Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas.

We recently reported the cloning of the REIC/Dkk-3 gene, whose expression was shown to be down-regulated in many human immortalized and tumor-derived cell lines [T. Tsuji et al. (2000) Biochem. Biophys. Res. Commun. 268, 20-24]. In the present study, we demonstrated that expression of the exogenous REIC/Dkk-3 gene in tumor cells inhibited cell growth. Furthermore, the level of REIC/Dkk-3 mRNA in normal human cells was lowest in the late G(1) phase during the cell cycle. Then we found that the expression of REIC/Dkk-3 was significantly down-regulated in surgically resected non-small-cell lung carcinomas. We determined the REIC/Dkk-3 locus on chromosome 11p15, where loss of heterozygosity has frequently been observed in human tumors. These findings indicate that REIC/Dkk-3 may function as a tumor suppressor.

[Changes of serum hepatocyte growth factor in coronary artery disease].

Hepatocyte growth factor (HGF) is an endothelial cell specific growth factor involved in the repair of endothelial cells and collateral formation, however, the role for coronary artery disease is still unknown. We measured serum HGF level in various coronary artery diseases to examine the clinical significance. Serum HGF level was measured using the enzyme-linked immunosorbent assay method in patients with stable effort angina pectoris (n = 26), old myocardial infarction (n = 18), unstable angina pectoris (UAP; n = 10) and acute myocardial infarction (AMI; n = 21). As a control group, we selected 11 patients with neurocirculatory asthenia. Blood samples from peripheral veins were collected at cardiac catheterization before heparin administration. In the AMI group, blood samples were also collected at 48, 72 hr, 1, 2, 3 and 4 weeks from the peripheral veins and 48 and 72 hr after reperfusion from the coronary sinus. Serum HGF level was significantly higher in the UAP (0.41 +/- 0.12 ng/ml, p < 0.001) and AMI groups (0.38 +/- 0.26 ng/ml, p < 0.05) compared to the control group (0.19 +/- 0.09 ng/ml). Serum HGF level peaked 48 hr after reperfusion in both the peripheral veins (0.42 +/- 0.16 ng/ml) and coronary sinus (0.58 +/- 0.23 ng/ml) in the AMI group, with a significantly higher level in the coronary sinus than the peripheral veins (p < 0.05). No significant correlation between peak HGF level in the peripheral veins and peak creatine kinase (CK), CK-MB, ejection fraction and cardiac index was observed. Serum HGF was elevated in acute coronary syndrome, indicating advanced endothelial cell damage. HGF is produced, at least partially, in the heart in patients with AMI. Serum HGF level may be useful to detect endothelial cell damage rather than myocardial cell damage.

Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma.

We have established a mouse model for scleroderma induced by repeated local injections of bleomycin (BLM). Daily injection of BLM at a dose of >10 microg per ml for 4 wk induced histologic changes of dermal sclerosis, but not fibrosis, with thickened and homogenous collagen bundles and cellular infiltrates in BALB/C mice, whereas clinical signs of scleroderma were not apparent. In addition, lung fibrosis was also induced preceding the cutaneous changes. Sclerotic changes were not found in other sites of the skin distant from the injection site. Dermal sclerosis could also be induced by injecting BLM only every other day. The sclerotic changes of the dermis were sustained after ceasing BLM applications for at least 6 wk. Mast cells gradually increased in number as the sclerotic changes developed. Marked degranulation of mast cells was observed with elevated histamine release. The amount of hydroxyproline in skin was significantly increased at 4 wk of BLM treatment as compared with that in untreated or phosphate-buffered saline-treated mice. Anti-nuclear antibody was detected in serum of BLM-treated mice. Transforming growth factor-beta1 mRNA was detected at an early phase, while transforming growth factor-beta2 mRNA was strongly expressed at 4 wk when the sclerotic features were prominent. These results suggest that dermal sclerosis induced by BLM closely resembles systemic sclerosis both histologically and biochemically. Our mouse model can provide a powerful tool of inducing dermal sclerosis to examine the pathogenesis and the therapeutic approach of scleroderma.

Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Tec is a non-receptor type tyrosine kinase which is tyrosine phosphorylated and activated upon stimulation of hematopoietic cells with various cytokines. The role of Tec in G protein-coupled receptor- and integrin-mediated signalings has not been elucidated. We therefore investigated the regulation of Tec in human blood platelets. Tec was rapidly tyrosine phosphorylated in response to platelet agonists which activate G protein-coupled receptors such as thromboxane A2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was significantly reduced under the conditions which abrogate fibrinogen binding to GP IIb-IIIa and subsequent platelet aggregation. However, TRAP induced significant levels of the phosphorylation even under these conditions and also in thrombasthenic platelets which lack functional GP IIb-IIIa molecules, suggesting that activation of G-protein-coupled receptor causes the phosphorylation. To clarify whether integrin engagement by itself causes the phosphorylation in Tec, we examined the state of the phosphorylation in platelets activated by integrin engagement. Platelet adhesion to immobilized fibrinogen or collagen induced significant levels of the phosphorylation. Furthermore, Tec was translocated to cytoskeleton in response to TRAP in a manner dependent on platelet aggregation, suggesting that Tec can be a component of integrin-mediated signalings. These results collectively indicate that Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Chronic myelogenous leukemia with a hypooctoploid cell population.

A patient with chronic myelogenous leukemia who underwent transformation from the accelerated phase to the chronic phase with interferon-alpha treatment is reported. On admission, the numbers of myeloblasts and promyelocytes in the peripheral blood and bone marrow were 7.0% and 12.8%, respectively. The platelet count was 633.6 x 10(4)/microliter, and megakaryocytes were frequent in the bone marrow. In the accelerated phase, cytogenetic analyses revealed a hypotetraploid and a hypooctoploid cell population with two and four Philadelphia chromosomes, respectively, in the bone marrow specimen. The hypooctoploid clone disappeared with administration of interferon-alpha and the hypotetraploid clone also subsequently disappeared.

Modulation of platelet activation in vitro by thrombopoietin.

Effect of human recombinant thrombopoietin (TPO) on platelet activation in vitro was studied. Although TPO itself did not cause platelet aggregation, it upregulated ADP-induced aggregation, especially the second wave of aggregation. This effect was dose-dependent for up to 5 ng/ml of TPO. When platelets were activated by epinephrine, collagen, or alpha-thrombin, similar effect was observed. However, TPO did not affect A23187- or PMA-induced aggregation, suggesting that TPO may have modulated the signal transduction pathway upstream of inositol 1,4,5-trisphosphate and diacylglycerol production. TPO also upregulated thrombin-induced alpha-granule secretion. To clarify the involvement of protein tyrosine phosphorylation, platelets were activated by TPO and/or suboptimal concentration of ADP, then tyrosine phosphorylation was detected by immunoblot analysis, using anti-phosphotyrosine monoclonal antibody. TPO by itself caused significant tyrosine phosphorylation of 146, 130, 122, 108, 97, 94, and 88 kDa proteins. Further, by using antibodies against signal transduction molecules for immunoprecipitation, we observed the significant tyrosine phosphorylation in Jak2 and Tyk2 molecules after TPO-stimulation. The results of the present experiment clearly indicate that TPO directly activated platelets and modulated intracellular signal transduction pathway.

Potent cholesterol-lowering effect by human granulocyte-macrophage colony-stimulating factor in rabbits. Possible implications of enhancement of macrophage functions and an increase in mRNA for VLDL receptor.

The mechanism by which granulocyte-macrophage colony-stimulating factor (GM-CSF) lowers plasma cholesterol levels is not well understood. We tested recombinant human GM-CSF (rhGM-CSF) on plasma cholesterol and triglycerides in rabbits and attempted to determine the mechanisms of the cholesterol-lowering effect. rhGM-CSF (20 micrograms.kg-1.d-1) was administered to normal and cholesterol-fed rabbits for 2 weeks and to Watanabe heritable hyperlipidemic (WHHL) rabbits for 1 week. The administration of rhGM-CSF markedly lowered cholesterol and triglycerides, an effect that persisted in normal and cholesterol-fed rabbits even after termination of treatment. The cholesterol-lowering effect of rhGM-CSF was also observed in WHHL rabbits. rhGM-CSF was capable of stimulating granulocyte-macrophage colony formation in vitro in rabbits with an effect comparable to that in humans. Northern blot analysis with rabbit very-low-density-lipoprotein (VLDL) receptor cDNA revealed that rhGM-CSF increased the levels of VLDL receptor mRNA in muscle of rabbits after only 1.5 hours of treatment compared with control (2.6-fold), with the 1.5-fold increase following a 5-day administration. No changes in the levels of LDL receptor mRNA in liver, spleen, and bone marrow were observed in the treated rabbits. These findings suggest that the cholesterol-lowering effect of rhGM-CSF may be mediated by enhancement of macrophage functions in lipid metabolism and the increase in mRNA for VLDL receptor in rabbits.

Coronary spasm in patients with coronary ectasia.

The incidence of coronary ectasia (CE) and the relationship between CE and coronary spasm that was said not to be apt to occur in patients with CE were studied. The study consisted of 1,373 patients (including 1,008 patients with ischemic heart disease) who underwent cardiac catheterization. In 74 patients with CE, coronary spasm was tested in 33 patients subjected to acetylcholine (ACh) provocation and in 17 patients subjected to ergonovine (Ergo) provocation. CE was found in 74 patients (5.4%), and its incidence was significantly higher (6.7%) in patients with ischemic heart disease. In addition, ACh and Ergo provocation tests gave positive results in 12 (36%) and 4 patients (24%), respectively. Spasm was not provoked as frequently in the ectatic portion as elsewhere. In addition, spasm was often found at the borders of the ectatic portion in both provocation tests. The incidence of coronary ectasia was higher than that described in previous reports, and coronary spasm was more often observed at the borders between ectatic and normal portions than elsewhere.

[Effect of REM sleep deprivation on the each seizure stage in feline amygdaloid kindling].

The effect of REM sleep deprivation (RD) on the each seizure stage in feline amygdaloid kindling (AM-K) was studied. RD for 12 hours (12-h RD) was performed by applying the platform procedure at stages 2 and 4, and immediately after RD the triggering threshold of each seizure stage and the duration of after discharge was measured. At stage 6, 12-h and 72-h RD were performed, and the generalized seizure triggering threshold (GST), the duration of after discharge, and the latency to generalized convulsive seizure (LGS) were measured. For the controls, a platform sufficiently large to allow a cat to lie down and sleep was used under the same conditions as those for RD (Non-RD). With the platform procedure employed in this study, the %REM decreased significantly to 2% of what it was before 12-h RD (p < 0.01). After the 12-h RD, the %REM and %S-2 increased significantly compared with those during the RD (p < 0.05-0.01), revealing a rebound phenomenon. After the 12-h RD, the %W-2 also showed a significant decrease (p < 0.05). The results were as follows: (1) The triggering seizure threshold of stage 2 after the 12-h RD showed a significant decrease (p < 0.01). Though the AD duration after RD prolonged than those after Non-RD, significant differences was not noted. (2) The triggering threshold and AD duration of stage 4 after 12-h RD showed no significant change. (3) GST, AD duration, LGS showed no significant change by 12-and 72-h RD. The results of present study led to the following conclusions: At stage 2 of AM-K, REM sleep increased temporarily, and consequently the REM pressure is elevated after RD so much that it lowers the triggering threshold at stage 2 of AM-K. On the other hand, at stages 4 and 6 of AM-K, REM sleep is temporarily decreased, and consequently the REM pressure is not elevated by RD so that the triggering threshold at stages 4 and 6 of AM-K were not lowered.

[Prevalence of anti-human parvovirus antibody in pregnant women].

Human parvovirus B19 (B19) has been shown to be associated with erythema infectiosum. Recently, it was reported that when a pregnant woman is infected with B19, the fetus in her uterus sometimes becomes hydropic and results in a stillbirth. But no epidemiologic study of pregnant women in Japan has been performed yet. We have established an in vitro propagation system of B19 virions and an assay system for detecting anti-B19 antibody by indirect immunofluorescence (IF) staining. We examined the positive rate of anti-B19 antibody among 337 normal blood donors, 329 normal pregnant women, 23 early aborted women and 24 non-immune hydrops fetalis cases by IF staining. The positive rate for anti-B19 IgG among normal blood donors increased with age. It was 22% aged 21 to 30, 44% aged 31 to 40, 65% aged 41 to 50, and 76% aged 51 to 60, respectively. Anti-B19 IgG was detected in 33% of 329 of normal pregnant women. The anti-B19 IgG positive rate was 26% in pregnant women in their twenties and 44% in those in their thirties. There was a significant difference between the two generations. The results show that more than half the women in their twenties and thirties risk B19-infection during pregnancy. Nine of twenty-three early aborted women were positive for anti-B19 antibody. Among six pregnant women who were positive for anti-B19 IgM, four delivered normal babies and the others aborted artificially or spontaneously. Anti-B19 IgG and IgM of twenty four non-immune hydrops fetalis (NIHF) were examined and four cases were found to be anti-B19 IgG positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a medicinal herbal liqueur, "yomeishu", on post-operative gynecological patients.

We administered 20 ml of Yomeishu (YMS) twice a day before meals for 12 weeks to 50 post-operative patients in gynecology and then inquired into their subjective 20 symptoms (sense of fatigue, insomnia, headache and heavy headedness, appetite, stomach-ache, abdominal inflation, vertigo, lumbago, etc.) The YMS group showed a significant improvement on 14 items compared with the control group. On the whole, a great improvement was observed in the YMS group with serious subjective symptoms as well, and there were significant differences for general condition, sense of fatigue, and coldness in extremities.

Histological, immunohistochemical, and chromosomal studies on non-adrenal neuroblastomas without increased excretion of catecholamines and their metabolites in urine.

We reviewed our records for the last 10 years and found three of the 36 patients with neuroblastoma did not excrete significantly increased quantities of catecholamine metabolites in urine. All of these tumors were histologically characterized by small round cells and possessed a few dense core granules on the electron microscopic examination. All of them were reacted with anti-neuron-specific enolase (NSE) antibodies, OKB2, PI153/3 monoclonal antibodies (MoAbs). The HNK-1, BA-1, and SJ-9A4 MoAbs reacted with two out of three. One of them demonstrated a reciprocal translocation involving the short arm deletion of chromosome 1. This multidisciplinary study has been helpful in making more accurate diagnoses for neuroblastoma without classical clinical characters.

[Influence of pregnancy and steroid hormones on interferon production of lymphocytes].

A study was done to investigate the involvement of interferon (IFN) in immunological acceptance of fetal allograft. IFN was induced by stimulating OK-432 from lymphocyte of pregnant and nonpregnant women as well as men. There was no difference in the IFN titer in these groups. In pregnant women there was no difference between trimesters, either. In addition, we examined the influence of sex steroid hormones such as progesterone and estrogen on IFN production by the lymphocytes of women and men. Generally, each steroid hormone suppressed the production of IFN. However, the extent of suppression varied with the concentration of hormones, resulting in different suppression curves for men and women in a physiological concentration. Differences in response by men and women were also noticed when interleukin 2 was added to examine whether it could reduce the suppression by steroid hormone. This unique sensitivity of female lymphocyte to steroid hormones may play a role in successfully taking a fetal allograft in pregnancy.

[Screening for chromosomal anomalies with maternal serum alpha-fetoprotein].

Between July 1, 1986 and January 31, 1988, genetic amniocentesis was performed on 205 patients. The maternal serum alpha-fetoprotein (AFP) and amniotic fluid AFP levels were measured by enzyme immunoassay. Gestational dates were confirmed by sonography, and AFP results were expressed as multiples of the median (MOM). The median of maternal serum AFP from 15 to 17 weeks of gestation was 43.4, 62.6 and 72.5 ng/ml. Three fetuses with chromosomal anomalies were diagnosed; trisomy 21, 4p trisomy, and trisomy 18 (trisomy 18 was in one fetus of a twin pregnancy; the other fetus was normal). Maternal serum AFP levels were, 0.41, 0.49 and 1.30 MOM. Maternal serum AFP less than 0.5 MOM in normal pregnancies was 1/205 (0.5%) and less than 0.6 MOM was 9/205 (4.4%). There was no relationship between maternal serum AFP and amniotic fluid AFP levels. Our results are in agreement with the majority of the results in the literature, showing that maternal serum AFP levels are lower in association with autosomal trisomy fetuses.

Adjuvant immunotherapy: two randomized controlled studies of patients with cervical cancer.

In order to prolong the survival of patients with cervical cancer, adjuvant immunotherapy was undertaken in Japan. The result of two randomized controlled studies using biological response modifiers such as OK-432 or sizofiran are described. The three-year recurrence-free rates of 221 patients in the OK-432 group and 161 patients in the control group were 71.9% and 58.6%, respectively. The intergroup difference was statistically significant. Of all the patients with Stage II or III cancer, time to recurrence and survival rate in 99 patients in the sizofiran group were significantly longer than in 96 patients in the control, evaluated at 5 years. Based on the results of the 2 randomized controlled studies, we conclude that adjuvant immunotherapy is very useful for prolonging the survival of patients with cervical cancer.

Nitroblue tetrazolium reduction by leukocytes in the cervix of pregnant women.

We examined nitroblue tetrazolium reduction activity in leukocytes from cervical mucus as well as peripheral blood from pregnant and nonpregnant women by histochemical methods. More than 90% of the cells examined were polymorphonuclear cells. Nitroblue tetrazolium reduction in polymorphonuclear cells from peripheral venous blood was significantly enhanced in pregnant women compared with that in nonpregnant women. However, there was no difference in activity between pregnancy periods. On the other hand, nitroblue tetrazolium reduction in cervical polymorphonuclear cells excluding women with cervicitis was also greater in pregnant women than in nonpregnant women. Moreover, activity in cervical polymorphonuclear cells gradually increased as pregnancy progressed. This enhanced activity in polymorphonuclear cells may be involved not only in the defense mechanism but also in cervical ripening in pregnancy.

Human cytotrophoblastic antigens defined by monoclonal antibodies.

Monoclonal antibodies have been raised against cytotrophoblast. Two different antigens, defined on cytotrophoblast but not on syncytiotrophoblast were designated ACT-1 and ACT-2, respectively. Chorionic villi were taken from normal early pregnancy and processed for immunization by two different procedures. ACT-1 was demonstrated to be present in lung alveolar cells, endothelial mucosa of the jejunum, colon, ureter, urinary bladder and the fallopian tube, and endometrial gland of the pregnant uterus. On the other hand, ACT-2 was present in the endothelial mucosa of the stomach, endothelium of the renal vessel, and the decidua of the pregnant uterus. Although the monoclonal antibodies did not react with such established cell lines as Bewo, SCH, OVK-18, HHUA, MK-01, FL, BHK and P3 X 63Ag 8 . 653, they did react with some of the cell lines when the cell membrane was destroyed with Triton X-100. Each antibody, therefore, may recognize the antigen not on the cell membrane but in the cytoplasm. The antigens might be shed or may disappear in the process of differentiation from cytotrophoblast to syncytiotrophoblast.