predicted problems with the installation costs.

Hematopoietic progenitor cell counts performed by the Sysmex SE-9000 analyzer can guide timing of peripheral blood stem cell harvest.

BACKGROUND: We studied whether the hematopoietic progenitor cell (HPC) count in peripheral blood as evaluated by an automated counter, the Sysmex SE-9000, correlated with CD34 positive (+) cell count and therefore could guide the timing of peripheral blood stem cell (PBSC) harvest. MATERIALS AND METHODS: HPC count and flow cytometric CD34+ cell count were measured in 90 peripheral blood samples and 30PBSC samples. The correlation between HPC count and apheretic CD34+ cell yield was examined in 19 patients. RESULTS: HPC count showed good correlations with CD34+ cell count in peripheral blood (r = 0.699) and PBSC (r = 0.892). The correlation between peripheral blood HPC count and apheretic CD34+ cell yield also was good (r = 0.789). CONCLUSION: Automated HPC counting can be used as a screening test to guide the timing of PBSC harvest.

Ammonia response to constant exercise: differences to the lactate response.

1. We evaluated the plasma ammonia response to constant exercise at different intensities. Ten healthy male volunteers were asked to perform constant exercise for 15 min at five different intensities: 80, 90, 100, 110 and 120% of their ventilatory threshold (VT). Blood concentrations of lactate, ammonia and hypoxanthine were measured during and after exercise. 2. The concentration of lactate increased continuously during exercise intensities equivalent to 100, 110 and 120% VT. Plasma ammonia began to increase at 6 min exercise and continued increasing during exercise at all five exercise intensities. Plasma hypoxanthine levels also increased continuously during exercise at all exercise intensities; however, they peaked at 5-10 min after exercise. The response of plasma ammonia and hypoxanthine increased with increasing intensities of exercise. 3. While the extent of the increase in lactate levels during exercise at 100, 110 and 120% VT was significantly higher than that at 80% VT, only the increase in ammonia and hypoxanthine levels at 120% VT were significantly higher than those at 80% VT. 4. In conclusion, the plasma ammonia response to constant exercise differed to the lactate and ammonia responses to short-term exhaustive exercise.

[Icteric-type hepatoma with liver cirrhosis successfully treated with hepatectomy after biliary drainage: case report].

A 67-year-old man with liver cirrhosis and an icteric-type hepatoma involving the left main portal vein underwent left hepatic lobectomy after percutaneous transhepatic biliary drainage. Surgery was successful because of effective biliary drainage and meticulous assessment of liver function tests, including 99mTC-galactosyl human serum albumin scintiphotography.

Left hepatic trisegmentectomy for intraductal papillary cholangiocarcinoma: report of a case.

We present a rare case of intraductal papillary cholangiocarcinoma in a 69 year-old man which was treated with left hepatic trisegmentectomy. The hepatic bile ducts were dilated by intraductal masses, which had extended into the intrahepatic bile ducts without involvement of the posterior inferior segmental duct (B6). The patient underwent left hepatic trisegmentectomy with hilar duct resection. The tumors in the posterior superior segmental duct (B7) were resected and biliary reconstruction was performed with a jejunal loop. Post-operative recovery was good, and the patient survived for 7 months after surgery.

Influence of superstition on the date of hospital discharge and medical cost in Japan: retrospective and descriptive study.

OBJECTIVES: To determine the influence of superstition about Taian (a lucky day)-Butsumetsu (an unlucky day) on decision to leave hospital. To estimate the costs of the effect of this superstition. DESIGN: Retrospective and descriptive study. SETTING: University hospital in Kyoto, Japan. SUBJECTS: Patients who were discharged alive from Kyoto University Hospital from 1 April 1992 to 31 March 1995. MAIN OUTCOME MEASURES: Mean number, age, and hospital stay of patients discharged on each day of six day cycle. RESULTS: The mean number, age, and hospital stay of discharged patients were highest on Taian and lowest on Butsumetsu (25.8 v 19.3 patients/day, P=0.0001; 43.9 v 41.4 years, P=0.0001; and 43.1 v 33.3 days, P=0.0001 respectively). The effect of this difference on the hospital's costs was estimated to be 7.4 million yen (¿31 000). CONCLUSION: The superstition influenced the decision to leave hospital, contributing to higher medical care costs in Japan. Although hospital stays need to be kept as short as possible to minimise costs, doctors should not ignore the possible psychological effects on patients' health caused by dismissing the superstition.

CYP21 pseudogene transcripts are much less abundant than those from the active gene in normal human adrenocortical cells under various conditions in culture.

The human steroid 21-hydroxylase pseudogene (CYP21P, also termed CYP21A) is transcribed in the adrenal cortex, but the relative abundance of transcripts from CYP21P and from the active CYP21 gene (also termed CYP21B) is not well established. In the present experiments we cultured primary human adrenocortical cells in defined medium and used RNase protection assays to examine whether there might be a selective increase in the relative abundance of CYP21P transcripts under any of the various regulatory factors known to affect expression of 21-hydroxylase. Differences between the sequences of intron 2 in CYP21P and CYP21 allowed the synthesis of gene-specific probes spanning exon 3 and parts of the adjacent introns. CYP21- and CYP21P-specific probes spanning the site of the start of transcription were also synthesized. CYP21 transcripts were readily detectable. In agreement with previous observations on 21-hydroxylase mRNA and enzyme activity in primary cultures of human adrenocortical cells, the abundance of CYP21 transcripts was increased by cyclic AMP analogues (N6-monobutyryl cyclic AMP and 8-bromo cyclic AMP), insulin, IGF-I and tetradecanoyl phorbol acetate (TPA). However, CYP21P transcripts were not detected in the presence of any of the various regulatory factors known to affect expression of 21-hydroxylase.

Effects of thyroid hormone (thyroxine) and testosterone on hepatic 11beta-hydroxysteroid dehydrogenase mRNA and activity in pubertal hypothyroid male rats.

To investigate the effects of thyroid hormone and testosterone on 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), we measured changes in hepatic 11beta-dehydrogenase activity and its mRNA levels in pubertal methimazole (MMI)-induced hypothyroid male rats following treatment with thyroxine ([T4] 50 microg/kg/d) or testosterone (250 microg/d) for 14 days. Hypothyroidism in male rats markedly reduced hepatic 11beta-HSD1 mRNA levels and serum testosterone concentrations (P < .01). Subcutaneous injection of T4 in the hypothyroid rats significantly (P < .01) increased hepatic 11beta-HSD1 mRNA to approximately normal levels and simultaneously increased serum testosterone levels. However, the same daily dose of T4 administered to castrated male hypothyroid rats for 14 days did not elevate hepatic 11beta-HSD1 activity. Treatment with testosterone for 14 days in castrated hypothyroid male rats and rats without gonadectomy significantly (P < .01) increased the enzyme activity without administration of T4. Variations in hepatic 11beta-HSD1 activity were demonstrated to be accompanied by changes in serum testosterone levels in the rats following alteration of the thyroid hormone state. These results suggest that the effect of T4 in increasing the subnormal 11beta-HSD1 gene expression in hypothyroid male rats is mediated by its ability to increase testosterone production in these rats, because in castrated hypothyroid rats, T4 does not elevate 11beta-HSD1 gene expression.

Induction of steroidogenic enzyme genes by insulin and IGF-I in cultured adult human adrenocortical cells.

Insulin and the insulin-like growth factors (IGFs) have multiple role in gene expression in steroidogenic cells. We investigated the regulation of steroidogenic enzyme gene expression by insulin and IGF-I in primary cultures of human adrenocortical cells from donors of ages 19-77 years. The effects of insulin and IGF-I observed here were independent of age and sex of the donor. After 5 days in serum-containing medium, cultures were exposed to insulin or IGF-I together with cyclic AMP analogs or ACTH in serum-free defined medium. Insulin and IGF-I at physiological concentrations increased mRNA levels for 17 alpha-hydroxylase and type II 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the absence of cyclic AMP or ACTH. They had lesser effects on 21-hydroxylase and cholesterol side-chain cleavage enzyme mRNA levels and were3 without effect on 11 beta-hydroxylase mRNA. All steroidogenic enzyme mRNAs were strongly increased by cyclic AMP or ACTH, and this increase was potentiated by insulin or IGF-I. These effects of insulin and IGF-I were accompanied by decreases in the ratio of dehydroepiandrosterone/cortisol synthesized from pregnenolone by the cultures. Induction of steroidogenic enzyme genes in adult human adrenocortical cells by insulin and IGF-I is unlikely to occur by means of a cyclic AMP-dependent mechanism. These data increase the evidence for an important regulation of steroidogenesis by these hormones.

The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex resulting from its low expression of 3 beta-hydroxysteroid dehydrogenase.

Based on indirect evidence, it has often been assumed that the zona reticularis of the adult human adrenal cortex is the source of the adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), but direct tests of this concept have been few. Using the techniques of cell culture, Northern blotting, and RIA, we compared the properties of separated adult zonal cells to those of fetal zone cells, a cell type well known to secrete large amounts of DHEA(S) due to its low expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In nine glands from donors of a wide age range, the zona fasciculata and zona reticularis were separated and dissociated, and the cells were placed in culture. After 5 days, serum was removed by a 24-h period in serum-free defined medium followed by a 24-h exposure to cAMP analogs, with the optional addition of insulin, also in serum-free medium. The separated fasciculata and reticularis cells showed large differences in the DHEA(S)/cortisol (F) production ratios from added pregnenolone precursor, consistent with the synthesis of only F and essentially no DHEA(S) by fasciculata cells and with the synthesis of mostly DHEA(S) with little or no F by both reticularis cells and fetal zone cells. The different patterns of steroidogenesis were accompanied by a much lower level of expression of type II 3 beta HSD in reticularis cells, similar to that in fetal zone cells. In contrast, other genes were similarly regulated in the two adult zones and in the fetal zone by both cAMP and insulin. The levels of messenger ribonucleic acids for 17 alpha-hydroxylase, cholesterol side-chain cleavage enzyme, 21-hydroxylase, and 11 beta-hydroxylase responded to cAMP and insulin in both reticularis cells and fetal zone cells in the same pattern as that previously established in fasciculata cells. The central role of the limited expression of 3 beta HSD in the DHEA(S)-synthesizing property of reticularis cells was established by inhibition of 3 beta HSD in fasciculata cells with trilostane, which caused them to increase their DHEA/F production ratio to a level exceeding even that in fetal zone cells. There did not appear to any age-related changes in gene expression that could account for the large age-related decline in DHEA(S) biosynthesis in humans in either reticularis or fasciculata cells. Thus, the most likely cause of the age-related decline in adrenal androgen biosynthesis is an age-related decline in the number of functional reticularis cells, without a major change in the differentiated properties of the zonal cells as a function of age.

Dual regulation of 21-hydroxylase activity by sex steroid hormones in rat hepatocytes.

In the rat liver, cytochrome P450 catalyzes the hydroxylation of steroid hormones. The expression and activity of some P450 isozymes are regulated by sex steroid hormones. Steroid 21-hydroxylase activity in rat liver is provided mainly by CYP2C6. We studied the regulation of 21-hydroxylase activity by sex steroid hormones in rat primary hepatocyte culture. We added estrogens (estrone, estradiol, estriol) and androgens (testosterone, dihydrotestosterone), (ranging from 10(-9) to 10(-5)M) to the culture. The 21-hydroxylase activity was stimulated by estrogens and was suppressed slightly by androgen in a dose-related manner. The results of our studies demonstrated that sex steroid hormones act differently on 21-hydroxylase activity in rat hepatocytes and, thus, support the hypothesis that the extra-adrenal production of deoxycorticosterone from circulating progesterone is increased during pregnancy by the massive presence of estrogens.