Prevention of breast tumour development in vivo by downregulation of the p185neu receptor.

Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.

T cell receptor-mediated recognition of self-ligand induces signaling in immature thymocytes before negative selection.

Shaping of the T cell repertoire by selection during intrathymic maturation involves T cell receptor (TCR) recognition of major histocompatibility complex/self-antigen complexes. In this communication, we studied the ability of minor lymphocyte stimulating (Mls) determinants to act as self-tolerogens in the selection of the T cell repertoire. We demonstrate that unprimed T cells from normal as well as TCR transgenic mice form Mls-specific conjugates with antigen-presenting cells, and that this TCR-ligand interaction leads to elevation of intercellular Ca2+ ([Ca2+]i). Peripheral T cells from TCR transgenic mice expressing receptors specific for self-Mls antigen show no reactivities to Mlsa. However, a proportion of immature thymocytes from these mice show specific binding and strong [Ca2+]i elevation in response to self-antigen-presenting cells, although these thymocytes do not proliferate. This self-reactivity of thymocytes is inhibited by antibodies specific for TCR, CD4, CD8, class II molecules, lymphocyte function-associated antigen 1, and intercellular adhesion molecule 1. These results demonstrate for the first time that before thymic negative selection, immature T cells can specifically interact with cells bearing self-antigen, and suggest that the resulting TCR-dependent signal transduction events provide a basis for negative selection of self-reactive T cells.

T cell receptor-induced calcineurin activation regulates T helper type 2 cell development by modifying the interleukin 4 receptor signaling complex.

The activation of downstream signaling pathways of both T cell receptor (TCR) and interleukin 4 receptor (IL-4R) is essential for T helper type 2 (Th2) cell development, which is central to understanding immune responses against helminthic parasites and in allergic and autoimmune diseases. However, little is known about how these two distinct signaling pathways cooperate with each other to induce Th2 cells. Here, we show that successful Th2 cell development depends on the effectiveness of TCR-induced activation of calcineurin. An inhibitor of calcineurin activation, FK506, inhibited the in vitro anti-TCR-induced Th2 cell generation in a dose-dependent manner. Furthermore, the development of Th2 cells was significantly impaired in naive T cells from dominant-negative calcineurin Aalpha transgenic mice, whereas that of Th1 cells was less affected. Efficient calcineurin activation in naive T cells upregulated Janus kinase (Jak)3 transcription and the amount of protein. The generation of Th2 cells induced in vitro by anti-TCR stimulation was inhibited significantly by the presence of Jak3 antisense oligonucleotides, suggesting that the Jak3 upregulation is an important event for the Th2 cell development. Interestingly, signal transducer and activator of transcription (STAT)5 became physically and functionally associated with the IL-4R in the anti-TCR-activated developing Th2 cells that received efficient calcineurin activation, and also in established cloned Th2 cells. In either cell population, the inhibition of STAT5 activation resulted in a diminished IL-4-induced proliferation. Moreover, our results suggest that IL-4-induced STAT5 activation is required for the expansion process of developing Th2 cells. Thus, Th2 cell development is controlled by TCR-mediated activation of the Ca(2+)/calcineurin pathway, at least in part, by modifying the functional structure of the IL-4R signaling complex.

A day trip to a forest park increases human natural killer activity and the expression of anti-cancer proteins in male subjects.

We previously reported that 2-night/3-day trips to forest parks enhanced human NK activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that this increased NK activity lasted for more than 7 days after the trip in both male and female subjects. In the present study, we investigated the effect of a day trip to a forest park on human NK activity in male subjects. Twelve healthy male subjects, aged 35-53 years, were selected after giving informed consent. The subjects experienced a day trip to a forest park in the suburbs of Tokyo. They walked for two hours in the morning and afternoon, respectively, in the forest park on Sunday. Blood and urine were sampled in the morning of the following day and 7 days after the trip, and the NK activity, numbers of NK and T cells, and granulysin, perforin, and granzyme A/B-expressing lymphocytes, the concentration of cortisol in blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trip on a weekend day as the control. Phytoncide concentrations in the forest were measured. The day trip to the forest park significantly increased NK activity and the numbers of CD16(+) and CD56(+) NK cells, perforin, granulysin, and granzyme A/B-expressing NK cells and significantly decreased CD4(+) T cells, the concentrations of cortisol in the blood and adrenaline in urine. The increased NK activity lasted for 7 days after the trip. Phytoncides, such as isoprene, alpha-pinene, and beta-pinene, were detected in the forest air. These findings indicate that the day trip to the forest park also increased the NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted for at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone levels may partially contribute to the increased NK activity.

Diphenylhydantoin: an alternative ligand of a glucocorticoid receptor affecting prostaglandin generation in A/J mice.

Evidence for the binding of 5,5-diphenylhydantoin and glucocorticoids to a common receptor is presented for pulmonary and hepatic cytosols and thymocytes of A/J female mice. The 5,5-diphenylhydantoin-protein complex is absorbed by DNA cellulose, and is incorporated into nuclei, 5,5-Diphenylhydantoin, like glucocorticoids, inhibits the production of prostaglandins in thymocytes. Thus a common receptor is probably responsible for the inhibitory and teratogenic effects of these drugs.

Effect of phytoncide from trees on human natural killer cell function.

We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37-60 years, who stayed at an urban hotel for 3 nights from 7.00 p.m. to 8.00 a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme A/B-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme A/B-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.

Effects of orally administered glycine on myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle in chicks.

We examined the effects of orally administered glycine on myofibrillar proteolysis in food-deprived chicks. Food-deprived (24 h) chicks were orally administered 57, 113, and 225 mg glycine/100 g body weight and killed after 2 h. The plasma N(tau)-methylhistidine concentration, used as myofibrillar proteolysis, was decreased by glycine. We also examined the expression of proteolytic-related genes by real-time PCR of cDNA from chick skeletal muscles. The mRNA expression of atrogin-1/MAFbx, proteasome C2 subunit, m-calpain large subunit, and cathepsin B was decreased by glycine in a dose-dependent manner. The plasma corticosterone concentration was also decreased by glycine, but the plasma insulin concentration was unaffected. These results indicate that orally administered glycine suppresses myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle by decreasing the plasma corticosterone concentration in chicks.

Visiting a forest, but not a city, increases human natural killer activity and expression of anti-cancer proteins.

We previously reported that a forest bathing trip enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes. In the present study, we investigated how long the increased NK activity lasts and compared the effect of a forest bathing trip on NK activity with a trip to places in a city without forests. Twelve healthy male subjects, age 35-56 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields and to a city, in which activity levels during both trips were matched. On day 1, subjects walked for two hours in the afternoon in a forest field; and on day 2, they walked for two hours in the morning and afternoon, respectively, in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood samples and completing the questionnaire. Blood and urine were sampled on the second and third days during the trips, and on days 7 and 30 after the trip, and NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trips on a normal working day as the control. Phytoncide concentrations in forest and city air were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzyme A/B-expressing cells and significantly decreased the concentration of adrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. In contrast, a city tourist visit did not increase NK activity, numbers of NK cells, nor the expression of selected intracellular anti-cancer proteins, and did not decrease the concentration of adrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air, but almost not in city air. These findings indicate that a forest bathing trip increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone may partially contribute to the increased NK activity.

A forest bathing trip increases human natural killer activity and expression of anti-cancer proteins in female subjects.

We previously reported that forest bathing trips enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after the trip in male subjects. In the present study, we investigated the effect of forest bathing trip on human NK activity in female subjects. Thirteen healthy nurses, age 25-43 years, professional career 4-18 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields. On day 1, the subjects walked for two hours in the afternoon in a forest field; on day 2, they walked for two hours each in the morning and afternoon in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood and completing a questionnaire. Blood and urine were sampled on the second and third days during the trip, and on days 7 and 30 after the trip. NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, the concentrations of estradiol and progesterone in serum, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the trip on a normal working day. The concentrations of phytoncides in the forests were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air. These findings indicate that a forest bathing trip also increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins in female subjects, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone levels may partially contribute to the increased NK activity.

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Forest bathing enhances human natural killer activity and expression of anti-cancer proteins.

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

Expression of bcl-2 in small cell lung carcinoma cells.

We have investigated the expression of bcl-2 protein in a panel of small cell lung carcinoma (SCLC) cells lines. Five of six cell lines examined expressed relatively high levels of bcl-2 protein and transcripts. The bcl-2 expression in SCLC cells, however, was not always associated with myc expression. Since dysregulation of bcl-2 may be involved in the course of tumorigenesis and/or in the acquisition of drug resistance of tumor cells, the expression of bcl-2 in SCLC cells may become an important indicator in the prognosis or treatment of SCLC.

Moloney murine leukemia virus infection accelerates lymphomagenesis in E mu-bcl-2 transgenic mice.

E mu-bcl-2 transgenic mice bearing the bcl-2 proto-oncogene linked to the immunoglobulin enhancer (E mu) sporadically develop B or T cell lymphomas after a long latent period. To identify genes that play important roles in development of lymphoid malignancies, proviral insertional mutagenesis with Moloney murine leukemia virus (MMuLV) was carried out in two lines of transgenic mice expressing the bcl-2 gene primarily in B or T cells. MMuLV infection of non-transgenic mice induced primarily mature T cell lymphomas. By contrast, infection of newborn E mu-blc-2 mice with the virus accelerated lymphomagenesis, and nearly all of the mice eventually succumbed to clonal pre-B, B, or mainly immature T cell lymphoma, indicating the active contribution of the bcl-2 gene in lymphomagenesis. Southern blot analysis of tumor DNA from MMuLV-infected transgenic mice revealed a proviral insertion at the c-myc gene in 26% (9/35) of tumors, at the pim-1 gene in 6% (2/35) and at the pim-2 (recently renamed tic-1) gene in 23% (8/35). Some tumors carried two activated oncogenes. No insertion was detected at the bmi-1 gene. These data suggest the usefulness of this transgenic system for analysis of lymphomagenesis involving the activated bcl-2 gene.

An H-2 linked difference in the binding of dexamethasone to murine hepatic cytosol receptor. A possible role of endogenous modifier(s).

The binding of dexamethasone to its receptor in hepatic cytosol preparations from pregnant mice of four congenic and recombinant strains, C57BL/10, B10.A, B10.A(2R) and B10.A(5R), which have almost identical genetic backgrounds other than the H-2 complex, on day 12 of gestation was analyzed by plotting the binding of ligand against cytosol concentration. The plots of C57BL/10 and B10.A(5R) mice were straight lines, but those of the strains B10.A and B10.A(2R) were upward concave curves. The curvature probably did not result from denaturation of the receptor, as indicated by the time course of the dexamethasone binding and by the fact that at a lower concentration of the ligand, at which the receptor would be less stable, there was less curvature than at a higher concentration of the ligand. The curvature can be explained by the presence of endogenous modifier(s) using an analogy from enzymology. Mathematical analysis, partial removal of the modifier(s) by gel filtration, and mixing of the cytosols from the two types of strains indicated the presence of an unsaturated amount of a modifier(s) in the cytosol of the B10.A and B10.A(2R) strains, and the presence of a saturated amount of the cytosol of the C57BL/10 and B10.A(5R) strains. Thus, the H-2 complex contains a gene(s) which regulates the level of a modifier(s) in hepatic cytosol which affects the binding of glucocorticoid to its hepatic cytosolic receptor.

Prepubertal diagnosis of steroid 5 alpha-reductase deficiency.

The diagnosis of 5 alpha-reductase deficiency was proven in two prepubertal patients with male pseudohermaphroditism (MPH). Both had a 46-XY karyotype and were reared as females; one child had been castrated in infancy. Clitoromegaly, urogenital sinus, and short vaginal pouch were present in both; inguinal gonads were palpable in one. The diagnosis was made biochemically by observing characteristic changes in five parameters: 1) abnormally high testosterone to dihydrotestosterone (T:DHT) ratio after hCG stimulation (35 and 53 vs. normal, 11 +/- 3), 2) abnormally high 5 beta-T metabolites in urine (8.1 and 6.0 vs. normal, less than 1),3) deficient conversion of T to DHT during [3H] T infusion (0.3 and 0.4% vs. normal, 5.3 +/- 3), 4) deficient conversion of [14 C] T to 5 alpha-reduced metabolites by nongenital skin fibroblasts (2.2 and 1.9 pmol/microgram DNA/nmol substrate vs. 68.4+/- 7.8 Pmol/microgram DNA/nmol substrate in normal controls), and 5) deficient conversion of [14C]T to DHT in genital skin slices. The fact that this syndrome represents a defect in T metabolism rather in in T binding is demonstrated by the observation that binding of [3H]DHT to cytosol of skin fibroblasts was normal (4.2 dpm/micrograms DNA vs. normal male values of 3.7 +/- 0.64). Thus, the present report suggests that 5 alpha-reductase deficiency can be diagnosed during childhood and even after castration by metabolic studies of nongenital skin fibroblasts and determination of the conversion ratio of [3H]T to [3H]DHT in plasma.

T-cell receptor and autoimmune disease.

Since the genes encoding the TCR have been cloned, their structure, organization, pattern of rearrangement, diversification and expression in ontogeny have been classified. However, there are still many important questions to be addressed, such as the nature of thymic education, tolerance, the mechanism of MHC-restricted antigen recognition and the relation between TCR repertoire and autoimmunity. In the future, new approaches to study these issues, such as transgenic mice, X-ray crystallography, and severe combined immune deficiency mice reconstituted with human hematopoietic cells will lead to a more profound understanding of these questions. This will hopefully allow us to manipulate the immune response in different and more effective ways than are currently available.

Relationship between bcl-2 and myc gene expression in human neuroblastoma.

We have investigated the relationship between the expression of bcl-2 and myc family genes in primary human neuroblastoma (NB) tumors and cell lines. Of 20 NB tumors examined, bcl-2 transcripts were expressed at variable levels in 16 tumors of all clinical stages. Of the 2 tumors with N-myc amplification, one expressed bcl-2 at a high level, whereas the other did so at a low level. In contrast, all NB tumors showed the expression of c-myc and/or N-myc transcripts. Similarly, of 9 NB cell lines with N-myc amplification examined, 6 expressed bcl-2 at high levels, whereas the other 3 expressed it at barely detectable levels. The 3 cell lines without N-myc amplification also expressed bcl-2 protein at high levels. All NB cell lines tested expressed either c-myc or N-myc protein. These data suggest that in NB, there is no significant association between bcl-2 expression and advanced tumor stages or N-myc amplification. The data also show that bcl-2 expression does not always coincide with myc expression in NB, suggesting that bcl-2- independent mechanisms may exist in the bcl-2-negative NB tumor cells in order to suppress the cell death promoting action of high myc expression.

Inhibitory action of chloramine on formate-metabolizing system. Studies suggested by an unusual case record.

We previously reported on a patient exposed simultaneously to methyl chloride and chloramine gas who developed metabolic acidosis and permanent blindness [M. Minami et al., Hum Exp Toxicol 11: 27-34, 1992]. The case report suggested the possibility of potentiation of methyl chloride toxicity by chloramine. The potentiating mechanism was investigated by exposing mice to methyl chloride followed by ammonia chloramine, and then the level of formate in urine samples was measured with an enzyme coupling method to detect disturbance of formate metabolism. Mice dosed with 0.05 mL 1.0 mM chloramine after methyl chloride exposure excreted a significantly larger amount of urinary formate than mice treated with only methyl chloride. There was no difference in urinary formate levels between mice treated with only 0.05 mL 1.0 mM chloramine and those given only the vehicle (0.1 M phosphate buffer pH 6.0) for chloramine. The underlying biochemical mechanism of deterioration of formate metabolism was found to be the inhibition of the enzyme, N10-formyl tetrahydrofolate (N10-f-THF) dehydrogenase by 0.56-3.35 microM chloramine in the in vitro experiment using the purified enzyme. Positive control mice, given orally 0.1 mL 10% methanol in 0.1 M phosphate buffer (pH 6.0) excreted the same amount of urinary formate as those receiving 0.05 mL 1.0 mM chloramine after methanol administration. This was ascribed to the inhibitory effect of chloramine on formaldehyde dehydrogenase and depletion of substrate for further metabolism. The inhibition of the enzyme by chloramine (2.7-100.8 microM) was confirmed by in vitro experiments, using the purified enzyme, formaldehyde dehydrogenase.

Elevated glucocorticoid receptor levels in lymphocytes of children with the fetal hydantoin syndrome (FHS).

Our recent studies of the teratogenic mechanisms of phenytoin (DPH) and glucocorticoids in mice have indicated that DPH utilizes the anti-inflammatory pathway of glucocorticoids in producing congenital defects, such as cleft palate. This pathway is influenced by H-2 and H-3 histocompatibility-linked genes in the mouse, such that congenic strains have H-2 or H-3 alleles that confer susceptibility to DPH-induced congenital defects, and susceptible H-2 congenic strains have high glucocorticoid receptor levels. However, other H-2 or H-3 alleles confer resistance to these defects in their otherwise genetically identical congenic partner strains, and "resistant" H-2 alleles are associated with low levels of these receptors. To determine whether this animal work is applicable to the human, we have sought to investigate whether the level of glucocorticoid receptors in circulating lymphocytes of children with the fetal hydantoin syndrome (FHS) is as it is in the animals. We found that children with FHS had glucocorticoid receptor levels significantly elevated above those of unaffected children with similar DPH exposure in control families. The receptor level of affected children was also significantly elevated above that of fathers of children with the FHS and of fathers and mothers of control children. These findings are consistent with those documented in the animal models and suggest that an elevated level of glucocorticoid receptors in lymphocytes may be a marker for susceptibility to the FHS syndrome.