伊藤 弘康

The effect of alpha-galactosylceramide (alpha-GalCer) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal cells was studied. alpha-GalCer augmented LPS-induced NO production in mouse peritoneal cells, but not in RAW 264.7 macrophage cells. alpha-GalCer augmented NO production, but not tumor necrosis factor (TNF)-alpha production in LPS-stimulated peritoneal cells. Peritoneal cells produced a significant level of interferon (IFN)-gamma in response to alpha-GalCer and anti-IFN-gamma antibody abolished the augmentation of LPS-induced NO production by alpha-GalCer. Moreover, anti-IFN-gamma antibody prevented the enhanced expression of an inducible type of NO synthase mRNA by alpha-GalCer. alpha-GalCer did not augment LPS-induced NO production in peritoneal cells from natural killer T (NKT)-deficient mice. Therefore, it was suggested that alpha-GalCer might augment LPS-induced NO production in peritoneal cells through release of IFN-gamma from NKT cells.

The effect Of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment Of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.

The role of p38 mitogen-activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS-induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)-kappaB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)-gamma-stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS-stimulated macrophages is discussed.

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by 11-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.

Recent studies have shown that tumor necrosis factor alpha (TNF-alpha) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-alpha knockout (TNF-alpha(-/-)) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-alpha(-/-) mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-alpha(-/-) mice after two sessions of immunization with the HBV plasmid; however, TNF-alpha was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-alpha(-/-) mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-alpha enhanced the proliferation of a TNF-alpha(-/-) clone but suppressed that of a TNF-alpha(+/+) clone in vitro, TNF-a. also has a direct effect on the proliferation of CTLs. In conclusion, TNF-alpha is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.

Background-The measurement of serum concentrations of cardiac troponin T (TnT) is a simple, useful method to detect myocyte injury that may be repeated multiple times to follow patients without interobserver variability. Methods and Results-Multiple measurements of TnT with a second-generation assay were performed in 60 patients with dilated cardiomyopathy confirmed by coronary angiography and endomyocardial biopsy between April 1996 and December 1999. Three evolutionary patterns of TnT concentrations were identified. Thirty-three patients had concentrations of TnT <0.02 ng/mL throughout the follow-up period (group 1). The remaining 27 patients had high initial serum concentrations of TnT (<greater than or equal to>0.02 ng/mL). In 10 of these 27 patients, TnT decreased to <0.02 ng/mL during follow-up (group 2), whereas 17 had persistently high serum TnT concentrations despite being conventionally treated for chronic congestive heart failure (group 3). Although the initial echocardiographic left ventricular diastolic dimension (LVDd) and left ventricular ejection fraction (LVEF) were not significantly different among the 3 groups, follow-up echocardiography showed significantly decreased LVDd and increased LVEF in group 1 (each P < 0.01) and group 2 teach P < 0.05) compared with increased LVDd and decreased LVEF in group 3 teach P < 0.05), The cardiac event-free rate was significantly lower in group 3 than in groups 1 and 2 teach P < 0.001), and the survival rate was lower in group 3 than in group 1 (P < 0.05). Conclusions-Persistently increased TnT concentrations in dilated cardiomyopathy suggest ongoing subclinical myocyte degeneration associated with deterioration of the patients' clinical status.

There is a type of arteriosclerosis with remodeling of middle-size arteries in which intimal hyperplasia of smooth muscle cells (SMCs) plays the main role, and there are few macrophages, T lymphocytes, and foam cells. It is unknown whether apoptosis and the expression of Bax, an inducer of apoptosis, are increased according to the progression of this stype of human arteriosclerosis, which is different from so-called atherosclerosis. Bax heterodimerizes with Bcl-2, an inhibitor of apoptosis, and the ratio of Bax to Bcl-2 determines cellular apoptosis or survival. Thus, we investigated apoptosis and the expression of Bax, bax mRNA, and Bcl-2 in human arteriovenous (AV) fistulas used for hemodialysis, a representative of arteriosclerosis of the aforementioned type. The material was 20 radial arteries obtained from 20 patients with chronic renal failure undergoing AV shunt surgery. SMCs, macrophages, and T lymphocytes were immunohistochemically identified at the light microscopic (LM) level. Apoptosis was detected by in situ terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-dUTP nick end labeling (TUNEL) at both the LM and electron microscopic (EM) level. Cell proliferating activity was estimated by proliferating cell nuclear antigen (PCNA). Bax and Bcl-2 were detected by immunohistochemistry and Western blot analysis. Expression of bax mRNA was detected by in situ hybridization. LM TUNEL-positive cells in both the intima and media were significantly increased according to the percent stenosis of the vessels. EM analysis revealed that ultrastructures of apoptotic SMCs were seen in both synthetic and contractile phenotypes. Their frequency of occurrence in the intima and media were greater in those vessels with &gt 50% stenosis than in those with &lt 50% stenosis (5.2±0.7% versus 1.0±0.7% versus 1.0±0.3% in the intima and 2.1±0.5% versus 0.2±0.1% in the media). The proportion of apoptotic SMCs with ruptured plasma membranes was greater than that of apoptotic SMCs with intact membranes in the intima of the former (4.1±0.6% versus 1.1±0.1%). Only those SMCs with apoptotic ultrastructures had TUNEL-positive nuclei with moderate or marked accumulation of immunogold particles at the EM level. However, ultrastructures of oncosis (primary necrosis) were not observed. Immunohistochemical analyses showed significantly positive correlations between percent stenosis of vessels and the percentage of either PCNA-positive intimal cells or Bax-positive areas in the intima and media. Bcl-2-positive cells were not observed in the intima but mainly in the outer media. The percentage of Bcl-2-positive medial cells was definitely decreased at an early stage after formation of the AV fistula but did not change according to the duration of hemodialysis or the progression of arteriosclerosis. Western blot analysis of Bax or Bcl-2 and in situ hybridization of bax mRNA confirmed the immunohistochemical data. Thus, regulation of cellularity in intimal hyperplasia of SMCs in human arteriosclerosis with remodeling is mediated by proliferation and apoptosis but not oncosis. The apoptosis is probably induced by an increase in the Bax to Bcl-2 ratio.

1. We investigated the acute effects of adrenaline infusion on plasma lipid levels in vehicle- and adriamycin-treated rabbits. Lipids were measured before and 30 and 60 min after the commencement of continuous intravenous administration of adrenaline (0.06 μg/kg per min) or saline in pentobarbital-anaesthetized rabbits. 2. Adrenaline infusion significantly increased plasma free fatty acid (P &lt 0.05) and noradrenaline (NA) levels (P &lt 0.05) in vehicle-treated control rabbits, but not in adriamycin-treated rabbits. However, adrenaline had no effect on plasma total cholesterol, free cholesterol, high-density lipoprotein-cholesterol, triglyceride or phospholipid levels. 3. Pretreatment with propranolol almost completely inhibited increased plasma free fatty acid and NA levels associated with adrenaline infusion, suggesting that adrenaline increases plasma free fatty acid and NA levels via the stimulation of β-adrenoceptors in vehicle-treated rabbits. 4. It is suggested that both the production of plasma free fatty acids and the release of NA via the activation of β-adrenoceptors is reduced in rabbits with adriamycin-induced cardiomyopathy. This may be related to the down-regulation of β-adrenoceptors caused by elevated plasma NA levels induced by cardiac failure.

We investigated the vasodilator effects of Captopril (CAP) on the systemic capacitance and resistance vessels by measuring changes in the mean circulatory pressure (MCP) and total peripheral resistance (TPR) that occurred in response to the intravenous injection of CAP (1 mg/kg) in open-chest dogs. The following five groups of dogs received CAP: (1) Group of untreated dogs, (2) TSA Group in which the dogs were subjected to total spinal anesthesia (TSA), (3) Ang II Group in which the dogs received a continuous intravenous infusion of angiotensin II (Ang II), (4) APR+Ang II Group in which the dogs received a continuous intravenous infusion of Ang II after pretreatment with aprotinin (APR, 25,000 K.I.E.), a kallikrein inhibitor, and (5) IND+APR+Ang II Group in which the dogs received a continuous intravenous infusion of Ang II after pretreatment with indomethacin (IND, 5 mg/kg), a prostaglandin synthesis inhibitor, and APR. 1) CAP significantly decreased TPR and MCP in the untreated Group. 2) CAP significantly decreased TPR and MCP in the TSA Group. There were no significant differences in percentage change of TPR (%ΔTPR) and percentage change of MCP (%ΔMCP) between the untreated and TSA Groups. 3) In the Ang II Group, CAP decreased the elevated TPR significantly, but hardly affected the elevated MCP. 4) CAP significantly decreased TPR without significant change in MCP in the APR+Ang II Group. The%ΔTPR was significantly smaller in this Group than in the Ang II Group. 5) CAP significantly decreased TPR without significant change in MCP in the IND+APR+Ang II Group. The%ΔTPR was significantly smaller in this Group than in the Ang II Group. When one compares the arterial system with the venous system, this study suggests that the kinin-kallikrein system activating action of CAP partly contributes to its vasodilator action on the systemic resistance vessels. © 1991, The Japanese Circulation Society. All rights reserved.