Impact of Green Tea Catechin Ingestion on the Pharmacokinetics of Lisinopril in Healthy Volunteers.

Lisinopril, a highly hydrophilic long-acting angiotensin-converting enzyme inhibitor, is frequently prescribed for the treatment of hypertension and congestive heart failure. Green tea consumption may reduce the risk of cardiovascular outcomes and total mortality, whereas green tea or its catechin components has been reported to decrease plasma concentrations of a hydrophilic ß blocker, nadolol, in humans. The aim of this study was to evaluate possible effects of green tea extract (GTE) on the lisinopril pharmacokinetics. In an open-label, randomized, single-center, 2-phase crossover study, 10 healthy subjects ingested 200 mL of an aqueous solution of GTE containing ~ 300 mg of (-)-epigallocatechin gallate, a major catechin component in green tea, or water (control) when receiving 10 mg of lisinopril after overnight fasting. The geometric mean ratio (GTE/control) for maximum plasma concentration and the area under the plasma concentration-time curve of lisinopril were 0.289 (90% confidence interval (CI) 0.226-0.352) and 0.337 (90% CI 0.269-0.405), respectively. In contrast, there were no significant differences in time to reach maximum lisinopril concentration (6 hours in both phases) and renal clearance of lisinopril (57.7 mL/minute in control vs. 56.9 mL/minute in GTE). These results suggest that the extent of intestinal absorption of lisinopril was significantly impaired in the presence of GTE, whereas it had no major effect on the absorption rate and renal excretion of lisinopril. Concomitant use of lisinopril and green tea may decrease oral exposure to lisinopril, and therefore result in reduced therapeutic efficacy.

Glucocorticoids attenuate the sensitivity of glucocorticoid-resistant lymphoid cells to doxorubicin via reduction in OCTN2.

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.

Role of (-)-epigallocatechin gallate in the pharmacokinetic interaction between nadolol and green tea in healthy volunteers.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Lack of pharmacokinetic interaction between fluvastatin and green tea in healthy volunteers.

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

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.

Hmga2 collaborates with JAK2V617F in the development of myeloproliferative neoplasms.

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.

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.

Reduction of c-Fos via Overexpression of miR-34a Results in Enhancement of TNF- Production by LPS in Neutrophils from Myelodysplastic Syndrome Patients.

Although increased TNF-α has been considered to cause ineffective hematopoiesis in myelodysplastic syndromes (MDS), the mechanisms of TNF-α elevation are not known. We recently found that c-Fos mRNA stabilization under translation-inhibiting stimuli was impaired in MDS-derived neutrophilic granulocytes. In the current study, we identified overexpression of c-Fos-targeting miR-34a and miR-155 as the cause of impairment. Expression levels of miR-34a but not miR-155 inversely correlated with ratios of c-Fos-positive cells in MDS-derived CD16+ neutrophils (r = -0.618, P<0.05), which were analyzed by flow cytometry. Among the seventeen patients, c-Fos was detectable in less than 60% of CD16+ cells in eight patients (Group A), while five (Group B) expressed c-Fos in more than 80% of CD16+ cells, which was consistent with the controls (88.6 ± 7.8%). Group A-derived granulocytes secreted more TNF-α in response to 1 µM LPS for 3 hours (735.4 ± 237.5 pg/mL) than Group B (143.5 ± 65.7 pg/mL, P<0.05) and healthy controls (150.8 ± 91.5 pg/mL, P<0.05). Knockdown of c-Fos in neutrophil-like differentiated HL60 increased the binding of NF-κB p65 to the promoter region of TNF-α DNA. Thus, c-Fos reduction via overexpression of miR-34a contributes to TNF-α overproduction under inflammatory stimuli in MDS.

A new method for maturity-dependent fractionation of neutrophil progenitors applicable for the study of myelodysplastic syndromes.

We applied our new method, maturity-dependent fractionation of bone marrow-derived neutrophil progenitors, to a study of gene expression profiles during granulopoiesis in myelodysplastic syndromes. CD34+ cells with low density [F1], CD11b-/CD16- [F2], CD11b+/CD16- [F3] and CD11b+/CD16low [F4] with intermediate density, CD11b+/CD16int [F5] and CD11b+/CD16high [F6] with high density were isolated from six patients. Although AML1 and C/EBP-ϵ mRNA peaked at F1 and F4, respectively, in healthy individuals, C/EBP-ϵ was maximized at F2/F3 in all patients, two of whom showed simultaneous peaks of AML1 at F2. Thus, this fractionation is useful to detect mistimed induction of granulopoiesis-regulating genes in myelodysplastic syndromes.

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

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<0.01). To our knowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

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 ß 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 ß 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.

Transcripts expressed using a bicistronic vector pIREShyg2 are sensitized to nonsense-mediated mRNA decay.

BACKGROUND: pIREShyg2 has been widely used as a bicistronic expression vector. However, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mRNA decay (NMD), an mRNA surveillance system that degrades mRNA with a premature termination codon (PTC). In mammalian cells, the induction of NMD requires either a long 3'UTR or the presence of an exon-junction complex downstream of a PTC. The efficiency of NMD is greater when a PTC generates longer 3'UTR. pIREShyg2 provides the first cistron gene with a long 3'UTR consisting of a downstream intervening sequence (IVS), an internal ribosomal entry site (IRES) and the second cistron. Therefore, we hypothesized that the first cistron genes in pIREShyg2 are sensitized to NMD, which affects their expression levels. To examine this hypothesis, cDNAs encoding human granulocyte-macrophage colony-stimulating factor receptor beta chain (betac) and its splice variant (betac79), in which the retention of a 79-base intron caused a frameshift generating 18 PTCs, were cloned into pIREShyg2 and stably expressed in a murine cell line, Ba/F3. RESULTS: Compared with wild-type betac, the mRNA levels of betac79 were less than one tenth and decayed faster. Both translation inhibition and Upf1 knockdown led to significantly greater up-regulation of betac79 than wild-type betac. However, the use of a monocistronic pMT21 vector abolished the up-regulatory effects of translation inhibition and Upf1 knockdown on both wild-type betac and betac79, suggesting that the NMD is attributable to a structural determinant in pIREShyg2. The elimination of the intron and the proximal 3' 17 PTCs did not alter the greater effects of translation inhibition on betac79, suggesting that the first PTC, which determines 3'UTR length, was sufficient to enhance NMD efficiency. Thus, transcripts of PTC-harboring genes with longer 3'UTR are more efficiently degraded by the vector-dependent NMD than those of wild-type genes with relatively shorter 3'UTR, resulting in minimized expression of truncated mutants. CONCLUSIONS: We conclude that pIREShyg2, which sensitizes its bicistronic transcripts to NMD, may be useful for studying NMD but should be avoided when maximum expressions of PTC-harboring genes are required.

Na+ -Ca2+ exchanger expression and its modulation.

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.

Terminally differentiated neutrophils predominantly express Survivin-2 alpha, 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-2 alpha, 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-2 alpha, not likely involved in apoptosis inhibition by GM-CSF/G-CSF.

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

We recently identified a reduction in the neutrophil surface expression of common beta chain (beta c) 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 beta c expression, beta c mRNA from neutrophilic granulocytes of MDS patients and healthy controls was analyzed by a combination of direct reverse transcriptase-polymerase chain reaction-based single-strand conformational polymorphism and sequencing. Nine different beta c transcripts were detected, but none was specific for MDS. However, one of the transcripts (beta c79) containing a 79-base intron insertion between exons V and VI was significantly increased in MDS. This 27-kd isoform consisted of the beta c N-terminal 182 amino acids followed by a new 84-amino-acid sequence. beta c79 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 alpha chain. Our study suggests that the accumulation of the abnormal beta c transcripts with intron V retention results in the reduction in cell-surface expression of beta c observed in MDS.

Transfusion-related acute lung injury following allogeneic bone marrow transplantation in a patient with acute lymphoblastic leukemia.

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.

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. 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) mM to 0.937(+/-0.081) mM (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.

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.