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AIMS: The aim of this study was to investigate the effects of a single green tea (GT), administered concomitantly or 1 hour before nadolol intake on nadolol pharmacokinetics. METHODS: In a randomized 3‐phase crossover study, 11 healthy volunteers received an oral administration of nadolol with, or 1 hour after preingestion of brewed GT, or with water in a volume of 150 mL. RESULTS: Geometric mean ratio with 90% confidence interval for nadolol AUC(0–48) was 0.371 (0.303–0.439) with concomitant GT. In addition, ingestion of GT 1 hour before nadolol administration resulted in a significant reduction of nadolol AUC(0–48) with geometric mean ratio of 0.536 (0.406–0.665). There were no differences in time to maximal plasma concentration and renal clearance of nadolol among groups. CONCLUSION: These results suggest that single concomitant ingestion of GT substantially decreases plasma concentrations of nadolol. Moreover, the reduction in nadolol bioavailability could persist for at least 1 hour after drinking a cup of GT.

Objectives: To clarify (1) the prevalence and associating factors of work-life conflict (WLC); (2) the details of gender-based discrimination; and (3) the association between WLC and gender-based discrimination among various professionals in a medical university organization. Methods: This cross-sectional study, conducted in 2017, included all employees working at a public medical university and two affiliated hospitals that lie in provincial cities in Japan. The outcome of interest was time-based WLC in the work-to-family or family-to-work direction, measured with a shortened version of an existing scale. Gender-based discrimination was measured according to a three-point scale. Results: Among the 3,347 employees, complete data sets were available for 2,285 (complete response rate, 68.3%). Of these, approximately 30% of respondents had perceived WLC. Multivariable logistic regression analysis showed that faculty members, nurses, and employees between 30 and 39 years old had a greater risk of WLC regardless of gender. Men were more likely to perceive gender-based discrimination in the contents of their work and the number of incidental tasks, while women were more likely to perceive discrimination with promotions and evaluation of academic achievements. Both men and women respondents who perceived gender-based discrimination had an increased risk of WLC. Conclusions: When promoting organizational well-being in a medical university, increased attention should be paid to faculty members, nurses and employees between 30 and 39 years old, as they have a greater risk of WLC. Our results also suggest that promoting gender equality is important to help achieve appropriate work-life balance.

Background Self-efficacy is crucial in improving medical student communication skills. This study aims to clarify whether the self-efficacy of medical students conducting medical interviews increased after simulated interviews or after feedback discussions. Methods A total of 162 medical students (109 men, 53 women) in their fourth or fifth year at a university in Japan participated in this study. The degree of self-efficacy in medical interviewing was measured before and after a medical interview with a simulated patient, and after the subsequent feedback session. Results ANOVA analysis revealed that self-efficacy for medical interviews was higher after both the interview and the feedback session than before the interview. Self-efficacy was highest after the feedback session among all three time points. Conclusions Feedback following a simulated interview with a simulated patient is important to improve the self-efficacy of medical students learning medical interviewing skills.

Background Professional identity formation is nurtured through socialization, driven by interaction with role models, and supported through early clinical exposure (ECE) programmes. Non-healthcare professionals form part of the hospital community but are external to the culture of medicine, with their potential as role models unexplored. We employed text mining of student reflective assignments to explore the impact of socialization with non-healthcare professionals during ECE. Methods Assignments from 259 first-year medical students at Fukushima Medical University, Japan, underwent hierarchical cluster analysis. Interrelationships between the most-frequently-occurring words were analysed to create coding rules, which were applied to elucidate underlying themes. Results A shift in terms describing professional characteristics was detected, from “knowledge/skill” towards “pride [in one’s work]” and “responsibility”. Seven themes emerged: contribution of non-healthcare professionals, diversity of occupation, pride, responsibility, teamwork, patient care and gratitude. Students mentioning ‘contribution of non-healthcare professionals’ spoke of altruistic dedication and strong sense of purpose. These students expressed gratitude towards non-healthcare professionals for supporting clinical work, from a doctor’s perspective. Conclusion Socialization with non-healthcare professionals provides important insights into the hospital working environment and cultural working norms. Through role modelling altruism and responsibility, non-healthcare professionals positively influenced student professional identity formation, promoting self-conceptualisation as a doctor.

In this month's "When I say…", Stanyon et al. frankly discuss 'cultural competence' in terms of power and privilege in the clinic and in the classroom.

The aim of the present study is to investigate a possible role of a single dose of (−)-epigallocatechin gallate (EGCG), the major catechin in green tea, for the pharmacokinetic interaction between green tea and nadolol in humans. In a randomized three-phase crossover study, 13 healthy volunteers received single doses of 30 mg nadolol orally with water (control), or an aqueous solution of EGCG-concentrated green tea extract (GTE) at low or high dose. Plasma concentrations and urinary excretion of nadolol were determined up to 48 h. In addition, blood pressure and pulse rate were monitored. In vitro transport kinetic experiments were performed using human embryonic kidney 293 cells stably expressing organic anion transporting polypeptide (OATP)1A2 to evaluate the inhibitory effect of EGCG on OATP1A2-mediated substrate transport. Single coadministration of low and high dose GTE significantly reduced the plasma concentrations of nadolol. The geometric mean ratios with 90% CI for area under the plasma concentration–time curves from 0 to infinity of nadolol were 0.72 (0.56–0.87) for the low and 0.60 (0.51–0.69) for the high dose. There were no significant differences in Tmax, elimination half-life, and renal clearance between GTE and water phases. No significant changes were observed for blood pressure and pulse rate between phases. EGCG competitively inhibited OATP1A2-mediated uptake of sulphobromophthalein and nadolol with Ki values of 21.6 and 19.4 μM, respectively. EGCG is suggested to be a key contributor to the interaction of green tea with nadolol. Moreover, even a single coadministration of green tea may significantly affect nadolol pharmacokinetics.

High-mobility group AT-hook 2 (HMGA2) is crucial for the self-renewal of fetal hematopoietic stem cells (HSCs) but is downregulated in adult HSCs via repression by MIRlet-7 and the polycomb-recessive complex 2 (PRC2) including EZH2. The HMGA2 messenger RNA (mRNA) level is often elevated in patients with myelofibrosis that exhibits an advanced myeloproliferative neoplasm (MPN) subtype, and deletion of Ezh2 promotes the progression of severe myelofibrosis in JAK2V617F mice with upregulation of several oncogenes such as Hmga2. However, the direct role of HMGA2 in the pathogenesis of MPNs remains unknown. To clarify the impact of HMGA2 on MPNs carrying the driver mutation, we generated ΔHmga2/JAK2V617F mice overexpressing Hmga2 due to deletion of the 3' untranslated region. Compared with JAK2V617F mice, ΔHmga2/JAK2V617F mice exhibited more severe leukocytosis, anemia and splenomegaly, and shortened survival, whereas severity of myelofibrosis was comparable. ΔHmga2/JAK2V617F cells showed a greater repopulating ability that reproduced the severe MPN compared with JAK2V617F cells in serial bone marrow transplants, indicating that Hmga2 promotes MPN progression at the HSC level. Hmga2 also enhanced apoptosis of JAK2V617F erythroblasts that may worsen anemia. Relative to JAK2V617F hematopoietic stem and progenitor cells (HSPCs), over 30% of genes upregulated in ΔHmga2/JAK2V617F HSPCs overlapped with those derepressed by Ezh2 loss in JAK2V617F/Ezh2Δ/Δ HSPCs, suggesting that Hmga2 may facilitate upregulation of Ezh2 targets. Correspondingly, deletion of Hmga2 ameliorated anemia and splenomegaly in JAK2V617F/Ezh2Δ/wild-type mice, and MIRlet-7 suppression and PRC2 mutations correlated with the elevated HMGA2 mRNA levels in patients with MPNs, especially myelofibrosis. These findings suggest the crucial role of HMGA2 in MPN progression.

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8–induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.

Glucocorticoid (GC) resistance is associated with poor response to the following chemotherapy in lymphoid malignancies, such as lymphoma and leukemia. However, it remains unclear whether GCs interfere with the cytotoxic effects of anti-cancer drugs on GC-resistant cells. In this study, we examined whether GCs affected the sensitivities to vincristine (VCR)/doxorubicin (DOX) and the expression of drug transporters in GC-resistant cells. The dexamethasone (DEX)/prednisolone (PSL)-resistant lymphoid and non-lymphoid cell lines Raji and HL60 were cultured with DEX for 7 days and then treated with VCR or DOX for 3 days. Seven days of DEX treatment increased the IC50s of both VCR and DOX in Raji cells but not in HL60 cells. The mRNA and protein expression levels of organic cation/carnitine transporter (OCTN) 2, one of the drug uptake transporters expressed in both cell lines, were decreased only in Raji cells. When Raji cells were cultured with PSL, the IC50 of DOX but not VCR increased as the expression of OCTN2 decreased. No significant increases in efflux transporter expression were induced by DEX or PSL. When siRNA against OCTN2 was introduced into Raji cells, the IC50 of DOX but not VCR increased significantly. These data suggested that both DEX and PSL decreased the sensitivity of the DEX/PSL-resistant Raji cells to DOX, a change that was at least partially due to reductions in OCTN2. Thus, the continuous usage of GCs may interfere with the effects of chemotherapy on GC-resistant lymphoid cells.

The objective of this study is to assess the effects of green tea and its major catechin component, (−)-epigallocatechin gallate (EGCG), on CYP2C9-mediated substrate metabolism in vitro, and the pharmacokinetics of fluvastatin in healthy volunteers. The metabolism of diclofenac and fluvastatin in human recombinant CYP2C9 was investigated in the presence of EGCG. In a randomized three-phase crossover study, 11 healthy volunteers ingested a single 20-mg dose of fluvastatin with green tea extract (GTE), containing 150 mg of EGCG, along with water (300 mL), brewed green tea (300 mL), or water (300 mL) after overnight fasting. Plasma concentrations of fluvastatin and EGCG were measured by ultra-performance liquid chromatography with fluorescence detection and a single mass spectrometer. EGCG inhibited diclofenac 4′-hydroxylation and fluvastatin degradation with IC50 of 2.23 and 48.04 μM, respectively. Brewed green tea used in the clinical study also dose-dependently inhibited the metabolism of diclofenac and fluvastatin in vitro. However, no significant effects of GTE and brewed green tea were observed in plasma concentrations of fluvastatin. The geometric mean ratios with 90% CI for area under the plasma concentration–time curve (AUC0–∞) of fluvastatin were 0.993 (0.963–1.024, vs. brewed green tea) and 0.977 (0.935–1.020, vs. GTE). Although in vitro studies indicated that EGCG and brewed green tea produce significant inhibitory effects on CYP2C9 activity, the concomitant administration of green tea and fluvastatin in healthy volunteers did not influence the pharmacokinetics of fluvastatin.

In patients with MPNs, repression of MIRlet-7 and mutations in the polycomb genes EZH2 and ASXL1 correlate with HMGA2 overexpression.Hmga2 overexpression collaborates with JAK2V617F to promote lethal MPN in mice, highlighting the crucial role of Hmga2.

To evaluate effects of itraconazole, rifampicin and grapefruit juice on pharmacokinetics and pharmacodynamics of a hydrophilic non-selective β-adrenoceptor blocker nadolol, we conducted an open-label, four-way crossover study in 10 healthy male volunteers. A single oral dose of 30 mg nadolol was administered with water (control), itraconazole (100 mg), or grapefruit juice (300 mL), or after a 6-day pretreatment with rifampicin (450 mg/day). Plasma concentrations and urinary excretions of nadolol were measured over 48 hours after its dosing. Systolic and diastolic blood pressures and pulse rate were periodically recorded after nadolol administration as pharmacodynamic parameters. Itraconazole increased the peak plasma concentration and the area under the plasma concentration-time curve (AUC0-∞ ) of nadolol by 468% and 224% of control, respectively (P < .001). A slight, but not statistically significant, decrease in AUC0-∞ of nadolol was observed in rifampicin and grapefruit juice phases as compared to control. Elimination half-life for nadolol did not differ among the four phases. During itraconazole phase, nadolol reduced pharmacodynamic parameters to a greater extent than the other phases. These results suggest that itraconazole substantially increases the oral availability of nadolol possibly by the inhibition of intestinal P-glycoprotein, whereas grapefruit juice has little effect on nadolol pharmacokinetics.

We previously reported a case of pulmonary hypertension, where the symptoms were improved by oral L-arginine (arginine) administration. Arginine may increase nitric oxide (NO) production in the pulmonary artery. Exhaled NO may reflect pulmonary artery NO production. It has been demonstrated that exhaled NO concentration is higher in patients with allergic diseases, but whether oral arginine administration alters exhaled NO is unknown. Therefore, in this study, we investigated whether oral arginine administration increases exhaled NO among healthy volun- teers with and without a history of allergy. Eleven subjects were given a single oral dose (200 mg/kg) of arginine, and their plasma arginine concentrations and exhaled NO were measured up to 150 minutes. Baseline values of exhaled NO concentration were significantly higher in those with a history of allergy (56.4±20.3 ppb, n=5, P

Background Pediatric apheresis for peripheral blood stem cell transplantation should be carried out with due concern for low corporeal blood volume and vulnerability to hypocalcemia-related complications, hypovolemic shock, and hypervolemic cardiac overload. Study Design And Methods We retrospectively investigated a total of 267 apheresis procedures from 1990 to 2013 on 93 children between 0 and 10 years old, including 89 patients and 4 healthy donors, with body weights of 6.3 to 44.0 kg. Results The median CD34+ cell yield per apheresis procedure was 2.3 × 10 6 CD34+ cells/kg (0.2–77.9 × 10 6 CD34+ cells/kg). Adverse events occurred in 11.6% of procedures (n = 31), including mild perivascular pain (n = 12), emesis (n = 9), hypotension (n = 3), urticaria (n = 2), numbness (n = 2), chest pain (n = 1), facial flush (n = 1), and abdominal pain (n = 1). Among hypotensive events, shock in a 9.6 kg one-year-old boy required emergency treatment in 1996. Thereafter, we adopted continuous injection of calcium gluconate, ionized calcium monitoring, central venous catheter access and circuit priming with albumin in addition to concentrated red cells. Since then we have had fewer complications: 16.4% per apheresis during 1990–1997 versus 5.8% during 1998–2013. No healthy pediatric donors suffered from any late-onset complications related to apheresis or G-CSF administration. Conclusion By employing appropriate measures, peripheral blood stem cell apheresis for small children can have an improved safety profile, even for children weighing

To investigate differentiation-dependent gene expression during granulopoiesis, we established a new method to isolate six sequential differentiation stages of neutrophil progenitors from bone marrow. Neutrophil progenitors were divided into three populations by density centrifugation, followed by depletion of other lineages, and further separated by fluorescence-activated cell sorting based on the expressions of CD34, CD11b, and CD16: CD34(+) fraction from a low-density population (F1), CD11b(-)/CD16(-) (F2), CD11b(+)/CD16(-) (F3), and CD11b(+)/CD16(low) (F4) fractions with intermediate density, and CD11b(+)/CD16(int) (F5) and CD11b(+)/CD16(high) (F6) fractions from a high-density population. To examine whether this fractionation was applicable to the study of in vivo gene expression profiles during granulopoiesis, we analyzed messenger RNA levels of AML-1 and CCAAT/enhancer binding protein (EBP)-ε and two target genes of C/EBP-ε, granulocyte-macrophage colony-stimulating factor receptor common β subunit and lactoferrin, in the six fractions and peripheral blood-derived neutrophils (F7). Expression of AML-1 and C/EBP-ε peaked at F1 and F4, respectively, followed by a gradual decrease. Although granulocyte-macrophage colony-stimulating factor receptor common β subunit messenger RNA levels remained low from F1 through F6 and elevated at F7, lactoferrin messenger RNA showed a drastic increase at F3 and dropped at F5. The difference in the expression profiles of the two C/EBP-ε target genes suggests the involvement of regulators other than C/EBP-ε in the induction of the two genes. The new fractionation method is able to provide new information on maturation-dependent gene expression during granulopoiesis.

MDS/MPN comprise a group of uncommon myeloid neoplasms with clinical and hematological features overlapping MDS and MPN. Pathogenesis remain elusive. HMGA2 on chromosome 12q plays a crucial role in self-renewal and proliferation of fetal hematopoietic stem cells (HSCs), but not adult HSCs because of repression by binding of MIRLET-7 with the 3'-untranslated region (3'UTR) of HMGA2 (Copley et al, Nat Cell Biol, 2013). Trimethylation of histone H3 at lysine 27, which is catalyzed by EZH2 - a member of polycomb recessive complex 2 (PRC2) - also suppresses HMGA2 (Sashida et al, JEM, 2016). We have demonstrated that expression of Hmga2 cDNA lacking 3'UTR caused proliferative hematopoiesis mimicking MPN in young mice and led to anemia with ineffective erythropoiesis and leukocytosis like MDS/MPN in old mice (Ikeda et al, Blood, 2011; Ueda et al, Blood Adv, 2017), suggesting that HMGA2 may contribute to the pathogenesis of MDS/MPN. In fact, we found high HMGA2 mRNA levels in granulocytes of MPN including >90% of patients with myelofibrosis (MF) and about 30% with essential thrombocythemia (ET), and either reduced MIRLET-7c levels or PRC2-related mutations were correlated with high HMGA2 mRNA in MF, while reduced MIRLET-7c with no particular mutation was correlated with high HMGA2 mRNA in ET (Harada-Shirado et al, BJH, 2015; Blood Adv, 2017). However, in MDS/MPN, only one case series has reported high HMGA2 mRNA due to the 12q rearrangement that removed the 3'UTR of HMGA2 in a few patients (Odero et al, Leukemia, 2005). So far, expression of HMGA2 has not been studied in MDS/MPN without the 12q rearrangement. To clarify the expression of HMGA2 in MDS/MPN in relation to clinical and other genetic findings, we investigated HMGA2 mRNA and MIRLET-7c levels by real-time RT-PCR and sought PRC2-related mutations by targeted deep sequencing in peripheral leukocytes of 11 patients with MDS/MPN [5 chronic myelomonocytic leukemia (CMML), 2 atypical chronic myeloid leukemia (aCML), and 4 unclassifiable MDS/MPN (MDS/MPN-u)]. In addition, to identify the features specific for MDS/MPN, we also evaluated 10 patients with low-risk MDS [MDS with multilineage dysplasia (MDS-MLD)]. No MDS/MPN or MDS-MLD patients had 12q rearrangement. In MDS/MPN, high HMGA2 mRNA [>1.0 relative to HPRT1 mRNA above mean + 2SD of healthy volunteers, per our previous study (Blood Adv, 2017)] was found in each subtype: 2 of 5 (40%) in CMML, 1 of 2 (50%) in aCML, and 3 of 4 (75%) in MDS/MPN-u. Therefore, more than half of patients with MDS/MPN (6 of 11, 54.5%) showed high HMGA2 mRNA levels. In contrast, high HMGA2 mRNA was found in only 1 of 10 (10%) patients with MDS-MLD (P = 0.06). Reduction in MIRLET-7c was seen in 5 of 11 (45.5%) MDS/MPN and 3 of 10 (30.0%) MDS-MLD (P = 0.16). Among these 5 MDS/MPN patients with low MIRLET-7c, only 2 patients had high HMGA2 mRNA. Thus, MIRLET-7c reduction did not correlate with deregulation of HMGA2 mRNA in MDS/MPN as frequently as in MPN. In contrast, HMGA2 mRNA levels were high in all 3 patients with MDS/MPN carrying EZH2 mutations, suggesting that loss of EZH2 might augment HMGA2 in MDS/MPN. Laboratory findings of MDS/MPN patients with high HMGA2 mRNA were not different from those without high HMGA2 mRNA. However, MDS/MPN patients with both high HMGA2 mRNA and an EZH2 mutation showed more severe anemia compared with those with high HMGA2 mRNA without an EZH2 mutation (hemoglobin level: 9.6±2.0 vs. 11.0±2.3 g/dL, P = 0.05). In conclusion, we demonstrated that MDS/MPN cases highly express HMGA2 mRNA even without 12q rearrangement, more often than MDS-MLD. Unlike MPN, HMGA2 mRNA levels did not correlate with reduced MIRLET-7c, but probably with mutations in EZH2, which may also contribute to anemia in MDS/MPN. Disclosures No relevant conflicts of interest to declare.

In response to injury and inflammation of tendons, tendon fibroblasts are activated, migrate to the wound, and eventually induce contraction of the extracellular matrices to repair the tissue. Under such conditions, Ca2+ signalling is involved in motility and contractility of tendon fibroblasts. Using cultured tendon fibroblasts isolated from rat Achilles tendons, we investigated functional expression of Na+/Ca2+ exchangers (NCX). The fluorometric study showed that the intracellular Ca2+ concentration ([Ca2+]i) was increased by reducing extracellular Na+ concentration ([Na+]o) in tendon fibroblasts. Selective NCX inhibitors, KB-R7943 and SEA0400, both attenuated [Na+]o-dependent [Ca2+]i elevation and the resting [Ca2+]i in tendon fibroblasts. RT-PCR, Western blots and sequence analyses revealed that NCX1.3 and NCX1.7 were expressed in cultured tendon fibroblasts. NCX2 mRNA was undetected. NCX3 expression was negligibly low. Immunofluorescence microscopy indicated that NCX1 protein localized in the plasma membrane especially at the microspikes of tendon fibroblasts. In the wound-healing scratch assay, the cells migrated toward the space created by a scratch and almost completely filled the space within 48 h. This phenomenon was significantly suppressed by KB-R7943 and SEA0400. Furthermore, the NCX inhibitors abrogated the tendon fibroblast-mediated collagen-matrix contractions. Two types of siRNAs for NCX1 also suppressed the migration and contraction of tendon fibroblasts. We conclude that NCX is expressed and mediates Ca2+ influx in cultured tendon fibroblasts. Since the pharmacological inhibitors and siRNA for NCX1 suppressed motility and contractility of tendon fibroblasts, NCX may play an important role in the function of tendon fibroblasts in the wound healing.

Here we reviewed our recent work on the chronic effects of nicotine on the Na+ -Ca2+ exchanger (NCX) gene and protein expressions in various organs of rats treated with nicotine in the drinking water for 4-12 weeks. Microarray analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) did not detect significant changes in NCX mRNA expression in cerebral cortex, hippocampus, heart and skeletal muscle. However, NCX1 protein was up-regulated by nicotine in cerebral cortex and hippocampus, but was down-regulated in the heart. NCX2 protein was up-regulated by nicotine in hippocampus. We suggest that although mRNA change was insignificant, NCX protein expression was altered by chronic nicotine administration in brain and heart in rats. We also reviewed our work on modulators of NCX gene expression and function in cardiac myocytes.

We recently identified a reduction in the neutrophil surface expression of common β chain (βc) of the receptor for granulocyte macrophage-colony stimulating factor (GM-CSF) in the patients with myelodysplastic syndromes (MDS). To determine the etiology of the impaired βc expression, βc mRNA from neutrophilic granulocytes of MDS patients and healthy controls was analyzed by a combination of direct reverse transcriptiase-polymerase chain reaction-based single-strand conformational polymorphism and sequencing. Nine different βc transcripts were detected, but none was specific for MDS. However, one of the transcripts (βc79) containing a 79-base intron insertion between exons V and VI was significantly increased in MDS. This 27-kd isoform consisted of the βc N-terminal 182 amino acids followed by a new 84-amino-acid sequence. βc79 was overexpressed in all MDS subtypes. No genomic mutations were detected within the intron or at the intron/exon boundaries. The isoform is predominantly located in the cytoplasm by Western blot analysis and was unable to generate high-affinity binding sites or transduce a signal for proliferation when coexpressed with the receptor for human GM-CSF α chain. Our study suggests that the accumulation of the abnormal βc transcripts with intron V retention results in the reduction in cell-surface expression of βc observed in MDS.

Transfusion-related acute lung injury (TRALI) is a clinical syndrome characterized by bilateral pulmonary edema in association with transfusions. We encountered a 23-year-old woman with acute lymphoblastic leukemia, in whom TRALI without anti-human leukocyte antigen class I and anti-granulocyte antibodies developed following allogeneic bone marrow transplantation. TRALI improved mainly in association with treatment of saline and ventilation support after several days, but graft-versus-host disease and thrombotic microangiopathy developed, resulting in death due to multiple organ failure. This case indicates that TRALI can also occur following allogeneic bone marrow transplantation.

BACKGROUND : ACD-A solution containing sodium citrate and citric acid is used as an anticoagulant agent during peripheral blood progenitor cell (PBPC) harvesting, and in rare cases can cause fatal citrate intoxication. The aim of this study was to establish effective methods for stabilizing ionized calcium (ICa) levels during PBPC harvesting. STUDY DESIGN AND METHODS : ICa was measured during 46 apheresis procedures conducted in 26 patients. Four patients in four procedures were infused with calcium gluconate solution before PBPC harvesting; three patients in six procedures were infused with calcium gluconate when symptoms of citrate intoxication appeared; and four patients in five procedures received a continuous infusion. Five patients in five procedures took an isotonic sports drink containing calcium when hypocalcemic symptoms appeared. The ICa level, blood pressure, and pulse rate were measured. RESULTS : ICa declined rapidly from the preapheresis level of 1.081(±0.092) m M to 0.937(±0.081) m M (13.3%, p < 0.0001) 10 minutes after the start of apheresis and continued to decline until the completion of the procedure. When patients received a continuous infusion of calcium during apheresis, ICa was relatively stabilized. ICa significantly rose (6.1 ± 3.6%, p < 0.02) within 2 to 5 minutes after oral intake of an isotonic sports drink containing calcium and was maintained within normal range for 31 to 55 minutes. CONCLUSION : An isotonic sports drink containing calcium has a quick stabilizing and a longer maintenance effect on ICa. Thus, we recommend the intake of an isotonic sports drink containing calcium as the easiest and best method for preventing hypocalcemia during apheresis.

Objective The aim of this study was to determine whether granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are more or less intrinsically sensitive to spontaneous apoptosis than granulocytes from healthy individuals. Resistance to apoptosis has been suggested as an explanation for the proliferation or selection of PNH clones. Patients and Methods Peripheral blood granulocytes from five patients with PNH, five patients with myelodysplastic syndrome (MDS), and five healthy volunteers were cultured in the absence of serum. Spontaneous apoptosis of the granulocytes was assessed every 6 hours by flow cytometry. The expression levels of CD16b, CD95, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor also were studied by flow cytometry, and caspase-3 activity was measured by fluorometry. Results There were no significant differences in the proportion or absolute numbers of apoptotic and apoptotic/dead granulocytes between the cells from PNH patients and healthy individuals, whereas those from MDS patients showed significantly lower frequencies of apoptotic granulocytes compared with normal controls. The proportion of CD16b − granulocytes was not significantly different among the three groups during in vitro culture. CD95 and GM-CSF receptor was not significantly increased in cultured granulocytes or noncultured granulocytes from, respectively, patients with PNH and normal controls. Caspase-3 activity significantly decreased in cultured granulocytes from MDS patients, but not in granulocytes from PNH patients. Conclusions Granulocytes from PNH patients did not display a reduced sensitivity to spontaneous apoptosis, suggesting that the apoptosis of blood cells in PNH may not be an important factor in proliferation or selection of PNH clones. These findings are in agreement with the normal lifespan of granulocytes in vivo.

Summary. The proliferative and differentiative response of neutrophils to granulocyte‐macrophage colony-stimulating factor (GM-CSF) is known to be impaired in patients with myelodysplastic syndromes (MDS). To investigate the mechanisms of the defective response in MDS, we examined expression levels of GM-CSF receptor a (GMRa) and common b (bc) subunits on CD16 1 neutrophils, CD14 1 monocytes and CD3 1 T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMRa was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, bc expression on neutrophils was significantly reduced in 14 out of 26 patients with a higher proportion occurring in the advanced stages of MDS including refractory anaemia with excess of blasts (RAEB), RAEB in transformation (RAEBt) and overt leukaemia compared with refractory anaemia (RA)/RA with ringed sideroblasts (RARS) or healthy controls. Decreased bc also correlated with the degree of hypogranular neutrophil morphology and increased infection. Expression of both subunits on T cells and monocytes in MDS was similar to normal controls. Polymerase chain reaction amplification of reversetranscribed mRNA isolated from the affected neutrophils suggests that the reduction of bc may result from decreased message levels. The observed reduction in GM-CSF receptor expression could account for the impaired proliferative and maturational responses in MDS.

Seventy-eight patients multitransfused using leukocyte-reduction filters at bedside were prospectively studied to determine the rate of post-transfusion alloimmunization and its risk to platelet transfusion. HLA- and HPA-antibodies were detected by standard lymphocytotoxicity test (LCT), antihuman globulin-lymphocytotoxicity test (AHG-LCT) and mixed passive haemagglutination method (MPHA).Of these 78 patients, all of whom had a negative antibody screening on admission, 11 (14%) developed HLA antibodies but only one (1%) developed an HPA antibody. HLA antibodies were detected in 9 (24%) of 38 patients with previous transfusion, pregnancy or both but in only 2 (5%) of 40 patients with no presensitization (p

As the volume of blood processed in harvesting peripheral blood stem cells (PBSCH) for autologous stem cell transplantation increases, clinical symptoms associated with hypocalcemia can occur. In this study, we evaluated several calcium supplementation methods for the prevention of hypocalcemia during PBSCH apheresis.Ionized calcium (ICa) level in peripheral blood was measured in 35 procedures in 19 patients. ICa in patients with hypocalcemic symptoms was lower than that in those without symptoms, but the difference was significant only at 120 minutes of apheresis. Continuous injection of calcium during the procedure kept ICa almost within the normal range in three patients. In six patients who took an isotonic drink per os at the time symptoms appeared, ICa value increased within 3 minutes after intake.As hypocalcemia-associated symptoms mostly appeared at 120 minutes after the start of the procedure, prophylactic treatment is not difficult. Further, continuous injection of pharmaceutical calcium into a venous port with measurement of ICa is an excellent method for infants and patients unable to take isotonic drinks orally. Oral administration of an isotonic drink is the most effective way to control ICa level, even though these drinks have a low Ca content.

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3′UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (P

The interaction of different members of the hematopoietic growth factor receptor family may be relevant to the increased proliferation and the failure of differentiation that characterizes the myeloid leukemias. We recently demonstrated that a chimeric receptor (GMER) that is composed of the extracellular and transmembrane domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (GMRα) and the cytoplasmic domain of the murine erythropoietin receptor mEpoR binds hGM-CSF with low affinity (3 nM) and confers both proliferative and differentiation signals to stably transfected murine Ba/F3 cells. To investigate whether the common β-subunit of the GM-CSF receptor (βc) can interact with GMER, either the entire β-subunit or a mutant, truncated β-subunit that completely lacks the cytoplasmic domain (βtr) was introduced into Ba/F3 cells that express GMER, and the binding of GM-CSF as well as proliferation and differentiation responses were measured. Scatchard analysis showed that both GMER + βc and GMER + βtr bound hGM-CSF with high affinity (Kd 40 pM to 65 pM). Proliferation assays showed that the maximum growth of cells expressing GMER + βc was identical to that of cells with GMER alone. However, proliferation of the cells that expressed GMER + βtr was reduced by 80–95% of GMER. Dose-response curves showed that the concentration of GM-CSF required for half-maximal growth was 0.5–5.0 pM for GMER + βc and 0.5–5 nM for GMER and GMER + βtr. The EpoR cytoplasmic domain of GMER also undergoes ligand-inducible tyrosine phosphorylation. However, the tyrosine phosphorylation did not correlate with growth in cells expressing βtr. Coexpression of βc with GMER in Ba/F3 cells grown in hGM-CSF markedly enhanced β-globin mRNA expression. These results indicate that βc can transduce a unique signal in association with GMER to influence both proliferative and differentiation signal pathways.

The high-affinity receptor for granulocyte-macrophage colony- stimulating factor (GMR) comprises at least 2 distinct subunits, alpha and beta common (beta c), whereas the normal erythropoietin receptor (nEpoR) comprises only one known subunit. An arginine to cysteine (R129C) mutation of the extracytoplasmic domain of the murine EpoR leads to Epo-independent growth in transduced cells (cEpoR). To investigate the proliferative functions of the cytoplasmic regions of each GMR subunit separately and the potential of the R129C EpoR mutation to induce factor-independent growth through heterologous receptor regions, we constructed four hybrid receptors: the extracellular region of either murine nEpoR or cEpoR linked to the transmembrane and cytoplasmic regions of either the human GMR alpha or beta c subunit (nE alpha, nE beta, cE alpha, and cE beta). We then expressed them in an interleukin-3-dependent murine cell line, Ba/F3. Expression of nE beta led to Epo-dependent growth, whereas expression of cE beta conferred factor-independent growth. Surprisingly, expression of cE alpha also resulted in factor-independent cell growth, whereas nE alpha did not respond to Epo. Furthermore, the functional hybrid receptors showed Epo-dependent (nE beta) or constitutive (cE alpha and cE beta) tyrosine phosphorylation of the cytoplasmic kinases JAK1 and JAK2. We reasoned that the proliferative signal of cE alpha was transduced either through the alpha tail itself or through an accessory protein such as the endogenous murine beta common subunit (mu beta c). To distinguish these possibilities, the chimeric receptor cE alpha was expressed in the interleukin-2-dependent murine cell line, CTLL-2, that does not express mu beta c. cE alpha did not induce cell growth in CTLL-2; however, when mu beta c was coexpressed with cE alpha in CTLL-2, factor-independent growth was reconstituted. In conclusion, the cytoplasmic domain of the GMR alpha subunit requires a beta chain for transduction of a proliferative signal. Furthermore, the R129C EpoR mutation can constitutively activate heterologous receptors to mediate factor-independent proliferation.

To evaluate effects of itraconazole, rifampicin and grapefruit juice on pharmacokinetics and pharmacodynamics of a hydrophilic non-selective β-adrenoceptor blocker nadolol, we conducted an open-label, four-way crossover study in 10 healthy male volunteers. A single oral dose of 30 mg nadolol was administered with water (control), itraconazole (100 mg), or grapefruit juice (300 mL), or after a 6-day pretreatment with rifampicin (450 mg/day). Plasma concentrations and urinary excretions of nadolol were measured over 48 hours after its dosing. Systolic and diastolic blood pressures and pulse rate were periodically recorded after nadolol administration as pharmacodynamic parameters. Itraconazole increased the peak plasma concentration and the area under the plasma concentration-time curve (AUC0-∞ ) of nadolol by 468% and 224% of control, respectively (P .001). A slight, but not statistically significant, decrease in AUC0-∞ of nadolol was observed in rifampicin and grapefruit juice phases as compared to control. Elimination half-life for nadolol did not differ among the four phases. During itraconazole phase, nadolol reduced pharmacodynamic parameters to a greater extent than the other phases. These results suggest that itraconazole substantially increases the oral availability of nadolol possibly by the inhibition of intestinal P-glycoprotein, whereas grapefruit juice has little effect on nadolol pharmacokinetics.

The molecular basis of ineffective hematopoiesis in myelodysplastic syndromes (MDS) has yet to be defined. Elevation of tumor necrosis factor (TNF)-α in bone marrow plasma has been considered to be a cause of apoptosis of hematopoietic progenitor cells, and this could result in ineffective hematopoiesis in MDS. However, the mechanism of TNF-α elevation has not been elucidated. We recently found aberrant regulation of c-fos mRNA stability in neutrophils isolated from MDS patients (Feng et al, PLoS ONE, 2013). Since the expression levels of RNA-binding proteins that regulated c-fos mRNA stability were not altered in the majority of the patients and no mutations were detected in c-fos mRNA, microRNAs were possibly involved in the insufficient stabilization of c-fos mRNA. In this study, we chose 20 microRNAs targeting c-fos according to four different databases and compared their expression levels in neutrophils between MDS patients and healthy volunteers. Among them, miR-34a and miR-155 were significantly increased in MDS (p Recently, it was reported that c-fos physically interacted with nuclear factor κB (NF-κB) p65 to inhibit transcription of TNF-α in response to pro-inflammatory cytokines such as lipopolysaccharide (LPS) in monocytic cells (Koga et al, Immunity, 2009). Therefore, we hypothesized that neutrophils with reduced c-fos would overproduce TNF-α in response to inflammatory stimuli in MDS. To test our hypothesis, siRNA for c-fos was introduced into HL60 cells that were differentiated toward a neutrophil-like phenotype by 1.25% DMSO. The treatment with siRNA decreased c-fos protein levels to 58.8 ± 19.6-fold of that in the control cells. When stimulated with 1 μM LPS for 3 hours, the increase rates of TNF-α mRNA were greater in c-fos siRNA-treated cells (41.2 ± 25.7-fold, p Disclosures No relevant conflicts of interest to declare.