Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

Mutations of the epigenetics-modifying gene (DNMT3a, TET2, IDH1/2) at diagnosis may induce FLT3-ITD at relapse in de novo acute myeloid leukemia.

Gene mutations were found in acute myeloid leukemia (AML) and their importance has been noted. To clarify the importance and stability of mutations, we examined gene mutations in paired samples at diagnosis and relapse of 34 adult AML patients. Five acquired gene mutations were detected at relapse. Of the 45 gene mutations at diagnosis, 11 of them were lost at relapse. The acquired mutations at relapse were all class I mutations as Fms-like tyrosine kinase 3 (FLT3) and rat sarcoma viral oncogene homolog (RAS) mutations. The disappeared mutations at relapse were 3 of 11 internal tandem duplications of FLT3 (FLT3-ITD) (27.3%), 3 of 3 FLT3 tyrosine kinase domain (FLT3-TKD) (100%), 3 of 13 Nucleophosmin 1 (23.1%) and 2 of 5 CCAAT/enhancer-binding protein-α (40%) mutations. However, epigenetics-modifying gene (DNMT3a, TET2 and IDH1/2) mutations had no change between diagnosis and relapse samples, and may become minimal residual disease marker. The frequency of FLT3-ITD at relapse in patients with DNMT3a mutation at diagnosis is significantly higher than those in patients without them (P=0.001). Moreover, the high frequency of FLT3-ITD at relapse is also seen in AML cases that initially present with any epigenetics-modifying gene mutations (P<0.001). Our results indicate that epigenetics-modifying gene mutations may cause genetic instability and induce FLT3-ITD, leading to resistance to therapy and relapse.

Importance of c-kit mutation detection method sensitivity in prognostic analyses of t(8;21)(q22;q22) acute myeloid leukemia.

Recently, c-kit mutations have been reported as a novel adverse prognostic factor of acute myeloid leukemia with t(8;21)(q22;q22) translocation (t(8;21) AML). However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive mutation detection method known as mutation-biased PCR (MB-PCR) and investigated the relationship between c-kit mutations and prognosis. When c-kit mutations were analyzed for 26 cases of t(8;21) AML using the direct sequence (DS) and MB-PCR, the latter had a much higher detection rate of c-kit mutations at initial presentation (DS 5/26(19.2%) vs MB-PCR 12/26(46.2%)). Interestingly for the three cases, in which c-kit mutations were observed only at relapse with the DS, c-kit mutations were detected at initial presentation using the MB-PCR. This result suggests that a minor leukemia clone with c-kit mutations have resistance to treatment and are involved in relapse. In univariate analyses, the presence of a c-kit mutation using DS was not an adverse prognostic factor (P = 0.355), but was a factor when using MB-PCR (P = 0.014). The presence of c-kit mutations with MB-PCR was also an independent adverse prognostic factor by multivariate analyses (P = 0.006). We conclude that sensitivity of c-kit mutation detection method is important to predict prognosis for t(8;21) AML.

Albumin-binding superoxide dismutase with a prolonged half-life reduces reperfusion brain injury.

To test the possible involvement of superoxide radicals in the pathogenesis of reperfusion injury, we synthesized a superoxide dismutase (SOD) derivative [poly(styrene-co-maleic acid) butyl ester (SM) covalently linked to SOD] that circulates bound to albumin, has a prolonged in vivo half-life, and accumulates in pH-decreased tissues. The protective effects of SM-SOD on regional cerebral blood flow, intracranial pressure, cardiac index, vascular permeability, and neurological outcome were investigated using a model of global brain ischemia in dogs. Intra-arterial injection of SM-SOD (10 mg/kg) just before reperfusion increased reactive hyperemia (SM-SOD, 160 +/- 36 ml.100 g-1.min-1, means +/- SD, n = 6; control, 100 +/- 34 ml.100 g-1.min-1, n = 6, P = 0.015), ameliorated delayed hypoperfusion (7 h after ischemia: SM-SOD, 40 +/- 14 ml.100 g-1.min-1; control 17 +/- 6 ml.100 g-1.min-1, P = 0.003), vascular permeability, and neurological outcome without affecting the cardiac index. These results indicate that superoxide radicals and/or their metabolite(s) might play a critical role in the pathogenesis of reperfusion injury in the brain.

Effectiveness of pressure support ventilation for mechanical ventilatory support in patients with status asthmaticus.

We compared the effects of pressure support ventilation (PSV) with those of assist control ventilation (ACV) on breathing patterns and blood gas exchange in six patients with status asthmaticus. Both PSV and ACV delivered adequate minute ventilation (PSV: 7.5 +/- 1.4 l/min/m2, ACV: 7.3 +/- 1.3 l/min/m2) to correct respiratory acidosis (pH = 7.33 +/- 0.12 during both PSV and ACV) and prevent hypoxia. Peak airway pressure during PSV was significantly lower with the same tidal volume than that during ACV (PSV: 30 +/- 10 cmH2O (2.9 +/- 1.0 kPa), ACV: 50 +/- 13 cmH2O (4.9 +/- 1.3 kPa)). The lower airway pressure during PSV was due to persistent inspiratory muscle activity. The oxygen cost of breathing estimated by oxygen consumption was equivalent in both modes. We conclude that PSV is effective in supplying tidal volumes adequate to improve hypercarbia at markedly lower airway pressures than ACV.

The effect of pressure support ventilation on auto-PEEP in a patient with asthma.

We report the effect of pressure support ventilation (PSV) on auto-PEEP in a patient with asthma. The patient showed a high level of auto-PEEP during spontaneous breathing through a T-piece. PSV effectively decreased auto-PEEP and inspiratory muscle effort with increasing levels of PSV.

Frequency- and length-dependent tension development in rat heart muscles exposed to isoflurane and halothane.

Using 22 isolated rat ventricular muscle preparations, we investigated whether or not the increase in preload and/or contraction frequency may counteract the negative inotropy of both isoflurane (2.0%) and halothane (1.0%). Increases in preload from 94% of Lmax (the length where muscles produce the maximal tension) to Lmax did not alter significantly the percent decrements in tension development caused by either isoflurane or halothane. The increases in contraction frequency from 0.1 to 0.6 Hz augmented the depressant effect of isoflurane significantly ( P < 0.001), while the depressant effect of halothane was not altered at these contraction frequencies. Small but significant counteraction occurred in the depressant effects of halothane at 0.8 and 1.6 Hz ( P = 0.002). These changes in intracellular mechanism(s), resulted from the increase in contraction frequency, interacted with the two anesthetics on tension development, while these may not be the case for the increase in preload.

Bacterial endotoxin-induced expression of metallothionein genes in rat brain, as revealed by in situ hybridization.

In order to clarify acute-phase response in brain, we investigated induction of metallothionein (MT) genes by administrating an endotoxin (lipopolysaccharide) in rat intraperitoneum. We performed in situ hybridization on the serial brain sections to identify the cells expressing the MT genes in acute-phase. After endotoxin administration, transcripts of MT genes were detected in the arachnoideal, ependymal cells and glial cells around the Purkinje cells of the cerebellum, while no significant induction of the MT genes by zinc ion was observed in brain. These results suggest that the acute-phase response occurs specifically in at least these 3 non-neuronal cells.

[Is airway occlusion pressure useful to predict successful weaning from mechanical ventilation in patients with acute respiratory failure?].

The purpose of this study was to evaluate whether airway occlusion pressure (P0.1) would be a useful predictor for successful weaning in mechanically ventilated patients with acute respiratory failure. We studied 23 marginal weaning candidates. Fourteen patients were able to be weaned from the ventilator, and 9 patients were not able to be weaned. P0.1 and other respiratory parameters were measured just prior to weaning and at the end of weaning or at the time of discontinuation of weaning. The mean value of P0.1 in the failed group was higher than that in the successful group both before and after weaning periods. However, P0.1 varied widely among patients and did not separate the failure group from the success group because of overlap between the two. There were significant differences between the two groups of the conventional weaning parameters, such as respiratory rate, minute ventilation, PaO2, and oxygen equivalent. We conclude that P0.1 is helpful to predict successful weaning. However, it can not be used as a single parameter for weaning because of the wide variations of absolute values among patients with acute respiratory failure.

Pretreatment with d-tubocurarine, vecuronium, and pancuronium attenuates succinylcholine-induced increases in plasma norepinephrine concentrations in humans.

We studied in patients the effect of d-tubocurarine, which has sympathetic ganglion blocking action, on succinylcholine-induced increases in plasma levels of catecholamines, and compared it with the effects of vecuronium and pancuronium, which have little sympathetic ganglion blocking action. Thirty-two patients were divided into five groups: seven were given 3 mL saline; seven received 1 mg/kg succinylcholine; and six, seven, and five patients were given 0.08 mg/kg d-tubocurarine, 0.01 mg/kg vecuronium, and 0.01 mg/kg pancuronium, respectively, all of which were injected 5 min before 1 mg/kg succinylcholine. Succinylcholine alone significantly increased plasma norepinephrine concentrations, systolic blood pressure, and heart rate from 187 +/- 39 pg/mL (mean +/- SEM), 93 +/- 2 mm Hg, and 77 +/- 4 beats/min to 429 +/- 61 pg/mL, 120 +/- 7 mm Hg, and 102 +/- 6 beats/min, respectively, with onset of fasciculations. Pretreatment with d-tubocurarine, vecuronium, and pancuronium significantly and equally attenuated both the fasciculations and the cardiovascular responses to succinylcholine. These results suggest that the sympathetic ganglion blocking action of neuromuscular relaxants when given before succinylcholine is not an important factor in attenuation of succinylcholine-induced increases in plasma levels of catecholamines.

Neuromuscular effects of respiratory and metabolic acid-base changes in vitro with and without nondepolarizing muscle relaxants.

The effects of metabolic (bicarbonate, [HCO3]) and respiratory (carbon dioxide, PCO2) acid-base changes on indirectly elicited twitch tension with and without nondepolarizing neuromuscular blocking agents were compared in a rat phrenic nerve-hemidiaphragm preparation. Ionized calcium [Ca2+] and magnesium [Mg2+] concentrations in the modified Krebs' solution were measured and kept constant. Likewise, twitch was altered when pH changes were produced by altering either PCO2 or [HCO3]. Decreasing pH either by increasing PCO2 or by decreasing [HCO3] significantly decreased (P less than 0.01) twitch, by 9.5 +/- 0.6 (SEM, n = 8) and 10.6 +/- 1.5%, respectively. Increasing pH by decreasing PCO2 or by increasing [HCO3] significantly increased (P less than 0.01) twitch, by 5.6 +/- 0.9 and 7.9 +/- 0.6%, respectively. After a partial depression of twitch by nondepolarizing neuromuscular blocking agents, the effects of PCO2 and [HCO3] changes were again assessed. Decreasing pH by increasing PCO2 or by decreasing [HCO3] intensified d-tubocurarine (dTc) (28.2 +/- 1.6 and 32.0 +/- 2.9%, respectively) and vecuronium (23.0 +/- 1.4 and 36.8 +/- 3.2%, respectively) block, whereas it reversed metocurine (1.2 +/- 2.2% NS and 2.9 +/- 1.3%, respectively) and pancuronium (8.3 +/- 1.5 and 11.5 +/- 3.0%, respectively) block. Conversely, increasing pH by decreasing PCO2 or by increasing [HCO3] antagonised dTc (12.8 +/- 2.2 and 13.6 +/- 1.8%, respectively) and vecuronium (25.3 +/- 1.7 and 25.0 +/- 3.0%, respectively) block, whereas it potentiated metocurine (4.2 +/- 0.6 and 8.0 +/- 1.1%, respectively) and pancuronium (11.0 +/- 1.2 and 17.5 +/- 2.0%, respectively) block. Except where indicated, all changes in block described above were statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic and additive induction of metallothionein in Chang liver cells. A possible mechanism of marked induction of metallothionein by stress.

We have studied metallothionein (MT) induction by three primary inducers; a heavy metal, a glucocorticoid and a cytokine, and by the combinations of these inducers in the cultured cells. MT-protein was synergistically induced by either a cytokine or a heavy metal with a glucocorticoid hormone and was additively induced by the combination of a cytokine and a heavy metal, but MT-mRNA levels were not completely correlated with MT-protein levels. These results suggest that posttranscriptional regulation may be involved in the synergistic induction of MT-protein. We propose a possible mechanism in which marked MT induction by stress in vivo is dependent on the combined effect of two or more inducers, because marked MT induction is not seen by an injection of a plausible dose of either a glucocorticoid hormone or a cytokine in vivo.

Interaction of sevoflurane, isoflurane, enflurane and halothane with non-depolarizing muscle relaxants and their prejunctional effects at the neuromuscular junction.

The interaction of four inhalational anesthetics (sevoflurane, isoflurane, enflurane and halothane) with pancuronium and vecuronium and also their prejunctional actions at the neuromuscular junction were quantitatively studied using rat phrenic nerve-hemidiaphragm preparations. To investigate the prejunctional effects of inhalational anesthetics, a train-of-four ratio (T4/T1) and the tetanus ratio (the ratio of the final response to the initial response during tetanus) were evaluated. All four inhalational anesthetics markedly potentiated the neuromuscular blockade of twitch response caused by either pancuronium or vecuronium with halothane and enflurane being the most potent both on a % concentration basis and on a MAC (minimum alveolar concentration) basis. Although none of the four inhalational anesthetics had any effects on the T4/T1 ratio, they produced variable effects on the tetanus ratio. Sevoflurane had little effect on the tetanus ratio, whereas 1 and 2% isoflurane and 1, 2 and 3% enflurane increased the tetanus ratio and 5% halothane and 5% enflurane significantly reduced the tetanus ratio. Halothane and enflurane had the most potent depressant action of the four inhalational anesthetics both on the % concentration basis and on the MAC basis. These results indicate that the main site of action of inhalational anesthetics is a postjunctional site at the neuromuscular junction and that they do not seem to act on prejunctional sites at the concentrations used in clinical situations.

Efficacy of flow-by during continuous positive airway pressure ventilation.

We investigated clinically the differences in respiratory work of patients imposed by three modes of one ventilator: the flow-by system, the demand valve system, and the pressure support system. Inspiratory work using flow-by and pressure support systems was reduced sufficiently when compared to the demand valve system. Moreover, fluctuation of the airway pressure was minimal with the flow-by mode. These results suggest that the flow-by mode is beneficial to patients breathing spontaneously with continuous positive airway pressure.

[The effect of pressure support ventilation on breathing patterns and the work of breathing].

We assessed breathing patterns during pressure support ventilation (PSV) and its relationship with the work of breathing in 10 postoperative patients. With increasing levels of pressure support, minute ventilation and tidal volume increased with a decrease in respiratory frequency. Increased minute ventilation was achieved by increased mean inspiratory flow. Duty cycle, however, decreased with PSV. This decrease might allow the diaphragm a longer rest period between contractions, which might decrease the risk of diaphragmatic fatigue. Furthermore, PSV reduced the inspiratory work added by a ventilator to near zero. Oxygen consumption was also decreased with PSV. We conclude that PSV improved the breathing patterns and minimized the work of breathing spontaneously via a ventilator.

Histological changes of neuronal damage in vegetative dogs induced by 18 minutes of complete global brain ischemia: two-phase damage of Purkinje cells and hippocampal CA1 pyramidal cells.

We have developed a functional vegetative model by an 18-min clamping of the ascending aorta combined with a bypass formation between the aorta to right atrium and the aorta to femoral vein. Complete global brain ischemia (CGBI) induced for 18 min with this model provided the following distinct advantages: cardiopulmonary functions were well preserved during postischemic recirculation, and all dogs survived without serious extracerebral complications. Neuronal damage in vegetative dog induced by an 18-min CGBI was studied by light and electron microscopy. The Purkinje cells and the hippocampal CA1 pyramidal cells showing clumping of nuclear chromatin and slightly increased stainability were observed after CGBI without recirculation. All these neurons showed transient increased stainability with microvacuolation 15 min after recirculation. Over 50% of these neurons showed virtually normal features 1 h after recirculation. Damage to these neurons progressed again slowly up to 6 h after recirculation. However, all these neurons had disintegrated 2-3 days after recirculation. A decrease in synaptic vesicles was observed in many presynaptic terminals in the molecular layers of the cerebellum after CGBI without recirculation. These changes in the presynaptic terminals progressed 15 min after recirculation. These results indicated that the damage to the Purkinje cells and the CA1 pyramidal cells induced by CGBI consisted of two phases, and that the change in the early phase was reversible. We speculate that the damage to the Purkinje cells in the early stage is related to the decrease of the synaptic vesicles in the presynaptic terminals.

Involvement of transforming growth factor-alpha secreted by macrophages in metallothionein induction by endotoxin.

The mechanism of metallothionein (MT) induction of the liver by endotoxin, which is mediated by a factor secreted by endotoxin-stimulated macrophages, was studied in vitro. MT induction of the liver cells by the endotoxin-stimulated macrophage conditioned medium was inhibited by a monoclonal antiepidermal growth factor (EGF) / transforming growth factor-alpha (TGF-alpha) receptor antibody, which acts as an antagonist of EGF and TGF-alpha. MT was induced by the substance, which was adsorbed by polyclonal antibody to TGF-alpha, but not by a monoclonal antibody to EGF, in the conditioned medium of endotoxin-stimulated macrophages. These results suggest that TGF-alpha secreted by macrophages is involved in MT induction by endotoxin.

[The effect of succinylcholine on vecuronium and pancuronium].

In 105 adult patients under halothane anesthesia, the neuromuscular blocking effects of vecuronium and pancuronium were determined with prior succinylcholine 1 mg.kg-1 administration and without. Force of the evoked twitch increased 123.7% of control after recovery from succinylcholine-induced block. Prior administration of succinylcholine was associated with a leftward shift of dose-response curve of vecuronium or pancuronium. Onset of the force reduction from initial dose (0.08 mg.kg-1) was faster and recovery from initial and maintenance doses (0.02 mg.kg-1) were slower. This potentiating effect persisted at least 2 hours.