Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.

S100A8/A9 is a major component of the acute phase of inflammation, and appears to regulate cell proliferation, redox regulation and chemotaxis. We previously reported that S100A8/S100A9 are upregulated in the premetastatic lung. However, the detailed mechanisms by which S100A8 contributes to tumor progression have not been elucidated. In this study, we investigated the TLR4/MD-2 dependency by S100A8 on tumor progression. We found that S100A8 (2-89) peptide stimulated cell migration in a manner dependent on TLR4, MD-2 and MyD88. The S100A8 (2-89) peptide also activated p38 and NF-κB in TLR4-dependent manner. The peptide induced the upregulation of both IL-6 and Ccl2 in peritoneal macrophages obtained from wild-type mice, but not TLR4-deficient mice. We then investigated the responsible region of S100A8 for TLR4/MD-2 binding by a binding assay, and found that C-terminal region of S100A8 binds to TLR4/MD-2 complex. To further evaluate the TLR4 dependency on tumor microenvironment, Lewis lung carcinoma-bearing mice were treated with Eritoran, an antagonist of TLR4/MD-2 complex. We found that both tumor volume and pulmonary recruitment of myeloid-derived suppressor cells were reduced with the treatment of Eritoran for five consecutive days. Eritoran reduced the development of tumor vasculature, and increased tumor-infiltration of CD8(+) T-cells. Taken together, S100A8 appears to play a crucial role in the activation of the TLR4/MD-2 pathway and the promotion of a tumor growth-enhancing immune microenvironment.

Anticoagulant activity in cell homogenate of adult T cell leukemia.

The hemostatic abnormality in 18 patients with adult T cell leukemia (ATL) was studied. Activated partial thromboplastin time (APTT) was slightly prolonged and prekallikrein activity was markedly low in these patients. The leukemic cell homogenate from these patients prolonged the recalcification time (RCT) of normal plasma; homogenates containing more than 3 x 10(3) cells/microliter prolonged it, although a lower cell concentration shortened it. The crude anticoagulant fraction from the gel filtration, with a molecular weight of about 34,000, prolonged RCT. The crude anticoagulant did not affect prothrombin time (PT), thrombin activity or activated X activity at any concentration, but prolonged the contact activation test, inhibited the activation of prekallikrein and prolonged RCT of Fletcher trait, Fitzgerald trait and F XII deficient plasma. These effects of ATL cell homogenate were stronger on platelet poor plasma than on platelet rich plasma. Although ATL cells had low procoagulant activity, increase of leukemic cells made anticoagulant activity predominant, might be the cause of hemostatic abnormality or amplify the bleeding tendency in patients with ATL.

Plasma level of IL-1 beta in disseminated intravascular coagulation.

The plasma level of interleukin-1 beta (IL-1 beta) was determined in normal individuals, patients with disseminated intravascular coagulation (DIC), patients in the pre-DIC period (within 7 days before the onset of DIC), and non-DIC patients to examine the relationship between DIC and the plasma IL-1 beta level. The plasma IL-1 beta level was 0-0.085 ng/ml in normal individuals, with little difference being seen according to related age. It was significantly higher in the DIC group (0.19 +/- 0.19 ng/ml) than in the pre-DIC group (0.05 +/- 0.08 ng/ml) or the non-DIC group (0.09 +/- 0.01 ng/ml). The plasma IL-1 beta level was not markedly elevated in leukemia patients, even in the DIC group, but it was significantly increased in the DIC group of solid cancer patients and was generally elevated in patients with sepsis. It was markedly elevated to 0.39 +/- 0.26 ng/ml in patients with organ failure. When mononuclear cells were incubated with lipopolysaccharide, it was found that IL-1 beta, tumor necrosis factor, and tissue factor (TF) were released into the medium, and there was an increase of TF release from endothelial cells incubated with this medium. These results suggest that the increase in IL-1 beta reflected the activation of monocytes and may be an important factor in DIC and its associated organ failure.

Inhibition of G1 cyclin expression in normal rat kidney cells by inostamycin, a phosphatidylinositol synthesis inhibitor.

We previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.

Changes in phenotypic expression of osteoblasts after X irradiation.

Changes in the phenotypic expression of osteoblasts after X irradiation were investigated. Osteoblast-like MC3T3-E1 cells at the actively proliferating, confluent and postproliferation stages were subjected to 10 Gy X irradiation. Irradiation at the confluent stage enhanced accumulation of type I collagen normalized to the DNA content. Irradiation at all stages down-regulated the expression of osteocalcin, but the levels of osteopontin and osteonectin mRNAs were unchanged from the control level. After irradiation at the later stages, the time-dependent increase in alkaline phosphatase activity per cell exceeded that in the control cells. The localization of alkaline phosphatase-positive cells was concordant with that of calcification. In addition, the quality of the calcium deposits was found to be similar to that in control cells as determined by energy dispersive spectrometry and the ratio of calcium to phosphorus, even if the cells were not exactly the same morphologically. The changes in phenotypic expression observed here are closely related to the enhancement of calcification observed in a previous study.

Induction of G1 arrest and selective growth inhibition by lactacystin in human umbilical vein endothelial cells.

In search for angiogenesis inhibitors, we tested protease and proteasome inhibitors for the induction of G1 arrest and selective inhibition of growth of human umbilical vein endothelial cells (HUVECs). Serine protease-, cysteine protease-, aspartate protease-, and aminopeptidase-inhibitors did not inhibit bFGF/FBS-induced S-phase induction in HUVECs, but a proteasome inhibitor, lactacystin did inhibit it reversibly. Lactacystin increased the cellular level of p53 and cdk2-associated p21WAF1/CIP1 leading to cdk2 inactivation. In addition to the angiogenesis inhibitor TNP-470, lactacystin also inhibited the growth of HUVECs selectively at about a 20 times lower concentration than that of other human cell lines, including normal fibroblasts and carcinoma cells. Lactacystin induced p53-dependent p21WAF1/CIP1 expression at lower concentrations in HUVECs than in other cells. These cellular effects were also observed with a tripeptide-type proteasome inhibitor, N-Ac-Leu-Leu-norleucinal.

Histopathologic comparison of anti-mitochondrial antibody-positive primary biliary cirrhosis and autoimmune cholangiopathy.

The present study aimed to investigate whether antimitochondrial antibody (AMA)-positive primary biliary cirrhosis (PBC) and AMA-negative PBC with autoantibodies differ histologically, especially with respect to infiltrating cells in portal tracts involved by chronic non-suppurative destructive cholangitis. Liver specimens were stained from 15 primary biliary cirrhosis with AMA (group 1), nine patients consistently negative for AMA but positive for antinuclear antibodies (ANA) (group 2). Group 2 showed overlapping features of PBC and autoimmune hepatitis type 1, in a pattern recently termed autoimmune cholangiopathy (AIC). We analyzed the cell population, including lymphocytes, plasma cells, large histiocytes, eosinophils and neutrophils, which had infiltrated portal tracts involved by destructive lesions. Although serum immunoglobulin M levels were higher in group 1 compared to those in group 2 (P=0.0282), patients of both groups were broadly comparable with respect to clinical features and laboratory data. Histologically, the number of plasma cell and its percentage among inflammatory infiltrating cells in the portal tract were higher in group 2 than in group 1 (P=0.0015, P=0.0070, respectively). The percentage of lymphocyte infiltration among inflammatory infiltrating cells in the portal tract were higher in group 1 than in group 2 (P=0.0052). The percentage of plasma cell infiltration among inflammatory infiltrating cells in the portal tract was correlated to immunoglobulin G levels in group 2 (r=0.949, P=0.0016). In conclusion, AMA-positive PBC and AIC showed differences in inflammatory cell population in involved portal tracts in this preliminary study.

Spa bathing activates fibrinolysis in patients with cerebral infarction.

The effects of spa bathing on blood coagulation and fibrinolysis were studied in 20 patients with chronic cerebral infarction. Blood was obtained before and after a 10-minute period of spa bathing at 41 degrees C. Prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII activity, von Willebrand factor activity, and antithrombin III activity did not show significant changes after bathing, but euglobulin lysis time was significantly reduced (p < 0.01) and fibrin lysis activity was increased (p < 0.05). These findings suggest that spa bathing activates fibrinolysis without markedly changing blood coagulation in patients with chronic cerebral infarction. It is thought that the activation of fibrinolysis without the activation of coagulation has a favorable effect on blood circulation. The results of fibrin-plate assays using C1 inactivator indicated that tissue-type plasminogen activator was the major contributor to the activation of fibrinolysis during spa bathing.

Nodular fasciitis of the upper labial fascia: cytometric and ultrastructural studies.

The rare case of nodular fasciitis in the upper labial fascia is reported. Light microscopic, electron microscopic and cytometric studies were performed. The histopathology corresponded to intermediate and reactive types in the Price and Bernstein classification. Cytometric study of evaluating DNA of fibroblasts revealed a high distinct peak in myxoid and inflammatory areas. Ultrastructurally, fibroblasts and myofibroblasts were identified in the lesion. Reported Japanese cases of orofacial nodular fasciitis are reviewed and compared with the European-American literature.

Improving symptoms of senile dementia by a night-time spa bathing.

In our medical and welfare facilities, many patients with senile dementia require aid in taking a bath. In most institutions, patients usually take a bath in the daytime within the working hours of the staff. However, most of these patients used to take a bath in the evening or at night at their homes. Some patients even fall asleep after daylight bathing. Thus, we studied the stabilizing effects of night-time spa bathing on symptoms associated with dementia. Ten patients (two male and eight females, aged 75-88) in special nursing institution for the aged, were enrolled in this study. They were all assessed as +4 on the Karasawa's clinical criteria for grading dementia. For 9 weeks, night-time spa bathing was performed at 18:00-19:00 twice a week. Except for the night-time spa bathing period, the bathing hour was 14:00-15:00 as usual. The observations of symptoms including restlessness, wandering and aggression were carried out ten times daily along with those on sleeping condition five times daily, to compare symptoms and conditions during 2 weeks of baseline daytime bathing periods, 9 weeks of night-time bathing periods and 2 weeks of daytime bathing periods, totaling 13 weeks. The results showed that sleeping conditions were ameliorated in more than 60-90% of the subjects. Their sleeping conditions began to improve 2 weeks after the start of night-time spa bathing with a remarkably improvement 4-6 weeks after the start. Restlessness was recognized in six subjects, wandering in eight and aggression in four at baseline, and 75-100% of the subjects with such symptoms improved markedly.

Plasma thrombomodulin values and hepatorenal function in the elderly.

To define the clinical significance of plasma thrombomodulin (TM) values in elderly, we examined plasma TM in healthy young subjects, healthy elderly subjects and patients with cerebral infarction sequelae. We also studied the relationship with effective renal plasma flow (ERPF) and with the liver's protein-production ability. The TM values of healthy elderly subjects were higher than those of healthy young subjects. There existed an inverse correlation between TM values and ERPF. Accordingly, high TM values might significantly influence renal arteriosclerosis. From the inverse correlation identified between TM and serum cholinesterase, it was estimated that high TM might appear in conjunction with the liver's protein production ability. Patients with cerebral infarction showed higher plasma TM values. It is thought that angiopathy has been maintained in patients as the anamnesis of cerebral infarction even though it occurred in the past. The TM values of patients with diabetes mellitus (DM) were higher than those without it. Moreover, the TM values of patients with DM complicated by retinopathy were higher than those uncomplicated by retinopathy. It is therefore estimated that increased TM might occur with angiopathy resulting from DM. A possibility thus exists that plasma TM could be utilized as one of the markers for endothelial injury.

[Coagulation/fibrinolysis disorders in the elderly].

To establish reference intervals for the interpretation of hemostatic tests in the elderly, 11 hemostatic tests were performed on 120 elderly individuals(26 males and 94 females) aged 78.6 years in average. The subjects lived in hostel for the elderly, walked without assistance, and were independent in activities in daily living. In 25.8% to 93.3% of the subjects, the results of the tests, except for prothrombin time and thrombin-antithrombin III complex, deviated from reference intervals obtained from young healthy subjects. This suggests the necessity of setting up reference intervals for the elderly. Fibrinolysis was more activated than coagulation, although coagulation had been considered to be more activated because thrombosis is common in the elderly. Wilcoxon test on the test values of the early elderly(65 to 74 years of age) and late elderly(75 to 92 years of age) as well as the correlation coefficient for age and test values revealed that separate reference intervals for antithrombin III, protein C, D-dimer, and thrombomodulin should be established for the early elderly(65 to 74 years) and late elderly(75 years or over). Our proposed reference intervals for the elderly are appropriate for the diagnosis of disseminated intravascular coagulation.

[Studies on C1 inactivator resistant fibrinolytic activity].

The fibrin lysis activity in the presence of enough quantity of C1 inactivator (C1.INA) is called C1.INA resistant fibrinolytic activity (C1.IRFA). C1.INA inhibits the activation of intrinsic fibrinolysis, therefore C1.IRFA is considered to reflect the extrinsic fibrinolytic activity mainly. We studied the effect of C1.INA using fibrin plate assay on fibrin lysis activity (FLA) and C1.IRFA in normal individuals and pregnant women, and studied the fluctuation of FLA and C1.IRFA during venous occlusion test (VO) or 1-deamino-8-D-arginine vasopressin (DDAVP) administration. After VO or DDAVP administration, the euglobulin lysis time (ELT) was reduced, FLA increased and C1.IRFA also increased. The increase in C1.IRFA accounted for most of the increase in FLA. Therefore, the increased fibrinolytic activity during VO or DDAVP administration is due to extrinsic fibrinolytic activity. In the pregnant women, the fibrinolytic activity was decreased during pregnancy and the low fibrinolytic activity is suggested to be involved in the low extrinsic fibrinolytic activity. The measurement of C1.IRFA is considered to be useful.

[Measurement of FDP-D-dimer in DIC and pre-DIC].

We measured FDP-D-dimer value in disseminated intravascular coagulation (DIC), pre-DIC (within 7 days before onset of DIC) and suspected DIC (not completely satisfying the DIC criteria). The level of FDP in many patients with pre-DIC was normal, but the level of FDP-D-dimer in most patients with pre-DIC was increased. FDP was markedly increased one day before onset of DIC but FDP-D-dimer was increased 7 days before the onset. FDP was significantly higher in DIC than in pre-DIC, but it was not higher in pre-DIC than in suspected DIC. In patients with hematological malignancies, FDP-D-dimer was statistically higher in DIC than in pre-DIC and in pre-DIC than in suspected DIC, but in non-hematological malignancies, FDP-D-dimer was not significantly different among the 3 groups. The peak increase of FDP-D-dimer was noted at a DIC score of 7 or 8. The correlation of FDP-D-dimer with FDP was better in pre-DIC than in DIC, and the ratio of FDP-D-dimer to FDP was higher in pre-DIC. FDP-D-dimer was not correlated with fibrinopeptide A or B beta 15-42 in pre-DIC. It is speculated that pre-DIC is a hypercoagulable state and FDP-D-dimer may be useful to the diagnosis of pre-DIC.

[Availability of DDAVP in hemostasis control for the breast tumor resection in a patient with thrombasthenia (type II)].

We reported a 33-year-old woman with thrombasthenia (type II) in whom 1-deamino-8-D-arginine vasopressin (DDAVP) was available for the hemostasis control during breast tumor resection. She has had recurrent nasal bleeding and purpura since 2 years old. On the first admission to our hospital because of hematuria at 18 years old, she was diagnosed as thrombasthenia (type II) from hemostatic studies. At 20 years old, she had a healthy baby by the cesarean section with transfusion of fresh blood and platelet concentrates. On the 5th admission for the breast tumor resection, defect of glycoprotein II b-III a of her platelets was confirmed by using SDS gel electrophoresis. Recently, there are some reports on availability of DDAVP for hemostatic control in platelet dysfunction of various etiologies as well as mild hemophilia and von Willebrand disease. So, DDAVP (0.4 microgram/kg) was used for hemostasis control. After 1 hour, the bleeding time was shortened from over 10 to 4 min, platelet adhesiveness to glass beads increased from 1.8 to 37%. Furthermore, the levels of Ristocetin cofactor and von Willebrand factor antigen (especially large multimer) also increased. But platelet aggregation with various inducers remained unchanged before and after infusion. Breast tumor (fibroadenoma) resection.

[Elevated levels of cytokines in plasma from patients with disseminated intravascular coagulation].

We measured blood concentrations of tumor necrosis factor (TNF), interleukin-1 beta (IL 1-beta), soluble interleukin 2 receptor (s-IL 2r), and interferon alpha (IFN alpha) in 30 patients with disseminated intravascular coagulation (DIC) and compared the results to those of 25 patients without DIC. Plasma levels of TNF, IL 1-beta, and s-IL 2r were higher in patients with DIC than in those without DIC. In one case of acute promyelocytic leukemia, plasma levels of TNF and IL-1 beta increased at the onset of DIC but decreased upon DIC improvement. These findings suggest that activation of the immune system is involved in the development of DIC. However, these concentrations were not markedly increased in patients with leukemia, although blood TNF and s-IL 2 r were markedly elevated in patients with solid cancers. Especially in patients with solid cancers, hyperactivation of the immune system may cause an increase in blood TNF and IL-1 beta and the development of DIC.

[Plasminogen activator in leukemic cell homogenate].

We examined fibrinolytic substances in homogenate of leukemic cells, normal granulocyte or mononuclear cells fraction. Plasminogen activators (PA) were significantly low in normal cells, but they were slightly increased in lymphoblastic leukemia cells and markedly increased in myeloblastic leukemia cells. In almost leukemic cells homogenate, the antigen ratio of tissue type PA (t-PA)/urokinase type PA (u-PA) was about 2.0. In especially AMMoL and AMoL, PA activity had discrepancy between euglobulin lysis time and amidolytic assay using chromogenic substrate. As PA inhibitor (PAI)-II was markedly increased in them. PAI might effect the PA assay. PA activity of leukemic cells homogenate was similar to that without t-PA stimulator and leukemic cells homogenate significantly stimulated t-PA. As both PA activity and antigen were statistically increased in leukemic cells homogenate of patient with disseminated intravascular coagulation (DIC), PA and PA stimulator in leukemic cell might play an important role in hypofibrinogenemia or DIC.

[Screening of phosphatidylinositol turnover inhibitors and regulation of cell cycle progression].

Phosphatidylinositol (PI) turnover is considered to be involved in the regulation of cell growth. The enzymes for PI turnover include phospholipase C (PLC), PI4-kinase and PI synthase. We have isolated pholipeptin and fluvirucin B2 from microorganisms and akaterpin from a marine sponge as PLC gamma inhibitors. We also isolated echiguanines from Streptomyces as PI4-kinase inhibitors. Since echiguanines did not inhibit the enzyme in situ, we synthesized their ribosylated derivatives that were effective in cultured cells. We previously isolated inostamycin from Streptomyces as an inhibitor of PI synthase. We found that inostamycin induced G1 block in cycling NRK cells. Inostamycin inhibited the serum-induced S-phase induction in quiescent NRK cells. Inostamycin was found to decrease serum-induced expression of cyclin D and cyclin E, without inhibiting the activation of MAP kinase. It also inhibited serum-induced activation of CDK2 and phosphorylation of pRB. Thus, PI synthesis was suggested to be involved in regulation of serum-induced S-phase induction by modulating G1 cyclin expression.

[Plasma thrombomodulin levels in elderly subjects].

Plasma levels of thrombomodulin (TM), fibrinogen, antithrombin III (ATIII) and thrombin ATIII complex (TAT) were studied in healthy young subjects (group A), healthy elderly subjects (group B) and patients with level of TM in group B tended to be higher than that in group A. Levels of TM, fibrinogen and TAT in group C suggested the presence of a hypercoagulable state. When group C was further divided into those with and without diabetes mellitus (DM), the TM level in the former tended to be higher than that in the latter. Furthermore, among the patients with DM, those with diabetic retinopathy showed significantly higher levels of TM than those without retinopathy. Thus, high TM levels indicate the presence of endothelial injury. In groups B and C, TM correlated positively with fibrinogen, and negatively with ATIII, which also indicates that a high TM level is related to a hypercoagulable state. In conclusion, the TM level is considered to be a potential marker of the presence of endothelial injury.