Early prognostication of neurological outcome by heart rate variability in adult patients with out-of-hospital sudden cardiac arrest.

BACKGROUND: Most deaths of comatose survivors of out-of-hospital sudden cardiac arrest result from withdrawal of life-sustaining treatment (WLST) decisions based on poor neurological prognostication and the family's intention. Thus, accurate prognostication is crucial to avoid premature WLST decisions. However, targeted temperature management (TTM) with sedation or neuromuscular blockade against shivering significantly affects early prognostication. In this study, we investigated whether heart rate variability (HRV) analysis could prognosticate poor neurological outcome in comatose patients undergoing hypothermic TTM. METHODS: Between January 2015 and December 2017, adult patients with out-of-hospital sudden cardiac arrest, successfully resuscitated in the emergency department and admitted to the intensive care unit of the Niigata University in Japan, were prospectively included. All patients had an initial Glasgow Coma Scale motor score of 1 and received hypothermic TTM (at 34 °C). Twenty HRV-related variables (deceleration capacity; 4 time-, 3 geometric-, and 7 frequency-domain; and 5 complexity variables) were computed based on RR intervals between 0:00 and 8:00 am within 24 h after return of spontaneous circulation (ROSC). Based on Glasgow Outcome Scale (GOS) at 2 weeks after ROSC, patients were divided into good outcome (GOS 1-2) and poor outcome (GOS 3-5) groups. RESULTS: Seventy-six patients were recruited and allocated to the good (n = 22) or poor (n = 54) outcome groups. Of the 20 HRV-related variables, ln very-low frequency (ln VLF) power, detrended fluctuation analysis (DFA) (α1), and multiscale entropy (MSE) index significantly differed between the groups (p = 0.001), with a statistically significant odds ratio (OR) by univariate logistic regression analysis (p = 0.001). Multivariate logistic regression analysis of the 3 variables identified ln VLF power and DFA (α1) as significant predictors for poor outcome (OR = 0.436, p = 0.006 and OR = 0.709, p = 0.024, respectively). The area under the receiver operating characteristic curve for ln VLF power and DFA (α1) in predicting poor outcome was 0.84 and 0.82, respectively. In addition, the minimum value of ln VLF power or DFA (α1) for the good outcome group predicted poor outcome with sensitivity = 61% and specificity = 100%. CONCLUSIONS: The present data indicate that HRV analysis could be useful for prognostication for comatose patients during hypothermic TTM.

Influence of Nutritional Management and Rehabilitation on Physical Outcome in Japanese Intensive Care Unit Patients: A Multicenter Observational Study.

BACKGROUND: There is lack of evidence regarding nutritional management among intensive care unit (ICU) patients in a population with relatively low body mass index. Therefore, we conducted an observational study to assess the nutritional management in Japanese ICUs. Also, we investigated the impact of nutritional management and rehabilitation on physical outcome. METHODS: The study population comprised 389 consecutive patients who received mechanical ventilation for at least 24 h and those admitted to the ICU for > 72 h in 13 hospitals. The primary outcomes were caloric and protein intake in ICU on days 3 and 7, and at ICU discharge. The secondary outcome was the impact of nutritional management and rehabilitation on physical status at ICU discharge. We defined good physical status as more than end sitting and poor physical status as bed rest and sitting. We divided the participants into 2 groups, namely, the good physical status group (Good group) and poor physical status group (Poor group) for analysis of the secondary outcome. Data were expressed as median (interquartile range). RESULTS: The median amount of caloric intake on days 3 and 7, and at ICU discharge via enteral and parenteral routes were 8.4 (3.1-15.6), 14.9 (7.5-22.0), and 11.2 (2.5-19.1) kcal/kg/day, respectively. The median amount of protein intake on days 3 and 7, and at ICU discharge were 0.2 (0-0.5), 0.4 (0.1-0.8), and 0.3 (0-0.7) g/kg/day, respectively. The amount of caloric intake on day 3 in the Poor group was significantly higher than that of the Good group (10.1 [5.8, 16.2] vs. 5.2 [1.9, 12.4] kcal/kg/day, p < 0.001). The proportion of patients who were received rehabilitation in ICU in the Good group was significantly higher than that of the Poor group (92 vs. 63%, p < 0.001). The multivariate analysis revealed that caloric intake on day 3 and rehabilitation in ICU were considered independent factors that affect physical status (OR 1.19; 95% CI 1.05-1.34; p = 0.005 and OR 0.07; 95% CI 0.01-0.34; p = 0.001). CONCLUSIONS: The caloric and protein intakes in Japanese ICUs were 15 kcal/kg/day and 0.4 g/kg/day, respectively. In addition, critically ill patients might benefit from low caloric intake (less than 10 kcal/kg/day) until day 3 and rehabilitation during ICU stay.

Importance of glutamine metabolism in leukemia cells by energy production through TCA cycle and by redox homeostasis.

Some cancer cells depend on glutamine despite of pronounced glycolysis. We examined the glutamine metabolism in leukemia cells, and found that HL-60 cells most depended on glutamine in the 4 acute myelogenous leukemia (AML) cell lines examined: growth of HL-60 cells was most suppressed by glutamine deprivation and by inhibition of glutaminolysis, which was rescued by tricarboxylic acid (TCA) cycle intermediate, oxaloacetic acid. Glutamine is also involved in antioxidant defense function by increasing glutathione. Glutamine deprivation suppressed the glutathione content and elevated reactive oxygen species most evidently in HL-60 cells. Glutamine metabolism might be a therapeutic target in some leukemia.

Adaptation of leukemia cells to hypoxic condition through switching the energy metabolism or avoiding the oxidative stress.

BACKGROUND: Like normal hematopoietic stem cells, leukemia cells proliferate in bone marrow, where oxygen supply is limited. However, the growth and energy metabolism of leukemia cells under hypoxia have not been well understood. Although it has been known that reactive oxygen species (ROS) is generated under hypoxic conditions, normal and leukemia stem cells were characterized by relatively low levels of ROS. Roles of ROS on leukemia cells under hypoxia also have not been well understood. METHODS: Four Leukemia cell lines were cultured under normoxia (21% O2) or hypoxia (1% O2), where NB4 and THP-1 were most extensively studied. To evaluate energy metabolism, we estimated whole cell number or apoptotic cells with or without a glycolysis inhibitor or an oxidative phosphorylation (OXPHOS) inhibitor. Glucose consumption and lactate production were also measured. To evaluate oxidative stress in hypoxic condition, the ROS level and GSH (reduced glutathione) / GSSG (oxidized glutathione) ratio was measured. In addition, pyruvate dehydrogenase kinase 1 (PDK1) and cytochrome c oxidase subunit 4 (COX4) were examined by western blotting or RT-PCR. RESULTS: NB4, which grows well under normoxia depending on glycolysis, demonstrated prominent apoptosis and growth suppression after 48 hours culture under hypoxia. NB4 cells cultured under hypoxia showed significantly increased ROS. Culture with a ROS scavenger resulted in decrease of apoptotic cell death of NB4 under hypoxia. NB4 cells cultured for longer period (7 days) under hypoxia did not come to extinction, but grew slowly by upregulating GSH synthesis to protect from ROS generated in hypoxic condition. By contrast, THP-1, which largely depends on OXPHOS in mitochondria under normoxia, demonstrated more growth under hypoxia by changing metabolism from OXPHOS to glycolysis through upregulating PDK1. Moreover, THP-1 avoided ROS generation by substituting COX 4 subunit (from COX 4-1 to COX 4-2) through upregulation of LON, a mitochondrial protease under hypoxia. CONCLUSIONS: We showed that leukemia cells survive and adapt to the hypoxic condition through various pathways. Our results will help understanding energy metabolism of leukemia cells and creating novel therapeutics.

Arsenic trioxide prevents nitric oxide production in lipopolysaccharide -stimulated RAW 264.7 by inhibiting a TRIF-dependent pathway.

Arsenic trioxide (ATO) is one of the most potent drugs in cancer chemotherapy, and is highly effective in treating both newly diagnosed and relapse patients with acute promyelocytic leukemia (APL). Despite a number of reports regarding the molecular mechanisms by which ATO promotes anti-tumor or pro-apoptotic activity in hematological and other solid malignancies, the effects of ATO on immune responses remain poorly understood. To further understand and clarify the effects of ATO on immune responses, we sought to examine whether ATO affects the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, RAW 264.7. Arsenic trioxide was found to prevent NO production in a dose-dependent manner. Arsenic trioxide significantly inhibited the increase in inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels. Furthermore, our analyses revealed that the inhibitory effect of ATO on iNOS expression was ascribed to the prevention of IRF3 phosphorylation, interferon (IFN)-ß expression, and STAT1 phosphorylation, but not the prevention of the MyD88-dependent pathway. Taken together, our results indicate that ATO prevents NO production by inhibiting the TIR-domain-containing adaptor protein inducing IFN-ß (TRIF)-dependent pathway, thus highlighting an anti-inflammatory property of ATO in innate immunity.

Establishment of a novel human myeloid leukemia cell line, AMU-AML1, carrying t(12;22)(p13;q11) without chimeric MN1-TEL and with high expression of MN1.

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.

Establishment of Down's syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT.

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.

Effects of evidence-based ICU care on long-term outcomes of patients with sepsis or septic shock (ILOSS): protocol for a multicentre prospective observational cohort study in Japan.

INTRODUCTION: Sepsis is not only the leading cause of death in the intensive care unit (ICU) but also a major risk factor for physical and cognitive impairment and mental disorders, known as postintensive care syndrome (PICS), reduced health-related quality of life (HRQoL) and even mental health disorders in patient families (PICS-family; PICS-F). The ABCDEF bundle is strongly recommended to overcome them, while the association between implementing the bundle and the long-term outcomes is also unknown. METHODS AND ANALYSIS: This is a multicentre prospective observational study at 26 ICUs. All consecutive patients between 1 November 2020 and 30 April 2022, who are 18 years old or older and expected to stay in an ICU for more than 48 hours due to sepsis or septic shock, are enrolled. Follow-up to evaluate survival and PICS/ PICS-F will be performed at 3, 6 and 12 months and additionally every 6 months up to 5 years after hospital discharge. Primary outcomes include survival at 12 months, which is the primary outcome, and the incidence of PICS defined as the presence of any physical impairment, cognitive impairment or mental disorders. PICS assessment scores, HRQoL and employment status are evaluated. The association between the implementation rate for the ABCDEF bundle and for each of the individual elements and long-term outcomes will be evaluated. The PICS-F, defined as the presence of mental disorders, and HRQoL of the family is also assessed. Additional analyses with data up to 5 years follow-up are planned. ETHICS AND DISSEMINATION: This study received ethics approvals from Saiseikai Utsunomiya Hospital (2020-42) and all other participating institutions and was registered in the University Hospital Medical Information Network Clinical Trials Registry. Informed consent will be obtained from all patients. The findings will be published in peer-reviewed journals and presented at scientific conferences. TRIAL REGISTRATION NUMBER: UMIN000041433.

The Asn505 mutation of the c-MPL gene, which causes familial essential thrombocythemia, induces autonomous homodimerization of the c-Mpl protein due to strong amino acid polarity.

We previously reported that a dominant-positive activating mutation (Asn505) in the transmembrane domain (TMD) of c-MPL, which encodes the thrombopoietin receptor, caused familial essential thrombocythemia. Here, we show that the Asn505 mutation induces both autonomous dimerization of c-Mpl and signal activation in the absence of its ligand. Signal activation was preserved in a truncated mutant of Asn505 that lacked the extracellular domain of c-MPL. We also found that the substitution of the amino acid (AA) residue at position 505 with others of strong polarity (Glu, Asp, or Gln) also resulted in activated dimerization without ligand stimulation. Overall, these data show that the Asn505 mutation transduced the signal through the autonomous dimerization of the c-MPL protein due to strong AA polarity. This finding provides a new insight into the mechanism of disease causation by mutations in the TMD of cytokine/hematopoietic receptors.

Lactate indices as predictors of in-hospital mortality or 90-day survival after admission to an intensive care unit in unselected critically ill patients.

BACKGROUND: We performed an exclusive study to investigate the associations between a total of 23 lactate-related indices during the first 24h in an intensive care unit (ICU) and in-hospital mortality. METHODS: Nine static and 14 dynamic lactate indices, including changes in lactate concentrations (Δ Lac) and slope (linear regression coefficient), were calculated from individual critically ill patient data extracted from the Multiparameter Intelligent Monitoring for Intensive Care (MIMIC) III database. RESULTS: Data from a total of 781 ICU patients were extracted, consisted of 523 survivors and 258 non-survivors. The in-hospital mortality rate for this cohort was 33.0%. A multivariate logistic regression model identified maximal lactate concentration at 24h after ICU admission (max lactate at T24) as a significant predictor of in-hospital mortality (odds ratio = 1.431, 95% confidence interval [CI] = 1.278-1.604, p<0.001) after adjusting for predefined confounders (age, gender, sepsis, Elixhauser comorbidity score, mechanical ventilation, renal replacement therapy, vasopressors, ICU severity scores). Area under curve (AUC) for max lactate at T24 was larger (AUC = 0.776, 95% CI = 0.740-0.812) than other indices (p<0.001), comparable to an APACHE III score of 0.771. When combining max lactate at T24 with APACHE III, the AUC was increased to 0.815 (95% CI:0.783-0.847). The sensitivity, specificity, and positive and negative predictive values for the cut-off value of 3.05 mmol/L were 64.3%, 77.4%, 58.5%, and 81.5%, respectively. Kaplan-Myer survival curves of the max lactate at T24 for 90-day survival after admission to ICU demonstrated a significant difference according to the cut-off value (p<0.001). CONCLUSIONS: These data indicate that the maximal arterial lactate concentration at T24 is a robust predictor of in-hospital mortality as well as 90-day survival in unselected ICU patients with predictive ability as comparable with APACHE III score.

Skeletal Muscle Index at Intensive Care Unit Admission Is a Predictor of Intensive Care Unit-Acquired Weakness in Patients With Sepsis.

BACKGROUND: Intensive care unit-acquired weakness (ICU-AW) can be diagnosed using the Medical Research Council (MRC) score. However, such scoring may not be possible in ICU patients who may be sedated or delirious or have encephalopathy. Currently, a quantitative assessment of the cross-sectional area of the muscle is available to assess changes in skeletal muscle mass using computed tomography (CT) images. This assessment calculates the skeletal muscle index (SMI) (cm2/m2) by dividing the cross-sectional area (cm2) of the skeletal muscle at the level of the third lumbar vertebra by the square of the patient's height (m2) on CT. This study assessed the effectiveness of SMI, as measured by abdominal CT scans, in predicting the onset of ICU-AW in patients with sepsis admitted to the ICU. METHODS: We examined septic ICU patients admitted to the Niigata University Hospital ICU during 2012 - 2017 under mechanical ventilation. Patients were retrospectively divided into two groups by MRC score at ICU discharge: group AW comprised patients with an MRC score < 48, and group non-AW (NAW) comprised the remaining patients. Clinicopathological factors at ICU admission such as age, gender, underlying disease, body mass index, and SMI were compared between the two groups. Statistical analyses were performed using the Mann-Whitney U test, Fisher's exact test, receiver operator characteristic (ROC) analysis and multivariate analysis. RESULTS: A total of 31 septic patients were examined, and 23 patients met the criteria for ICU-AW. The prevalence of women was significantly higher in group AW (P < 0.05). All clinical factors, except for gender, were not significantly different between the two groups. SMI was significantly lower in group AW than in group NAW (P < 0.05). ROC analysis revealed that the cut-off value of SMI for predicting ICU-AW was 44.1, and the multivariate analysis revealed that only low SMI was a significant factor in predicting ICU-AW (P < 0.05). CONCLUSIONS: Our results show that SMI measurement at ICU admission is a valid predictive factor for ICU-AW progression in septic patients.

Identification of two distinct populations of endothelial progenitor cells differing in size and antigen expression from human umbilical cord blood.

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood, bone marrow, and umbilical cord blood (CB) and determined to be in heterogeneous populations; however, specific variations in their characteristics remain to be clarified. In this study, we observed that mononuclear cells (MNCs) of CB change in morphology to differentiate into mature endothelial cells (EC) after 6 weeks of culture. In early days of culture along with the differentiation, two distinct populations of EPCs were detected, defined by two-dimensional dot plots (forward scatter vs side scatter) with flow cytometry, namely, relatively small cells (S-EPCs) and relatively large cells (L-EPCs). S-EPCs were found to express CD34 but not CD14, while the converse was the case for L-EPCs. When CD34(+)/CD14(-) cells and CD34(-)/CD14(+) cells were isolated from original MNCs of CB and cultured independently, S-EPCs and L-EPCs were derived from CD34(+)/CD14(-) and from CD34(-)/CD14(+) cells, respectively. Furthermore, when the two EPCs at day 7 were separated by cell sorter and recultured, there was no crossover in terms of CD34 and CD14 expression. While expression of VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) on L-EPCs was significantly greater than on S-EPCs, levels of CD31 were lower. In addition, L-EPCs exhibited greater proliferative ability on stimulation with VEGF. Although these two EPCs expressed different phenotypes, including growth factor receptors, and had different proliferative ability, they both eventually differentiated into mature ECs after more than 3 weeks of culture.

Establishment and characterization of a novel vincristine-resistant diffuse large B-cell lymphoma cell line containing the 8q24 homogeneously staining region.

Chromosome band 8q24 is the most frequently amplified locus in various types of cancers. MYC has been identified as the primary oncogene at the 8q24 locus, whereas a long noncoding gene, PVT1, which lies adjacent to MYC, has recently emerged as another potential oncogenic regulator at this position. In this study, we established and characterized a novel cell line, AMU-ML2, from a patient with diffuse large B-cell lymphoma (DLBCL), displaying homogeneously staining regions at the 8q24 locus. Fluorescence in situ hybridization clearly detected an elevation in MYC copy numbers corresponding to the homogenously staining region. In addition, a comparative genomic hybridization analysis using high-resolution arrays revealed that the 8q24 amplicon size was 1.4 Mb, containing the entire MYC and PVT1 regions. We also demonstrated a loss of heterozygosity for TP53 at 17p13 in conjunction with a TP53 frameshift mutation. Notably, AMU-ML2 cells exhibited resistance to vincristine, and cell proliferation was markedly inhibited by MYC-shRNA-mediated knockdown. Furthermore, genes involved in cyclin D, mTOR, and Ras signaling were downregulated following MYC knockdown, suggesting that MYC expression was closely associated with tumor cell growth. In conclusion, AMU-ML2 cells are uniquely characterized by homogenously staining regions at the 8q24 locus, thus providing useful insights into the pathogenesis of DLBCL with 8q24 abnormalities.

Accidental falls related to clearing heavy snow on rooftops in a rural heavy snow area in Japan.

Aim: The purpose of this study is to describe our experience with patients who fell from rooftops while clearing snow. The falls occurred in rural areas that receive heavy snowfall and are undergoing depopulation and an increasing proportion of elderly residents. Methods: A retrospective observational chart review was carried out at the sole hospital providing emergency services in a rural heavy snow area in Japan. Results: A total of 70 patients were enrolled during four winter seasons between December 2009 and March 2013. Their mean age was 61 years, and 90% were male. The mean vertical height of falls was 4.1 m. A total of 174 injuries was observed, averaging 2.5 injuries per patient. Fractures accounted for 78% of all injuries, and main fractures included vertebra with lower extremities or rib fractures; 86% of patients sustained a maximum abbreviated injury scale score of 2-3. Conclusions: In a rural heavy snow area in Japan, the incidence of accidental falls related to clearing snow was high, and the victims were elderly. Fractures accounted for 78% of all injuries, and most patients suffered from moderate to serious injuries.

Low p53 expression of acute myelocytic leukemia cells with t(8;21) chromosome abnormality: association with low p14(ARF) expression.

In this study, the mRNA expression of p14(ARF) in t(8;21)AML cells was found to be significantly lower than acute myelocytic leukemia (AML) cells without t(8;21) chromosome abnormality, which was concordant with previous observation by Linggi et al. that AML1-MTG8 represses the transcription of p14(ARF). Although p53 mRNA expression level of t(8;21)AML cells was not low, p53 protein expression was reduced in t(8;21)AML cells. Genotoxic damage by ionizing radiation did not induce p53 upregulation in t(8;21)AML cells. Since p14(ARF) has been demonstrated to inhibit p53 degradation by binding to MDM2, repression of p14(ARF) expression in t(8;21)AML may facilitate the degradation of p53 by MDM2. Low p14(ARF) in t(8;21)AML may also account for the absence of upregulation of p53 by ionizing radiation. Then, we have shown that p53 expression level was inversely correlated with S/G2/M population of cell cycle in AML cells. Most of the t(8;21)AML are considered to be in p53(low) S/G2/M(high). It is now widely known that formation of AML1-MTG8 by t(8;21) translocation is a very early event in leukemogenesis, and AML1-MTG8 alone might have limited proliferative potential. Then, secondary oncogenic events such as activated receptor tyrosine kinase (like c-kit mutation), is necessary to become full-blown leukemia. Low p53 protein expression and insufficient induction of p53 by genotoxic damage might increase the opportunity to obtain additional oncogenic events, since genome guard function of p53 does not work in t(8;21)AML cells.

Global real-time quantification/reverse transcription-polymerase chain reaction for detecting proto-oncogenes associated with 14q32 chromosomal translocation in multiple myeloma.

A global real-time quantitative/reverse transcription-polymerase chain reaction technique for detecting the expression of six 14q32 chromosomal translocation-associated proto-oncogenes in marrow plasma cells was established and applied to myeloma specimens. This technique is an alternative method of detecting 14q32 rearrangements and allows investigation of the relationship between proto-oncogene expression and clinical features.

Overexpression of salivary-type amylase reduces the sensitivity to bortezomib in multiple myeloma cells.

Amylase-producing myeloma exhibits refractoriness to chemotherapy and a dismal prognosis. In this study, we established a human myeloma cell line, 8226/AMY1, in which a lentivirally transfected AMY1 gene was stably expressed and explored its biological characteristics. 8226/AMY1 showed a survival advantage over mock control when treated with dexamethasone, bortezomib, and lenalidomide in vitro partly through inhibition of apoptosis induced by these reagents. In a xenograft murine model, 8226/AMY1 showed rapid tumor growth and reduced sensitivity to bortezomib compared with mock. A microarray gene expression analysis identified TCL1A, which functions as a coactivator of the cell survival kinase Akt, differentially up-regulated in 8226/AMY1. The expression of phosphorylated Akt was increased in the 8226/AMY1 cells following bortezomib treatment, but not in the mock cells. In addition, treatment with perifosine, an inhibitor of Akt phosphorylation, enhanced the anti-myeloma effect of bortezomib in the 8226/AMY1 cells. Our data suggest that amylase-producing myeloma reduced the sensitivity to bortezomib in vitro and in vivo, and the up-regulation of TCL1A may influence the drug susceptibility of 8226/AMY1 via the phosphorylation of Akt. These findings provide clues for developing treatment approaches for not only amylase-producing myeloma, but also relapsed and refractory myelomas.

Lymphoid blastic crisis of chronic myelogenous leukaemia with inv(16)(p13;q22).

We report a case of chronic myelogeneous leukaemia (CML) in B-lineage lymphoid blastic crisis (BC) having chromosome abnormality, inv(16)(p13;q22) in addition to Philadelphia chromosome, in 20/20 marrow metaphase. Inv(16)(p13;q22) was not observed in cells of chronic phase or accelerate phase. Abnormalities of chromosome 16, including inv(16)(p13;q22), del(16)(q22) and t(16;16)(p13;q22), have been reported mostly in acute myelomonocytic leukaemia (AML), (FAB M4-Eo), and some in CML-BC and myelodysplastic syndrome (MDS) cases. Most of the cases showed increase of myelomonocytic components and abnormal eosinophils with dysplastic granules in the bone marrow (BM). However, our case was diagnosed as lymphoid BC without increase of myelomonocytic components, although some abnormal eosinophilia was seen. To date, lymphoid BC of CML having inv(16)(p13;q22) abnormality has not been reported. The case presented here could be a clue to understand the pathophysiology of inv(16)(p13;q22) leukaemia.

Augmented expression of P-gp/multi-drug resistance gene by all-trans retinoic acid in monocytic leukemic cells.

P-glycoprotein (P-gp)/multi-drug resistance 1 (MDR1) gene is recognized to be, at least in part, responsible for the refractoriness to chemotherapy of leukemia. The transcriptional mechanism of MDR1 gene is largely unknown. However, recent reports have clarified that early growth response 1 gene (Egr1) positively regulates MDR1 transcription, while Wilms' tumor suppressor gene (WT1) does negative regulation of MDR1 gene expression in 12-O-tetradecanoylphorbol-13-acetate treated K562 cells. In addition, Egr1 and WT1 are structurally related transcription factors and bind to quite similar DNA sequences. Our study of mRNA expression profile of Egr1, WT1 and MDR1 in fresh AML samples demonstrated that there are disease-specific patterns. Egr1 mRNA was frequently and strongly expressed in monocytic leukemia cells, especially in AML M4 cells. WT1 mRNA was undetectable in t(8;21) AML cells. mRNA expression of MDR1 was frequent in AML M1 and t(8;21) AML cells, in which the expression level was highest in AML M1 and was low in monocytic leukemia (M4 and M5). Then, expression level of MDR1 was inversely correlated with Egr1. By liquid culture of leukemia cell lines and fresh AML cells with the addition of all-trans retinoic acid (ATRA), modulation of P-gp/MDR1 and Egr1 was observed and the pattern of modulation was divided into four groups: (1) blastic AML type, in which distinct expression of P-gp/MDR1 and CD34 was not influenced by ATRA; (2) t(8;21)AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 was kept high; (3) AML M3 type, in which P-gp/MDR1 expression was reduced with granulocytic differentiation by ATRA; (4) monocytic AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 expression decreased, and strong Egr1 expression was downregulated just prior to the augmentation of P-gp/MDR1 expression. WT1 expression was not influenced by the addition of ATRA in each group. Previous reports have suggested that P-gp/MDR1 plays an important role in resistance to chemotherapy, and is recognized as one of the stem cell marker. However, P-gp/MDR1 expression augmented by ATRA, which was observed in monocytic AML, was recognized as a functional molecule of mature monocyte/macrophage, because CD34 expression decreased and CD13 expression increased by ATRA. Finally, expression of P-gp/MDR1 in monocytic leukemia, which was functionally confirmed by Rh123 efflux study, was thought to be closely related to the characteristic modulation of Egr1 expression by ATRA.