Diverse underlying proliferation response to growth factors in imatinib-treated Philadelphia chromosome-positive leukemias.

Since BCR-ABL plays an essential role in the growth factor-independent proliferation of Philadelphia chromosome (Ph)+ leukemia cells, imatinib treatment of Ph+ leukemia cells inactivates signaling pathways of BCR-ABL, and subsequent addition of growth factors (GFs) could restore the signaling pathways without reactivating BCR-ABL. Here we demonstrated that non-lymphoid Ph+ leukemia cell lines responded to diverse GFs depending on their immunophenotype and gene expression of transcription factors and GF receptors, while lymphoid Ph+ leukemia cell lines restrictively responded to flit3 ligand and interleukin-7, suggesting that GF sensitivity of imatinib-treated Ph+ leukemia cells could be powerful for specifying their distinctive lineage.

Resistance of T-cell acute lymphoblastic leukemia to tumor necrosis factor--related apoptosis-inducing ligand-mediated apoptosis.

OBJECTIVE: Cytotoxic ligands are involved in tumor immunity and graft-vs.-leukemia effect after allogeneic stem cell transplantation for leukemia. To clarify the susceptibility of T-cell acute lymphoblastic leukemia (T-ALL) to tumor immunity, sensitivity to recombinant human soluble Fas ligand (rhsFasL) and tumor necrosis factor-related apoptosis-inducing ligand (rhsTRAIL) was determined. MATERIALS AND METHODS: Sensitivity to rhsFasL and rhsTRAIL and cell surface expression of their receptors were tested in T-ALL cell lines (n = 7) and patients' samples (n = 17) and compared with those in B-precursor ALL cell lines (n = 30). Expression of components of the death-inducing signaling complex and the TRAIL receptor genes (DR4/DR5), and the methylation status and promoter activity of the DR4/DR5 gene were tested in T-ALL cell lines. RESULTS: T-ALL cell lines showed higher level of Fas expression and higher sensitivity to rhsFasL than did B-precursor ALL cell lines. Despite comparable expression of components of death-inducing signaling complex, cell lines and patients' samples of T-ALL showed TRAIL-resistance associated with low cell surface expression of DR4/DR5. Gene expression of DR4/DR5 in T-ALL cell lines was significantly lower than that in B-precursor ALL cell lines, and the methylation status of the gene promoter in T-ALL cell lines was associated with the gene expression level at least for DR4. The demethylating agent, 5-aza 2'deoxycytidine, upregulated the gene expression of DR4/DR5, but was insufficient for their surface expression due to low basal promoter activity. CONCLUSIONS: In contrast to higher sensitivity to FasL, T-ALL showed resistance to TRAIL, which might be responsible for resistance to TRAIL-mediated cellular immunity.

Fms-like tyrosine kinase 3 ligand stimulation induces MLL-rearranged leukemia cells into quiescence resistant to antileukemic agents.

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in acute lymphoblastic leukemia with the mixed-lineage leukemia (MLL) gene rearrangement refractory to chemotherapy. We examined the biological effect of FLT3-ligand (FL) on 18 B-precursor leukemic cell lines with variable karyotypic abnormalities, and found that nine of nine MLL-rearranged cell lines with wild-type FLT3, in contrast to other leukemic cell lines, are significantly inhibited in their proliferation in a dose-dependent manner by FL. This inhibition was due to induction of the G0-G1 arrest. A marked up-regulation of p27 by suppression of its protein degradation and an abrogation of constitutive signal transducers and activators of transcription 5 phosphorylation were revealed in arrested leukemia cells after FL stimulation. Importantly, FL treatment rendered not only cell lines but also primary leukemia cells with MLL rearrangement resistant to chemotherapeutic agents. MLL-rearranged leukemia cells adhering to the bone marrow stromal cell line, which expresses FL as the membrane-bound form, were induced to quiescent state resistant to chemotherapeutic agents, but their chemosensitivity was significantly restored in the presence of neutralizing anti-FL antibody. The FL/FLT3 interaction between leukemia cells and bone marrow stromal cells expressing FL at high levels should contribute, at least in part, to persistent minimal-residual disease of MLL-rearranged leukemia in bone marrow.

Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening.

The Na(+)/H(+) exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca(2+) overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca(2+) overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and the mitochondrial membrane potential (DeltaPsi(m)) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 microM) prevented ouabain-induced hypercontracture (from 40 +/- 2 to 24 +/- 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca(2+)](m) overload (from 149 +/- 6 to 121 +/- 5% of the baseline value, P < 0.05) but did not affect DeltaPsi(m). These results indicate that cariporide attenuates the [Ca(2+)](m) overload without the accompanying depolarization of DeltaPsi(m). Moreover, cariporide increased the time taken to induce the MPT (from 79 +/- 11 to 137 +/- 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 +/- 3 to 50 +/- 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca(2+)](m) overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.

Monitoring neutrophil engraftment in allogeneic stem cell transplantation by flow cytometric analysis of neutrophil-specific antigens NA1 and NA2.

Neutrophil-specific antigen (NA) expression on neutrophils was analysed in 18 Japanese children before and after allogeneic stem cell transplantation (allo-SCT) with myeloablative regimen. Donor-recipient NA-incompatibility was present in one of eight NA1/NA2 heterozygous patients and eight of 10 NA1/NA1 or NA2/NA2 homozygous patients. After allo-SCTs from NA-incompatible donors, a neutrophil recipient-to-donor conversion was confirmed in all cases. Conversion to donor NA type was complete before the absolute neutrophil count reached 0.1 x 10(9)/l. These observations indicate that flow cytometric analysis of NA antigens is a simple and useful method for monitoring neutrophil engraftment in NA-incompatible allo-SCT.

Stanniocalcin 1 prevents cytosolic Ca2+ overload and cell hypercontracture in cardiomyocytes.

BACKGROUND: The aim of the present study was to examine whether stanniocalcin 1 (STC1) affects cardiomyocytes under physiological or pathophysiological conditions. METHODS AND RESULTS: Using fresh isolated rat cardiomyocytes, the effects of STC1 on cell hypercontracture, cell shortening and Ca(2+) transients were measured after exposing the cells to ouabain. STC1 alone did not affect cell shortening or the Ca(2+) transient. Exposure to ouabain significantly increased the fraction of hypercontractured cells (40.5+/-1.4% vs 3.5+/-1.7% in the control, p<0.01). However, treatment with STC1 decreased the percentage of cell hypercontracture that was induced by ouabain, in a concentration-dependent manner (17.4+/-2.6% at 2.5 nmol/L STC1, p<0.01). Moreover, STC1 prevented the increase in diastolic intracellular Ca(2+) level that was induced by ouabain (-5.3+/-2.7% vs 7.9+/-3.7% induced by ouabain, p<0.05; -15.3+/-5.1% in the control) in the cardiomyocytes. CONCLUSIONS: STC1 prevented the increase in diastolic Ca(2+) overload and ouabain-induced cell hypercontracture, which suggests that STC1 could effectively prevent cytosolic Ca(2+) overload and protect cardiomyocytes from pathophysiological conditions such as in the failing heart.

Genetic abnormalities involved in t(12;21) TEL-AML1 acute lymphoblastic leukemia: analysis by means of array-based comparative genomic hybridization.

The TEL (ETV6)-AML1 (RUNX1) chimeric gene fusion is the most common genetic abnormality in childhood acute lymphoblastic leukemias. Evidence suggests that this chimeric gene fusion constitutes an initiating mutation that is necessary but insufficient for the development of leukemia. In a search for additional genetic events that could be linked to the development of leukemia, we applied a genome-wide array-comparative genomic hybridization technique to 24 TEL-AML1 leukemia samples and two cell lines. It was found that at least two chromosomal imbalances were involved in all samples. Recurrent regions of chromosomal imbalance (>10% of cases) and representative involved genes were gain of chromosomes 10 (17%) and 21q (25%; RUNX1) and loss of 12p13.2 (87%; TEL), 9p21.3 (29%; p16INK4a/ARF), 9p13.2 (25%; PAX5), 12q21.3 (25%; BTG1), 3p21 (21%; LIMD1), 6q21 (17%; AIM1 and BLIMP1), 4q31.23 (17%; NR3C2), 11q22-q23 (13%; ATM) and 19q13.11-q13.12 (13%; PDCD5). Enforced expression of TEL and to a lesser extent BTG1, both single genes known to be located in their respective minimum common region of loss, inhibited proliferation of the TEL-AML1 cell line Reh. Together, these findings suggest that some of the genes identified as lost by array-comparative genomic hybridization may partly account for the development of leukemia.

Double-orifice mitral valve associated with noncompaction of left ventricular myocardium.

Double-orifice mitral valve (DOMV) is a rare anomaly commonly associated with other congenital heart diseases. We present two patients with DOMV and noncompaction of the left ventricular myocardium (NLVM). Case 1 was a 5-year-old male diagnosed with dilated cardiomyopathy. His echocardiogram showed thin myocardium with dilatation at the basal of the left ventricle, thick noncompacted myocardium around the apex of the left ventricle, and DOMV (complete bridge type) with mild mitral regurgitation. Case 2 was an 11-year-old male diagnosed with complete atrioventricular block. His echocardiogram showed thick noncompacted myocardium with mild hypokinesis from the posterior to lateral wall and DOMV (complete bridge type) with mild mitral regurgitation. DOMV is commonly associated with congenital anomaly and always has an abnormal subvalvar apparatus. The mitral valve and its apparatus embryologically originate from the endomyocardium, which is thought to be the origin of noncompacted myocardium. We speculate that patients with DOMV may have NLVM.

Induction of impaired membrane phospholipid asymmetry in mature erythrocytes after chemotherapy.

Senescent erythrocytes undergo a loss of phospholipid asymmetry in the plasma membrane and are removed from the circulation by phagocytosis.To examine the loss of phospholipid asymmetry in mature erythrocytes after chemotherapy, we monitored phosphatidylserine (PS)-exposing erythrocytes by using flow cytometry to detect annexin V-bound erythrocytes in the circulation of acute lymphoblastic leukemia patients after consolidation chemotherapy. Both the percentage and the absolute number of annexin V-positive erythrocytes gradually increased immediately after chemotherapy. This result paralleled the change in the serum levels of bilirubin, suggesting a direct induction of PS-externalization in mature erythrocytes and a subsequent increase in erythrocyte clearance by splenic macrophages. The PS-exposing erythrocyte counts showed negative correlations to platelet and reticulocyte counts, which reflect the hematopoietic potential in the bone marrow. This result suggests that bone marrow suppression could be responsible for the enlarged senescent population in the circulation. Moreover, PS-exposing erythrocytes in the circulation remained at relatively high levels even after the serum level of bilirubin normalized, indicating that the decreased clearance of senescent erythrocytes as a result of impaired phagocytosis following bone marrow suppression might also be involved in the increase in PS-exposing erythrocytes in the circulation late after chemotherapy.

An MCA/MR syndrome with hypocholesterolemia due to familial hypobetalipoproteinemia: report of a second patient with Nguyen syndrome.

[Nguyen et al. (2003); Am J Med Genet 121A: 109-112] reported a boy with severe hypocholesterolemia due to autosomal dominant hypobetalipoproteinemia (ADBHL) associated with severe growth retardation, mental deficiency, epicanthal folds, a short nose with low nasal bridge and anteverted nares and bilateral partial cutaneous syndactyly of toes 2 and 3. Many of these manifestations resembled those in a mild form of Smith-Lemli-Opitz syndrome (SLOS). We report on a 13-year-old boy with ADHBL, who manifested a SLOS-like phenotype, including mental retardation and a characteristic face, similar to that of a patient reported by [Nguyen et al. (2003); Am J Med Genet 121A: 109-112]. Our patient supports the hypothesis by [Nguyen et al. (2003); Am J Med Genet 121A: 109-112] that ADHBL induced cholesterol deficiency has a significant effect on morphogenesis during embryogenesis, although additional genetic or environmental factors may be required to develop an SLOS-like phenotype in individuals with ADHLB. This is a second case of Nguyen syndrome.

Lateralization of the frontal lobe functions elicited by a cognitive bias task is a fundamental process. Lesion study.

Cognitive neuroscience researchers have hypothesized that context-dependent and context-independent response selection is associated with the left and right frontal lobe, respectively, in right-handed adult males. Patients with left frontal lobe lesions show context-independent reasoning in a cognitive bias task (CBT), while those with right frontal lesions show context-dependent reasoning. Young children show more context-independent responses in a modified CBT (mCBT), while adolescents and adults show more context-dependent responses. We investigated the cognitive bias of right-handed children with unilateral frontal lobe lesions/epileptic foci to explore the plasticity of lateralization in the frontal lobes. The study included eight children with left frontal lobe lesions/epileptic foci (LLF) and four children with right frontal lobe lesions/epileptic foci (RLF). Twenty-three right-handed age-matched males served as controls. A computer presented version of the original card-choice task that was simplified and modified for children was used (mCBT). Simple visual stimuli differed dichotomously in shape, color, number, and shading. A target object presented alone was followed by two choices from which subjects made selection based on preference. Considering all four characteristics, the degree of similarity between the target and the subjects' choice was scored for 30 trials. A high score indicated a context-dependent response selection bias and a low score indicated a context-independent bias. The RLF subjects had a higher converted score (mean: 26.8+/-2.2), while LLF subjects showed a lower converted score (mean: 7.75+/-6.3). There were highly significant differences between LLF subjects and the other groups (P<0.001 vs. controls or RLF subjects). No significant correlations were observed between the converted scores and the age at onset, time since insult, or IQ in either LLF or RLF subjects. These findings suggest that the lateralization of frontal lobe function elicited by mCBT is fundamental and independent of language lateralization, rather than secondary to it. Furthermore, these findings also indicate that the timetable for the development of lateralized frontal lobe functions depends upon biologic factors.

Sequential 3-D MRI frontal volume changes in subacute sclerosing panencephalitis.

In patients with subacute sclerosing panencephalitis, clinical stages defined according to Jabbour correlate strongly with frontal lobe dysfunctions. Non-quantitative radiologic investigations in these patients have confirmed cerebral atrophy without identifying predominantly affected regions. We addressed this issue over an 8-year-old boy's course using volumetry based on three-dimensional T1-weighted gradient echo magnetic resonance imaging. Seven normal 6-12-year-old subjects served as controls. Whole-brain volume declined as Jabbour stage advanced from I to III. Frontal lobe volume and frontal-to-whole-brain volume ratios fell significantly as clinical stage progressed. Thus, cerebral atrophy in this SSPE patient was predominantly frontal, and paralleled clinical progression.

[Study of saccadic eye movement in children with attention deficit/hyperactivity disorder].

Recent studies have postulated that impairment of behavior inhibition, leading to executive dysfunction in future, is the fundamental pathophysiology of attention deficit/hyperactivity disorder (AD/HD). To evaluate this, we studied two saccadic eye movement tasks: visual- and memory-guided, in 8 children with AD/HD (combined type;mean age, 9.1 years, range 6 approximately 11 years) and 16 healthy children (mean age, 9.2 years, range 6 approximately 12 years). In the memory-guided saccade task, reflexive anticipatory errors were more frequently observed in AD/HD than in controls. The children with AD/HD showed longer response latency in the memory-guided saccade task, and greater coefficient of variation in both the latency and amplitude in both the tasks. These results suggested that saccade tasks, especially the memory-guided task, may be useful in clarifying pathophysiology of AD/HD.

TEF, an antiapoptotic bZIP transcription factor related to the oncogenic E2A-HLF chimera, inhibits cell growth by down-regulating expression of the common beta chain of cytokine receptors.

Gain and/or loss of function mediated by chimeric transcription factors generated by nonrandom translocations in leukemia is a key to understanding oncogenesis. E2A-hepatic leukemia factor (HLF), a chimeric basic region/leucine zipper (bZIP) transcription factor expressed in t(17;19)-positive leukemia cells, contributes to leukemogenesis through its potential to inhibit apoptosis. To identify physiologic counterparts of this chimera, we investigated the function of other bZIP factors that bind to the same DNA sequence recognized by E2A-HLF. Here, we show that thyrotroph embryonic factor (TEF), which shares a high level of sequence identity with HLF and recognizes the same DNA sequence, is expressed in a small fraction of each subset of hematolymphoid progenitors. When TEF was introduced into FL5.12 interleukin 3 (IL-3)-dependent cells, TEF protected the cells from apoptosis due to IL-3 deprivation. Unexpectedly, TEF also almost completely down-regulated expression of the common beta (betac) chain of cytokine receptors. Consequently, TEF-expressing cells accumulated in G(0)/G(1) phase without undergoing apoptosis. These findings suggest that TEF is one of the apoptotic regulators in hematopoietic progenitors and controls hematopoietic-cell proliferation by regulating the expression of the betac chain. In contrast, E2A-HLF promoted cell survival more efficiently than TEF but did not down-regulate betac chain expression, suggesting that E2A-HLF retains ideal properties for driving leukemic transformation.

[Sympathetic skin response and emotional changes of visual stimuli].

We studied sympathetic skin response (SSR) to visual emotional stimuli in 11 normal children and 13 normal adults. The arousal and valence levels of original pictures were previously assessed by 20 normal children and adults. SSR appearance ratio tended to be high for pictures with high arousal and those with either high or low valence, and was higher in children than in adults. The response for pictures with high arousal and low valence was different between children and adults. These pictures were classified into 2 groups; one consisted of disgusting creatures such as worms and roaches, and another of violent scenes, symbolizing physiological and social unpleasure, respectively. In children, SSRs were evoked with the same ratio for these two groups of pictures. In adults, however, SSRs for social unpleasure pictures were evoked with a significantly higher ratio compared to those for physiological ones. These results indicate that children are more susceptible to emotional stimuli and that this susceptibility diminishes with development, suggesting the association between emotional signal and moral development.

[Case of severe motor and intellectual disabilities presenting with partial central diabetes insipidus triggered by infection].

We report a 42-year-old woman with severe motor and intellectual disabilities (SMID) who showed partial central diabetes insipidus during severe pneumonia. Serum sodium levels were previously within the upper normal range from 140 to 147 mEq/L. During pneumonia, however, serum sodium rose rapidly to reach 185 mEq/L. The daily urinary volume exceeded the daily intake of water. Nasal administration of 1-desamino-8-D-arginine vasopressin (DDAVP) reduced the daily urinary volume and the serum sodium level to normal ranges. Consequently, we diagnosed her as having central diabetes insipidus (DI). She required a smaller dose of DDAVP (2.5 microg/day) than usual DI (5-15 microg/day) to maintain normal urinary volume and the serum sodium level for seven months. After the nasal administration of DDAVP was discontinued, the serum sodium levels again returned to within the upper normal range. A water deprivation study demonstrated poor elevation of both plasma antidiuretic hormone (ADH) level (range, 0.5-2.0 pg/ml) and urine osmolarity (peak level, 552 mOsm/kgH2O) despite the elevation of plasma osomolarity, suggesting latent partial central DI. Water balance and serum electrolyte levels should be closely monitored in cases of SMID.

Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors.

Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.

[Prevention of hepatic veno-occlusive disease by a combination of heparin and prostaglandin E1 in children undergoing hematopoietic stem cell transplantation].

The effect and safety of combination prophylaxis for hepatic veno-occlusive disease (VOD) following stem cell transplantation (SCT) was retrospectively evaluated in a total of 53 children who survived until day 30. Prophylaxis was started on the day before conditioning, and heparin (10 unit/kg/hr) alone was used in 6 patients, PGE1 (5 ng/kg/min) plus heparin plus PTX (200 mg/day) in 17 patients, lipo-PGE1 (0.5 ng/kg/min) plus heparin plus PTX in 7 patients, and lipo-PGE1 plus heparin in 23 patients. Diagnosis of VOD was made based on McDonald's criteria in 5 cases (9.4%), but not on Jones' criteria. No statistically significant difference was observed in the incidence of VOD among each prophylaxis group. The degree of VOD was mild in all of 5 cases, and all recovered with continuation of the prophylactic procedure. Each prophylactic procedure was performed without any significant adverse effect except for seizure induced by lipo-PGE1 in one patient with Lennox syndrome. Since both the incidence and fatality rate of VOD in children undergoing SCT are approximately 20% according to most of the previous reports, the present study suggests the effectiveness of combination prophylaxis.

Hepatocyte growth factor protects small airway epithelial cells from apoptosis induced by tumor necrosis factor-alpha or oxidative stress.

Involvement of hepatocyte growth factor (HGF) in lung morphogenesis and regeneration has been established by in vitro and in vivo experiments in animals. In the present study, the protective activity of HGF against tumor necrosis factor (TNF)-alpha or hydrogen peroxide (H2O2)-induced damage of pulmonary epithelial cells was examined using the human small airway epithelial cell line (SAEC). Western blot analysis revealed that the receptor for HGF (c-Met) was highly expressed on the surface of SAEC and its downstream signal transduction pathway was functional. The SAEC was induced into apoptosis by the treatment with TNF-alpha or H2O2 in a dose-dependant manner, but was significantly rescued from apoptosis in the presence of HGF. The HGF effect was evident when added not only at the same time but also within several hours after treatment. This protective activity of HGF against the TNF-alpha- or H2O2-induced apoptosis was mediated, at least in part, by up-regulating the nuclear factor kappaB activity and an increase in the ratio of apoptosis-suppressing to apoptosis-inducing proteins. These results suggest that administration of HGF might exhibit a potent function in vivo for protection and improvement of acute and chronic lung injuries induced by inflammation and/or oxidative stress.