Differential expression of CD45RO (UCHL1) and its functional relevance in two subpopulations of circulating TCR-gamma/delta+ lymphocytes.

We examined the developmental profile of TCR-gamma/delta+ cells with respect to CD45RO expression. Although total TCR-gamma/delta+ cells were negligible in the neonatal blood and increased with advancing age, most blood TCR-gamma/delta+ cells markedly expressed CD45RO without a distinction of age, probably reflecting a different CD45RO expression of two subsets defined by BB3 and delta TCS1 mAbs. The vast majority of BB3+ cells expressed CD45RO, whereas expression of CD45RO was virtually absent in the delta TCS1+ population. Functional studies revealed that, while both TCR-gamma/delta+ cell subsets showed CD3-mediated activation, only BB3+ (or Ti gamma A+) cells, but not delta TCS1+ cells, appeared to proliferate in response to PPD in PPD-reactive individuals. The results suggested that the CD45RO+ (BB3+ or Ti gamma A+) subset among blood TCR-gamma/delta+ cells may be mainly involved in the memory or primed component of the immune system responding to some foreign antigens.

Interleukin-10 inhibits apoptotic cell death in infectious mononucleosis T cells.

T lymphocytes from patients with acute EBV-induced infectious mononucleosis rapidly die by apoptosis in vitro. Because human and viral IL-10 are likely to be induced during acute EBV infection and display a variety of functions on human T cells, we examined IL-10 effects on infectious mononucleosis T cell death. After 12 h of incubation in medium alone, only 35.6 (+/- 8.2%) of the originally seeded infectious mononucleosis T cells were viable. Addition of human IL-10 (100 U/ml) to T cell cultures significantly improved recovery of viable cells (71.3 +/- 6.2%, P = 0.0156). Viral IL-10 had comparable effects to human IL-10 in this system. Protection from death by human and viral IL-10 (100 U/ml) was dose dependent and continued over a 6-d culture period. The human IL-10 effect was neutralized by the anti-human IL-10 mAb 19F1. Morphology and analysis of DNA after separation on agarose gels showed that IL-10 inhibits loss of cell volume, chromatin condensation, and DNA fragmentation, characteristics of death by apoptosis. As assessed by [3H]thymidine incorporation, the T cells were not induced to proliferate by IL-10 above the level exhibited when first removed from blood. T cells protected from death by IL-10 proliferated to IL-2 and spontaneously killed sensitive targets as effectively as medium-precultured T cells. Thus, IL-10 promotes the survival of infectious mononucleosis T cells otherwise destined to die by apoptosis and may be critical for the establishment of immunologic memory after resolution of the illness.

Proton flow along lipid bilayer surfaces: effect of halothane on the lateral surface conductance and membrane hydration.

Impedance dispersion in liposomes measures the lateral charge transfer of lipid membrane surfaces. Depending on the choice of frequency between 1 kHz and 100 GHz, relaxation of the counterions at the interface, orientation of the head group, and relaxation of the bound and free water are revealed. This study measured the impedance dispersion in dipalmitoylphosphatidylcholine (DPPC) liposomes at 10 kHz. The surface conductance and capacitance showed breaks at pre- and main transition temperatures. Below the pre-transition temperature, the activation energy of the ion movement was 18.1 kJ.mol-1, which corresponded to that of the spin-lattice relaxation time of water (18.0 kJ.mol-1). At temperatures between pre- and main transition it increased to 51.3 kJ.mol-1, and agreed with 46.2-58.0 kJ.mol-1 of the activation energy of the dielectric relaxation of ice. Because the present system was salt-free, the ions were H3O+ and OH-, hence, their behavior represents that of water. The above results show that below the pre-transition temperature, the conductance is regulated by the mobility of free ions, or the number of free water molecules near the interface. On the other hand when the temperature exceeded pre-transition, melting of the surface-bound water crystals became the rate-limiting step for the proton flow. Halothane did not show any effect on the ion movement when the temperature was below pre-transition. When the temperature exceeded pre-transition, 0.35 mM halothane (equilibrium concentration) decreased the activation energy of the ion movement to 29.3 kJ.mol-1. This decrease indicates that halothane enhanced the release of the surface-bound water molecules at pre-transition. The surface-disordering effect of halothane was also shown by depression of the pre-transition temperature and decrease of the association energy among head groups from 9.7 kJ.mol-1 of the control to 5.2 kJ.mol-1 at 0.35 mM.

Changes in surface capacitance and conductance parallel to phospholipid membranes associated with phase transition: effects of halothane.

The effects of phase transition on the surface capacitance and conductance parallel to dipalmitoyl- (DPPC) and dimyristoyl-phosphatidylcholine (DMPC) membranes were studied by impedance dispersion. The phospholipid aggregates were embedded into pores of a polycarbonate filter and the impedance dispersions were measured at a frequency range from 30 Hz to 1.0 MHz. When the frequency was below 120 kHz, the capacitance showed a peak at the pretransition temperature and a steep rise at the main-transition temperature. In this system, the observed capacitance consists of frequency-dependent and -independent parts. The frequency-dependent part is a surface phenomenon and arises from the lateral motion of counterions at the membrane/water interface. The frequency-independent part represents mainly the properties of the bulk lipid phase. Addition of halothane decreased the total capacitance of the DPPC aggregates at the low frequency range to 1/2 to 1/8 of the control depending upon the temperature. The surface component was solely responsible for this capacitance decrease, because the non-surface component was slightly increased instead. The data suggest that halothane inhibited the lateral ionic flow parallel to the interface.

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.

Involvement of presurgical pain in preemptive analgesia for orthopedic surgery: a randomized double blind study.

Preemptive analgesia (PA) is effective in animal models but its clinical effectiveness remains controversial. We examined the effect of preexisting pain on PA. Subjects were recruited from patients needing orthopedic surgery. Some had presurgical pain (fracture surgery and arthritic surgery), while others had no presurgical pain (removal surgery for a tumor, nail or plate). Epidural morphine or a saline control was given preemptively before surgery and maintained until skin closure. Following skin closure, naloxone or placebo was injected intravenously to erase the aftereffects of the morphine. After total recovery, the PCA pump was set to inject epidural morphine. Pain intensity after surgery was measured by a visual analogue scale (VAS), and the amount of morphine used within 48h after surgery. PA was significantly effective for removal surgery, but ineffective for fracture or arthritic surgery. For the fracture and arthritic surgery PA treatment groups, there was a significant correlation between pre- and postsurgical (6h) spontaneous pain, while the corresponding control groups showed no significant correlation. Postsurgical VAS values in the fracture and arthritic surgery control groups increased significantly compared with presurgical VAS values. PA was effective when presurgical pain was absent, but ineffective when presurgical pain was present. We propose that central sensitization is already established by presurgical pain, and preserved until the termination of surgery. The ineffectiveness of PA did not depend on whether the pain was acute (fracture surgery) or chronic (arthritic surgery).

Ischemic tolerance preserves propagation of membrane depolarization.

The functional integrity of the synaptic connections within the hippocampus in gerbils that had acquired ischemic tolerance was investigated. The propagation of membrane depolarization across the hippocampus in response to electrical stimulation of CA1 was monitored with the use of a high speed optical recording technique. In comparison to control slices, propagation was significantly depressed and depolarization was shortened in slices from gerbils subjected to 5 min of ischemia. Hippocampal slices from gerbils who were preconditioned with prior sublethal ischemia demonstrated only a slight reduction in propagation. The duration of depolarization was longer than that of ischemia group. These findings suggest that ischemia induces a functional disturbance of synaptic transmission and membrane depolarization. Ischemic preconditioning significantly reduced the extent of this functional disturbance.

A forebrain ischemic preconditioning model established in C57Black/Crj6 mice.

Although many kinds of rat and gerbil cerebral ischemic preconditioning models are available, only a focal ischemic preconditioning model in mice has been reported. As most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice. In the present study, we developed a forebrain ischemic preconditioning model in C57Black/Crj6 (C57BL/6) mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO. On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 immunohistochemistry and quantified by cresyl violet staining. Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) was performed 72 h after reperfusion to detect DNA fragmentation. Ischemia for 18 min resulted in injury to the striatum, cortex and hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia itself caused neither noticeable striatal neuronal damage nor DNA fragmentation, it significantly reduced striatal neuronal damage and DNA fragmentation caused by the subsequent 18-min ischemia. These results indicate that striatal neuronal injury after transient BCCAO can be strongly reduced by a sublethal ischemic episode in C57BL/6 mice. As many kinds of gene-altered C57BL/6 mice are available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice.

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices.

As alterations in intracellular pH (pH(i)) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH(i) in rat hippocampal slices using the BCECF fluorescent technique. The 'resting' pH(i) in the CA1 pyramidal cell layers was 6.93 +/- 0.07 (mean +/- S.D., n = 72 slices) in 25 mM HCO3-/5% CO2-buffered solution at 37 degrees C. Exposure of hippocampal slices to NMDA in the range of 10-1000 microM produced a biphasic change in pH(i): an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 microM) but not CNQX (40 microM) blocked the NMDA-induced changes in pH(i). In 0 Ca medium (0 mM Ca2+ supplemented 1 mM EGTA, referred to as 0 Ca), pH(i) acid shift caused by NMDA (20 microM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca2+ ([Ca2+]i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH(i) acid shift by two-thirds, while the NMDA-induced pH(i) alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH(i) acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH(i). The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca2+]i. The pH(i) acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.

Effects of pentobarbital and ketamine on brain injury-induced anti-ischemic activity.

Survival rates following incomplete brain ischemia induced during pentobarbital anesthesia were significantly higher in mice with a minor brain injury, inflicted one week before, than in those given a sham operation. Improvement of the survival rates in mice with brain injury, however, became insignificant when brain ischemia was imposed during ketamine anesthesia, suggesting that the actions of certain factors or protective mechanisms against brain ischemia, developed by brain injury, are antagonized by ketamine and/or potentiated by barbiturate anesthesia.

Prior brain injury protects death from local anaesthetic-induced convulsion.

Minor brain injury was inflicted with a small hypodermic needle at four sites from the scalp 7 days before the production of convulsion by i.p. injection of 100 mg/kg lidocaine in mice. The latency to convulsion and survival rate were significantly longer and higher, respectively, in the brain-injured group than in the sham-operated one. Thus, the results suggest that a protective mechanism develops in the injured brain against asphyxia caused by lidocaine convulsion.

Prostaglandin E1 modifies porcine cerebral arterial tone through cyclooxygenase related eicosanoid(s) release.

The effects of prostaglandin E1 (PGE1) on porcine cerebral arteries were studied in the absence and presence of cyclooxygenase inhibitors, 5 x 10(-6) M indomethacin or 10(-4) M aspirin. In preparations placed at a resting tension, 3 x 10(-9) to 10(-6) M PGE1 caused dose-dependent contractions. In prostaglandin F2 alpha (PGF2 alpha)- contracted preparations, low concentrations of 10(-9) and 3 x 10(-9) M PGE1 did not modify arterial tone and higher concentrations of 10(-8) to 10(-6) M PGE1 further increased the arterial tone in all preparations tested. In contrast, in the presence of the cyclooxygenase inhibitors, low concentration of 10(-9) and 3 x 10(-9) M PGE1 markedly decreased the arterial tone induced by PGF2 alpha and higher dose of 10(-8) to 10(-6) M PGE1 increased the arterial tone in 15 of 18 preparations (83%). These findings suggest that PGE1 modifies porcine cerebral vascular tone at least partly through cyclooxygenase-related eicosanoid(s) production.

Arginine induces apoptosis and gene expression of pancreatitis-associated protein (PAP) in rat pancreatic acinar AR4-2J cells.

Arginine-induced pancreatic acinar cell injury has been reported in vivo, but the mechanism involved is unknown. In this study we investigated the effects of arginine on the cell morphology and pancreatitis-associated protein (PAP) gene expression in rat pancreatic acinar AR4-2J cells in vitro. Arginine inhibited the proliferation of AR4-2J cells in a dose-dependent manner. This decrease in proliferation was due to an increase in apoptosis, as assessed by cell morphology and DNA fragmentation. PAP messenger RNA (mRNA) was expressed at doses of 2.5 and 5.0 mg/ml of arginine, and a time-course study showed that the expression started 2 h after arginine addition and peaked at 6 h. Apoptosis was rarely seen when PAP mRNA was highly expressed, but occurred when PAP mRNA expression was decreased. These results suggest that arginine induces apoptosis and PAP gene expression in pancreatic acinar cells and that PAP might inhibit the induction of apoptosis.

Target cell-induced apoptosis in IL-2-activated human natural killer cells.

We demonstrated that tumor cells induce cell death in lymphokine-activated NK (LAK) cells, but not in non-activated NK cells. Cell death in LAK cells involves nuclear condensation and DNA cleavage, all of which are characteristic features of apoptosis. The mechanism involves signaling through integrins and requires src family tyrosine kinases and protease activities. Engagement of an apoptotic signal molecule, Fas, may also trigger LAK cell death by apoptosis. It appears that LAK cells rapidly die by apoptosis after attacking tumor cells. This phenomenon may provide a means for potential tumor target cells to escape from natural immunosurveillance during therapeutic interventions such as those using IL-2 or LAK cells.

Sublethal cerebral ischemia inhibits caspase-3 activation induced by subsequent prolonged ischemia in the C57Black/Crj6 strain mouse.

Caspase-3 activation has been implicated in ischemic neuronal death. In the present study, we examined if cerebral ischemic tolerance induced by sublethal ischemia is associated with an attenuation of caspase-3 activation in a mouse forebrain ischemia model. Forebrain ischemia in C57Black/Crj6 strain mice was induced by bilateral common carotid artery occlusion (BCCAO) for 18 min. Two episodes of 6-min ischemia were carried out as preconditioning 48 and 72 h before the 18-min BCCAO. Caspase-3-like activity was determined by fluorescently monitoring the release of amino-4-methylcoumarin from N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin in the striatal protein extracts at 4, 24, and 72 h after reperfusion. The results showed that the ischemic preconditioning significantly attenuated caspase-3 activation at 4, 24, and 72 h after reperfusion, and reduced neuronal loss caused by the 18-min ischemia as examined on the 7th day after reperfusion. The present results suggest that the neuroprotection achieved by ischemic preconditioning is related to an attenuation of caspase-3 activation.

The effects of anesthetics on cortical spreading depression elicitation and c-fos expression in rats.

The effects of anesthetics on the generation of cortical spreading depression (CSD) were investigated. Volatile anesthetics halothane, isoflurane, sevoflurane (0.5, 1.0, and 2.0 MAC), and the intravenous anesthetic pentobarbital were studied. Cortical spreading depression was induced by 3M-KCl applied to a surface of brain cortex for 30 minutes. Direct current (DC) potential was recorded, and the number, amplitude, and duration of CSDs were observed. With increasing concentrations of each volatile anesthetic, there was a dose-related reduction in CSD frequency but not in CSD amplitude. At 2.0 MAC of sevoflurane the suppression of CSD was less than with the other volatile anesthetics. In addition, the influence of anesthetics on expression of c-fos mRNA was investigated. Additional animals anesthetized by isoflurane or sevoflurane were studied. Five CSDs were elicited by electric stimulation (0.5 mV, 1 second) in each animal. In situ hybridization with 35S-labeled oligonucleotides was used to evaluate the level of c-fos mRNA. The expression of c-fos was observed in the hemisphere in which CSD was elicited, but there was no difference in expression of c-fos among the groups. We conclude that volatile anesthetics can induce suppression of CSD elicitation in a dose dependent manner, but that at high concentrations sevoflurane is significantly less effective than other volatile agents. Pentobarbital has the least effect on KCl-induced CSD. These data suggest that the choice of anesthetics can impact the results of studies examining membrane depolarization and the ionic changes initiated by CSD.

[Isolation of Vibrio cholerae in imported frozen seafood and their cholera-enterotoxin production].

A survey study for Vibrio cholerae in imported seafood was conducted during January 1991 to December 1994. A total of 7,439 specimens (approximately 20% of all imported food) were randomly picked up and examined for contamination of V. cholerae. Among these, V. cholerae O1 were isolated from 9 specimens, but they were all cholerae enterotoxin (CT)-negative. In terms of V. cholerae non-O1, a total of 2,803 specimens (37.4%) were contaminated with this vibrio. Shrimp, especially the ones still in their shells and imported from Asian countries such as India and Indonesia, were highly contaminated with V. cholerae. Although no strains of V. cholerae O1 isolated in this study produced CT, 2 strains of V. cholerae non-O1 were proved to be CT-producers. Taking together the high contamination of V. cholerae in imported seafood and a part of those strains producing CT, we believe that careful survey for the possible contamination of V. choleare in imported seafood is necessary.

[Effects of halothane and isoflurane on the canine duodenal paraneurons].

Administration of amino acid solution (50 mM tryptophane and phenylalanine in saline) into the canine duodenum is known to cause an increase in pancreatic secretion. This response is mediated by the excitation of duodenal endocrine cells, paraneurons, which release cholecystokinin (CCK) into the systemic circulation in response to intraluminal amino acid stimuli. Pancreatic secretory cells are then evoked by the CCK in the blood to secrete the juice into the duodenum. The authors investigated the effects of two general anesthetics, halothane and isoflurane, on this response. Nine mongrel dogs were subjected to this study. Each dog underwent laparotomy under nitrous oxide (75%)-oxygen (25%) anesthesia with pancuronium (GO-Pb). The duodenal loop was exposed and two polyethylene cannulae (18Fr) were introduced into the loop. Proximal cannula was for the administration of the amino acid solution into the loop, and distal one was for drainage of the solution. The pancreatic duct was inserted with a polyethylene catheter, through which pancreatic juice was collected and measured for the volume and protein output by spectrophotometry. After these surgical procedures, the pancreatic secretory response to intraluminal amino acid stimuli was examined under GO-Pb (Control). Then halothane (1.0%) (Group 1, four dogs) or isoflurane (2.0%) (Group 2, five dogs) was administered for 30 min and the same response was tested. The pancreatic secretory response to intraluminal amino acid stimulus was suppressed by the surgical concentrations of both halothane (1.0%) and isoflurane (2.0%). Neither halothane nor isoflurane suppressed the pancreatic secretory response evoked by intravenous CCK infusion (10 Ivy Dog Units.(ABSTRACT TRUNCATED AT 250 WORDS)

[Digital imaging microscopy for intracellular Ca2+ in cultured single rat vascular smooth muscle cells using fluorescent Ca2+ indicator "fura-2"].

We presented the principle and methodology of digital imaging microscopy for intracellular Ca2+ in cultured single vascular smooth muscle cells of the rat using fluorescent Ca2+ indicator "fura-2". The methods seemed useful for studying the physiological and pathological phenomena in a single smooth muscle cell. Analysis of the spatial and temporal dynamics of intracellular Ca2+ might be crucial for studying the active sites of vasoactive or anesthetic drugs in a vascular smooth muscle cell as well as for understanding pathophysiology of the arterial spasm.

[Anesthetic management for pericardial fenestration in a hypertrophic cardiomyopathy (HCM) patient with massive pericardial effusion].

A 65 year-old male with HCM had progressively increased pericardial effusion. He also had atrial fibrillation (af), cardiac systolic dysfunction and chronic renal failure needing hemofiltration. Pericardial fenestration was carried out to improve diastolic function. Anesthetic management with fentanyl plus low-dose propofol infusion and postoperative analgesia with epidural morphine were effective for hemodynamic stability to prevent myocardial depression and to control ventricular response to atrial fibrillation. Intraoperative trans-esophageal echocardiography (TEE) monitoring was very useful for fluid therapy, inotropic support and estimation of systolic and diastolic function.