Therapeutic experience with hepatoblastoma associated with trisomy 18.

Trisomy 18 is often fatal, but patients with this disease can now have longer survival due to proactive treatment intervention. However, hepatoblastomas may develop in these patients. In this study, we report four cases of hepatoblastoma associated with trisomy 18. All of the patients had congenital heart disease and three had undergone intracardiac surgical repair. Tumor growth was relatively slow in all cases, and there were no problems with chemotherapy tolerability and surgical resection. Three of the patients are currently disease-free and the fourth is alive with remaining of the tumor. These cases suggest that combined chemotherapy and surgical resection may be an option to treat hepatoblastoma associated with trisomy 18 when cardiac pulmonary function is relatively stable.

Fatal cardiac anomaly of unguarded mitral orifice with asplenia syndrome.

We report the case of a newborn baby with an unguarded mitral orifice associated with asplenia syndrome, double-outlet right ventricle, dysplastic tricuspid valve, and pulmonary stenosis. This case was accompanied by severe tricuspid regurgitation and severe right ventricular hypertrophy. The patient had a fatal clinical course due to severe hypoxia and congestive heart failure. Unguarded mitral orifice is a rare disease in which there has been no previous report of lethal clinical course during the neonatal period. Prior reports stated that unguarded mitral orifice was a new constellation of defects and that its etiology and embryology could be classified in the same category because of similar associated malformations of double-outlet right ventricle and pulmonary stenosis or atresia. However, the present case was diagnosed on autopsy as also having asplenia syndrome. Therefore, it is possible that the genetic etiology of unguarded mitral orifice in this case was different from cases of non-heterotaxy. .

A child with acute encephalopathy associated with quadruple viral infection.

UNLABELLED: Pediatric acute encephalopathy (AE) was sometimes attributed to virus infection. However, viral infection does not always result in AE. The risk factors for developing infantile AE upon virus infection remain to be determined. Here, we report an infant with AE co-infected with human herpesvirus-6 (HHV-6) and three picornaviruses, including coxsackievirus A6 (CVA6), Enterovirus D68 (EV-D68), and human parechovirus (HPeV). EV-D68 was vertically transmitted to the infant from his mother. CVA6 and HPeV were likely transmitted to the infant at the nursery school. HHV-6 might be re-activated in the patient. It remained undetermined, which pathogen played the central role in the AE pathogenesis. However, active, simultaneous infection of four viruses should have evoked the cytokine storm, leading to the pathogenesis of AE. CONCLUSION: an infant case with active quadruple infection of potentially AE-causing viruses was seldom reported partly because systematic nucleic acid-based laboratory tests on picornaviruses were not common. We propose that simultaneous viral infection may serve as a risk factor for the development of AE.

Differential diagnosis of delirious behavior in children with influenza.

Delirious behavior (DB) in children infected with influenza virus is an important symptom associated with encephalopathy. As children with influenza-associated DB with encephalopathy may require therapy whereas children with influenza-associated DB without encephalopathy do not, distinguishing between these conditions is essential. To clarify these differences and identify the most common features of acute encephalopathy, we retrospectively reviewed the clinical course, laboratory data, magnetic resonance imaging (MRI) and electroencephalography (EEG) findings, therapy, and prognosis of 48 children with influenza exhibiting DB. Of the 48 children, 37 and 11 were diagnosed with influenza A and B, respectively. Moreover, 40 were diagnosed with DB without encephalopathy (DBNE group) and 8, with DB with encephalopathy (DBE group). Reversible splenial lesion (RESLE) was detected in 7 patients in the DBNE group, mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) in 2 patients, and a mild form of acute encephalopathy with biphasic seizures and late reduced diffusion in 1 patient in the DBE group. Serum sodium levels <136mEq/L were observed in 28 cases. Disturbance of consciousness was observed in 25 cases, seizure in 20, and slow waves on EEG in 22. Methylprednisolone pulse therapy was administered in 8 cases. No cases of neurological sequelae were observed. Although most of the clinico-radiological features of the DBNE and DBE groups did not differ substantially, marked differences were observed in the age at onset, initial neurological symptoms, duration of DB, rate of seizure, and slowing of background activity on EEG. These differences should be considered when distinguishing between DBNE and DBE in children.

Consecutive oral administration of Bifidobacterium longum MM-2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model.

Bifidobacterium, one of the major components of intestinal microflora, shows anti-influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM-2 (MM-2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM-2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro-inflammatory cytokines such as IL-6 and TNF-α in bronchoalveolar lavage fluid. The anti-IFV mechanism of MM-2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN-γ, IL-2, IL-12 and IL-18. Even in non-infected mice, MM-2 administration also induced significant enhancement of both IFN-γ production by Peyer's patch cells (PPs) and splenetic NK cell activity. Oral administration of MM-2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti-IFV effect. MM-2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.

N-glycolylneuraminic acid on human epithelial cells prevents entry of influenza A viruses that possess N-glycolylneuraminic acid binding ability.

UNLABELLED: Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. IMPORTANCE: Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.

Binding kinetics of sulfatide with influenza A virus hemagglutinin.

Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k on) of 1.41 × 10(4) M(-1) sec(-1), dissociation rate constant (k off) of 2.03 × 10(-4) sec(-1) and K d of 1.44 × 10(-8) M to sulfatide immobilized on a sensor chip. The k off values of sHA were similar for sulfatide and GD1a, whereas the k on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.

[Measurement of NK cell and macrophage activation].

FcgammaRIII(CD16), one of the low-affinity IgG Fc receptors exists in two forms. FcgammaRIIIa is expressed on NK cells, a subset of T lymphocytes, a subpopulation of monocytes and macrophages, and shows a cell type specific glycosylation pattern. FcgammaRIIIb is expressed exclusively on neutrophils in two allotypes, NA1/2, and it can be induced on eosinophils. Both FcyRIIIs are released from the cell surface. FcgammaRIIIa is released by the action of a metalloprotease upon the in vitro activation of NK cells and macrophages. FcgammaRIIIb is released upon activation and during the apoptosis of neutrophils by proteolytic activity. So, the amount of each type of soluble FcgammaRIII means the activation of each type of cell. We measured three types of soluble FcgammaRIII in plasma and also urine with newly developed anti-FcgammaRIIIa and anti-FcgammaRIIIa(Mphi) monoclonal antibodies. We found that sFcgammaRIIIa may be a novel marker of inflammatory activity in rheumatoid arthritis. sFcgammaRIIIa(M phi) may serve as predictive marker for atherosclerosis. Further, urinary sFcgammaRIIIa and sFcgammaRIIIa(Mphi) may be novel markers for the assessment of disease activity in nephritis.

Pitavastatin further decreases serum high-sensitive C-reactive protein levels in hypertensive patients with hypercholesterolemia treated with angiotensin II, type-1 receptor antagonists.

Lipid-lowering therapy with a statin not only powerfully lowers cholesterol but also exerts anti-inflammatory effects by decreasing serum C-reactive protein (CRP). Since an angiotensin II, type-1 receptor antagonist (ARB) also decreases CRP levels, the add-on effect of statins on CRP may be worth exploring. We determined the effect of pitavastatin on serum levels of highly sensitive CRP (hs-CRP) in 30 patients with hypercholesterolemia undergoing treatment with anti-hypertensive medication including ARBs. Pitavastatin, 2 mg daily, was given. The control group consisted of hypertensive patients without hyperlipidemia. The low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and hs-CRP were measured at baseline, 1, 3, 6, and 12 months after treatment. For the atherosclerotic index, LDL-C/HDL-C ratios at 12 months were calculated. The LDL-C level was markedly reduced at 1 month and thereafter. The baseline level of hs-CRP in the hyperlipidemia group was significantly higher than that in the control group (1.647 ± 0.210 mg/L vs. 0.666 ± 0.097 mg/L p < 0.0001). After 3 months, the percentage of reduction of hs-CRP was significantly higher than that in the control group. The absolute values of hs-CRP were significantly decreased to a level similar to the control group, and the hs-CRP in both groups was remained at the same level for 12 months. Although the LDL-C/HDL-C ratios of the pitavastatin group was significantly reduced from 3.3 to 1.8, those of the control group were not changed. In conclusion, pitavastatin was found to have powerful anti-inflammatory, add-on effects over the similar effects of ARB as assessed by hs-CRP.

[Case of spuriously high level of squamous cell carcinoma antigen in patient without carcinoma--HPLC and ultrafiltration are useful to analyze the true value].

Squamous cell carcinoma antigen (SCCA) is a diagnostic tumor marker for the advanced uterine, cervix and lung tumor. Although SCCA is a prognostic indicator for some tumors, recent progress of this marker has shown that the SCCA could also be found in the serum of nonmalignant disease such as renal failure and others. Here, we report a case of spuriously high level of SCCA in patient without carcinoma, renal failure, head-and-neck disease and lung disease. An early fifties female who had been undergone the diagnostic conization for high-grade cervical intraepithelial neoplasia ten years ago and observed without special treatment with around 20ng/ml level of SCCA. She has no signs of tumor, renal failure, head-and-neck disease or lung disease until now. The high performance liquid chromatography with Superdex 200 showed the molecular weight of the major part of SCCA of the patient is more than 160 kDa and the part of 45 kDa, the same molecular weight as lung tumor, is trace amount. Moreover, the ultrafiltration analysis showed the SCCA of the present case did not penetrate the 100 kDa cut-filter, but SCCAs with other patients with uterine, cervix, lung tumor and renal failure did penetrate the filter. In this case, the analysis of molecular weight of SCCA using HPLC gel filtration and ultrafiltration is useful to rule out spuriously elevated SCCA.

Phagocytosis of heat-killed Staphylococcus aureus by eosinophils: comparison with neutrophils.

Eosinophils are characterized by several functional properties, such as chemotaxis, adhesion, superoxide anion production, and degranulation. In this article, we have studied the role of bacterial ingestion by eosinophils in comparison with that by neutrophils. Eosinophils and neutrophils were purified by using the Percoll gradient method followed by selection with CD16-coated immunomagnetic beads and centrifugation through a Ficoll-Hypaque gradient combined with dextran sedimentation, respectively. Both cells were preincubated with anti-FcgammaRIIa mAb (CD32 mAb), anti-FcgammaRIIIb mAb (CD16 mAb), anti-CR3 (CD11b mAb), or anti-CR1 (CD35 mAb) before being examined for phagocytosis of opsonized heat-killed Staphylococcus aureus (S. aureus). Phagocytosis and production of hydrogen peroxide were simultaneously measured by flow cytometry using S. aureus labeled with propidium iodide and stained with 2',7'-dichlorofluorescein diacetate. Eosinophils showed significantly lower activity than neutrophils in both phagocytosis and hydrogen peroxide production. Phagocytosis by both cells was decreased by heat-inactivated serum. Phagocytosis by neutrophils was significantly inhibited by CD16 mAb and CD32 mAb, whereas that by eosinophils was only inhibited by CD35 mAb. Whereas the mechanism of phagocytosis by neutrophils was mediated by CD16 and CD32, that of eosinophils was modulated by complement receptor 1 (CD35).

Soluble FcgammaRIIIa(Mphi) levels in plasma correlate with carotid maximum intima-media thickness (IMT) in subjects undergoing an annual medical checkup.

Macrophages play a major role in the development of vascular lesions in atherogenesis. The cells express FcgammaRIIIa (CD16) identical to that in NK cells, but with a cell type-specific glycosylation, and these soluble forms (sFcgammaRIIIa) are present in plasma. We measured sFcgammaRIIIa(Mphi) derived from macrophages in plasma from subjects undergoing an annual medical checkup. The levels of sFcgammaRIIIa(Mphi) increased with age, and correlated positively with body mass index, blood pressure, LDL cholesterol to HDL cholesterol ratio, triglycerides, hemoglobin A1c, and creatinine, but negatively with HDL-cholesterol levels. The sFcgammaRIIIa(Mphi) levels were related to the number of risk factors for atherosclerosis: such as aging, current smoking, diabetes, hypertension, hyper-LDL-cholesterolemia, hypo-HDL-cholesterolemia, and family history of atherosclerotic diseases. In addition, the sFcgammaRIIIa(Mphi) levels were correlated with carotid maximum intima-media thickness (IMT). These findings indicate the macrophages are activated during the incipient stage of atherosclerosis, and suggest sFcgammaRIIIa(Mphi) may be used as a predictive marker for atherosclerosis.

Isolation of bisphenol A-tolerant/degrading Pseudomonas monteilii strain N-502.

Bisphenol A (BPA) is a highly biotoxic compound that kills many microorganisms at a low concentration (1,000 ppm). We isolated BPA-tolerant/degrading Pseudomonas monteilii strain N-502 from about 1,000 samples collected from a field, sewage, and pond water. The isolated strain had strong BPA tolerance and high BPA-degrading activity. This strain was able to grow in a minimum medium containing BPA as the sole carbon source. Strain N-502 is an aerobic, motile, gram-negative, nonspore-forming, rod-shaped bacterium and was identified as P. monteilii, based on 16 S rRNA gene analysis. Strain N-502 completely degraded BPA 500 ppm in a 10-day, in culture system and was able to degrade BPA 100 ppm in a 2-h resting cell system. This strain also showed potent ability to degrade BPA 500 and 1,000 ppm in the resting cell system. Moreover, the initial BPA degradation rate was accelerated with the addition of Ca(2+), Mg(2+), and folic acid.

Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats.

Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

Increased soluble FcgammaRIIIa(Mphi) in plasma from patients with coronary artery diseases.

Atherosclerosis is the underlying disease process in patients affected with coronary artery diseases (CAD). Macrophages play a major role in the development of vascular lesions in atherogenesis. The cells express Fcgamma receptor type IIIa (FcgammaRIIIa: CD16) identical to that in natural killer cells (NK cells), but with a cell type-specific glycosylation. In contrast, neutrophils express FcgammaRIIIb. These FcgammaRIIIs are released from the cell surface on activation, and these soluble forms (sFcgammaRIII) are present in the plasma. We measured sFcgammaRIIIa(Mphi) in the plasma with a newly developed anti-FcgammaRIII mAb, MKGR14, which recognizes FcgammaRIIIa(Mphi) specifically. The level of sFcgammaRIIIa(Mphi), as well as the level of sFcgammaRIIIa (sFcgammaRIIIa(Mphi) plus sFcgammaRIIIa(NK)) or the level of total sFcgammaRIII (sFcgammaRIIIa plus sFcgammaRIIIb), were significantly increased in patients with CAD, but not in patients with vasospastic angina (VSA) or intact coronary arteries, compared with age-matched healthy donors. The sFcgammaRIIIa(Mphi) level was related to the number of significantly affected coronary arteries, and positively correlated with LDL-cholesterol to HDL-cholesterol ratios, but negatively with HDL-cholesterol. No correlation among the levels of three sFcgammaRIIIs was observed in CAD patients, as well as in healthy donors. The macrophages are activated during the process of atherosclerosis, and sFcgammaRIIIa(Mphi) may serve as a novel marker for atherosclerosis.

The role of a mutation of the CXCR4 gene in WHIM syndrome.

We investigated the role of a mutation of the CXCR4 gene in 11-year-old twin sisters with WHIM syndrome. The mutated gene may result in production of the mutant CXCR4 protein causing abnormal apoptosis and migratory function, which are thought to be related to the cause of chronic neutropenia in WHIM syndrome.

Polymorphism of Fc gamma RIIa may affect the efficacy of gamma-globulin therapy in Kawasaki disease.

We evaluated whether there is a possible relationship between the effectiveness of gamma-globulin treatment for patients with Kawasaki disease (KD) and the polymorphism of Fcgamma RIIa, IIIb, and IIIa. Genomic DNA was extracted from whole blood collected from 56 patients with KD who received gamma-globulin treatment. The genotypes for Fcgamma RIIIb-NA(1, 2), Fcgamma RIIa-H/R131, and FcgammaRIIIa-F/V158 were determined to investigate the association between these polymorphisms and the development of coronary lesions (CALs). Twenty-three percent of patients with the HH allele for the Fcgamma RIIa polymorphism progressed to CALs, compared with 60% with the HR and RR alleles. HR and RR alleles may be a predictor of the progression of CALs in KD before the initiation of gamma-globulin therapy.

A novel endogenous digitalis, telocinobufagin, exhibits elevated plasma levels in patients with terminal renal failure.

BACKGROUND AND OBJECTIVES: There are several potential endogenous digitalis-like factors (EDLF) in mammalian body fluids, and marinobufagenin (MBG) may be the most potent EDLF. Improved assays are needed to confirm the potency of these metabolites. In the present study, we have identified MBG and telocinobufagin (TCB) in human plasma by high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR). METHODS AND RESULTS: The high-resolution MS analysis revealed the molecular masses of TCB and MBG to be the same as their respective theoretical values. Using a tandem mass spectrometer, the mass-charge ratio for TCB was determined to be 403.2 for the parent ion and 349.2 for the daughter ion. The mass-charge ratio for MBG was m/z 383.2 and m/z 401.2. The NMR study revealed that the signals for MBG and TCB were the same as those obtained by MS analysis. In human blood, MBG and TCB were also identified by liquid chromatography (LC) as well as MS. In the LC/MS assay, proscillaridin A was used as an internal standard. The plasma was pretreated with Sep-Pak C18, and then 50 microL was applied to the C8 high-performance liquid chromatography (HPLC) column. The mean plasma concentration of MBG in healthy volunteers (0.94 +/- 0.28 ng/mL) was significantly lower than that in patients undergoing regular hemodialysis (3.81 +/- 1.92 ng/mL). The concentration of TCB in the healthy volunteers (1.80 +/- 0.55 ng/mL) was also significantly lower than that in patients with terminal renal failure (6.86 +/- 4.30 ng/mL). CONCLUSION: These results indicate that the major EDLF is TCB because its plasma concentration is the highest among the reported endogenous digitalis candidates.

Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells.

Changes in [Ca(2+)](i) across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity. In the present study, we investigated the effect of ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, on NO release and [Ca(2+)](i) movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca(2+)](i) using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t((1/2)) (half-time of the decline of [Ca(2+)](i) to basal levels after stimulation with 10 micro mol/L bradykinin) was used as an index of [Ca(2+)](i) extrusion. A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose-response concentration induced by bradykinin. The same dose of ouabain affected [Ca(2+)](i) movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t((1/2)) was significantly increased. Pretreatment of RAEC with Na(+)-free solution, an inhibitor of the Na(+)/Ca(2+) exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na(+)/K(+)-ATPase activity and an increase in intracellular Na(+) concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca(2+)](i) by forward mode Na(+)/Ca(2+) exchange to prolong the Ca(2+) signal time. From these results, we suggest that nanomolar levels of ouabain modulate [Ca(2+)](i) movements and NO production in RAEC.

[Soluble Fc gamma RIIIa derived from macrophages].

Macrophages play a major role in the development of vascular lesions in atherogenesis. The cells express Fc gamma RIIIa (CD16) identical to that in NK cells, but with a cell type-specific glycosylation. The Fc gamma RIIIa molecules are released from the cell surface on activation, and these soluble forms (sFc gamma RIIIa) are present in plasma. We measured sFc gamma RIIIaM phi in plasma by Immuno-PCR with newly-developed anti-Fc gamma RIII mAb, MKGR14 (mIgM), which recognizes Fc gamma RIIIaM phi specifically. In healthy donors, the level of sFc gamma RIIIaM phi increased with age, and was positively correlated with LDL-cholesterol to HDL-cholesterol ratios and LDL-cholesterol, but negatively with HDL-cholesterol. In addition, the sFc gamma RIIIaM phi level was related to the number of risk factors for atherosclerosis and correlated with carotid maximum intima-media thickness in subjects for annual medical checkup. The sFc gamma RIIIaM phi level was significantly increased in patients with coronary artery disease (CAD) with atherosclerosis, but not in patients with vasospastic angina (VSA) or intact coronary artery, compared with age-matched healthy donors. The sFc gamma RIIIaM phi level was related to the number of significant coronary artery stenoses, and positively correlated with LDL-cholesterol to HDL-cholesterol ratios and lipoprotein (a), but negatively with HDL-cholesterol. These findings suggest that the macrophages are activated during the incipient stage of atherosclerosis, and that sFc gamma RIIIaM phi may serve as predictive marker for atherosclerosis.