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.

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.

Effect of KC-9432, a new hypolipidemic compound, on high density lipoprotein cholesterol in rats.

KC-9432 (a new hypolipidemic compound, alpha-[p-(p-chlorobenzoyl) phenoxy-alpha-cyclohexyl acetic acid ethyl ester) was studied in rats for its effect on plasma lipid and lipoprotein levels. KC-9432 was particularly effective in hyperlipidemia in rats fed with a diet containing 2% cholesterol and 1% sodium cholate. The hypocholesterolemic activity of KC-9432 was far more potent than that of clofibrate. KC-9432 markedly increased the reduced HDL cholesterol level in dietary-induced hyperlipidemia in rats.

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.

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.

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.

A new type of laryngotracheoesophageal cleft with extended bronchoesophageal cleft.

A case of total laryngotracheoesophageal cleft with extended fronchoesophageal cleft is reported. The baby had a long cleft not only on the whole length of the laryngotracheoesophagus but it also extended to the right lower bronchus and esophagus. It was associated with right upper bronchial stenosis, sequestration of right lower lobe, and a complex cardiovascular anomaly. There have been no reports of this extended type of cleft in the 59 reported cases.

The immunological relationship between filtrable agent, Salmonella and murine leukosis.

Salmonella typhimurium was invariably isolated from our J strain murine leukosis. Immunization of D103 mice with either inactivated Salmonella typhimurium or the cell-free extract of leukosis inhibited the transplantation of leukosis. The adoptive immunization of D103 mice with spleen cells of Strong A mice immunized with either Salmonella or the cell-free extract of leukosis inhibited the transplantation of leukosis. The addition of either Salmonella or the cell-free extract of leukosis inhibited the migration of macrophages of leukosis spleen in tissue culture. Strong A mice is non-susceptible to J strain leukosis. However, inoculation of neonatal Strong A mice with the cell-free extract of leukosis produced a susceptibility to the transplantation of leukosis. These results suggest that both a filtrable agent and Salmonella typhimurium are present in cells of this leukosis and might be etiologically related to the leukosis.

Tumor induction in Swiss mice by filtrable agent and Salmonella typhimurium.

Combined inoculation of a cell-free extract of leukotic tissue of D103 mice and Salmonella typhimurium into adult Swiss mice induced leukosis and solid tumors. The induced solid tumors were histologically multifarious, and were transplantable in Swiss mice, but not in other strains of mice.

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.

[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.

[Interferon-like substance in pregnancy sera].

Interferon(IFN) can be produced from lymphocytes in vitro as well as in vivo using various immune stimuli. This paper shows that the amount of IFN or IFN-like substance increases in pregnancy sera as pregnancy proceeds. 1) IFN activity was detected in 45% of 97 pregnant women overall and more frequently as pregnancy went on. 2) The IFN was trypsin sensitive, beat labile, acid sensitive and species specific. 3) Maternal lymphocytes produced approximately twice as much interferon as cord blood lymphocytes did by phytohemagglutinin stimulation. 4) IFN was detected in half of the mixed lymphocyte cultures(MLR) containing cord blood and maternal blood. Whether or not they can produce IFN may be an indication of the immunological competence of pregnant women and the interferon may contribute to the immunoregulation of fetal acceptance.

The effect of various hormones on the humoral immune response of murine lymphocytes in vitro.

Human chorionic gonadotropin (HCG) and steroid hormones were tested for their direct effects on murine lymphocyte humoral response to sheep RBCs using an in vitro culture system. A significant increase in plaque forming cells (PFC) was induced by the addition of HCG at a dose of either 50 i.u. or 500 i.u. However, no increase was observed by the addition of steroid hormones. HCG may contribute to the success of fetal allografts by increasing the number of blocking antibodies in the early stages of pregnancy.

Pharmacological and toxicological studies of a new non-steroidal anti-inflammatory drug: KC-8973.

A benzophenone derivative, 4-butyl-2'-fluorobenzophenone (KC-8973), having anti-inflammatory effects has been studied pharmacologically and toxicologically. On the carrageenin-induced paw edema, KC-8973 had the most potent activity of the tested commercial non-steroidal anti-inflammatory drugs. KC-8973 had a moderate effect on bradykinin-induced capillary permeability and lesser effect on dextran-, egg white- and serotonin-induced paw edemas in rats, respectively. From the detailed studies of prophylactic and therapeutic effects on the adjuvant-induced arthritis in rats, it was demonstrated that KC-8973 has an immuno-suppressive activity in addition to an anti-inflammatory effect. KC-8973 had a local-analgesic and antipyretic effect on the rat paw edema induced by brewer's yeast. KC-8973 caused no undesirable side effect at pharmacologically effective dose in the 1-month sub-acute toxicological study.