Yayoi Shikama

Treatment for the decline of ionized calcium levels during peripheral blood progenitor cell harvesting

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.

Involvement of Na+/Ca2+ exchanger in migration and contraction of rat cultured tendon fibroblasts

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.

Terminally differentiated neutrophils predominantly express Survivin-2α, a dominant-negative isoform of Survivin

Survivin, which is a member of the inhibitor of apoptosis protein family, was found originally in immature cells and cancer cells but not in non‐neoplastic adult tissues. The subsequent identification of four other alternative splice variants that possess distinct functions and localizations suggested the diverse roles of survivin isoforms. An unspecified isoform of survivin was found recently to be induced in terminally differentiated neutrophils by cytokines that prolong the neutrophil lifespan, such as GM‐CSF and G‐CSF, suggesting the importance of survivin in blocking apoptosis in neutrophils. To examine the mechanism by which survivin inhibits neutrophil apoptosis, we attempted to induce survivin by GM‐CSF/G‐CSF in an HL60 cell line that was differentiated into neutrophils by all‐trans retinoic acid and DMSO and freshly isolated human neutrophils. The antiapoptotic isoform “Survivin,” which was decreased during differentiation, was re‐induced by GM‐CSF in neutrophil‐like, differentiated HL60. In contrast, in normal neutrophils, survivin mRNA was observed to increase spontaneously after 24 h incubation, and no additional elevation was induced by GM‐CSF/G‐CSF, which exerted their antiapoptotic effects on the neutrophils in 6 h, despite the lack of survivin induction. PCR and Western blotting detected Survivin‐2α, a dominant‐negative of antiapoptotic Survivin, with no other isoforms in the freshly isolated or incubated neutrophils. Our study revealed that the expressed isoforms and the response to GM‐CSF were different between the HL60‐derived and normal neutrophils, which predominantly expressed Survivin‐2α, not likely involved in apoptosis inhibition by GM‐CSF/G‐CSF.

Granulocytes from patients with paroxysmal nocturnal hemoglobinuria and normal individuals have the same sensitivity to spontaneous apoptosis

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.

Pharmacokinetic and Pharmacodynamic Interaction of Nadolol With Itraconazole, Rifampicin and Grapefruit Juice in Healthy Volunteers

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.

Neutrophil-specific reduction in the expression of granulocyte--macrophage colony-stimulating factor receptor subunits in myelodysplastic syndromes.

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 α (GMRα) and common β (βc) subunits on CD16+ neutrophils, CD14+ monocytes and CD3+ T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMRα was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, βc 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 βc 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 reverse‐transcribed mRNA isolated from the affected neutrophils suggests that the reduction of βc 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.

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes

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.

Maturity-dependent fractionation of neutrophil progenitors: A new method to examine in vivo expression profiles of differentiation-regulating genes

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.

Accumulation of an intron-retained mRNA for granulocyte macrophage-colony stimulating factor receptor common β chain in neutrophils of myelodysplastic syndromes

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.

Comprehensive technical and patient-care optimization in the management of pediatric apheresis for peripheral blood stem cell harvesting

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 × 106 CD34+ cells/kg (0.2-77.9 × 106 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 <10 kg.