Yukitoshi Hayase

Radiation Sensitivity of Megakaryocyte Colony-Forming Cells in Human Placental and Umbilical Cord Blood

Abstract Kashiwakura, I., Kuwabara, M., Inanami, O., Murakami, M., Hayase, Y., Takahashi, T.A. and Takagi Y. Radiation Sensitivity of Megakaryocyte Colony-Forming Cells in Human Placental and Umbilical Cord Blood. The in vitro radiation sensitivity of CFU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34+ cells were irradiated with X rays at a dose rate of 73 cGy/min. The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CFU-Meg) and small colonies (mature CFU-Meg). Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D0 for immature CFU-Meg = 56–77 cGy, D0 for mature CFU-Meg = 86 cGy–1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D0 for immature CFU-Meg = 89–98 cGy; D0 for mature CFU-Meg = 1.25–1.31 Gy). Our results showed that the immature CFU-Meg were more radiosensitive than the mature CFU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11.

Chronopharmacokinetics of Tacrolimus in Kidney Transplant Recipients: Occurrence of Acute Rejection

The circadian variation of clinical pharmacokinetics of tacrolimus was studied using 16 adult renal transplant recipients 1 month after the operation. The recipients were administered tacrolimus twice a day (9 a.m. and 9 p.m.), and whole‐blood samples were obtained just prior to and 1, 2, 3, 6, 9, and 12 hours after oral administration. Histological specimens of transplant kidney were collected by an allograft core biopsy on day 28 after the transplantation. There were no circadian changes in the area under the concentration‐time curve (AUC0–12) (214 ng•h/mL during daytime vs. 223 ng•h/mL during nighttime) resulting from morning and night doses. A slight delay in mean residence time (MRT0–12) and time to the peak concentration (tmax) was found after night doses, but there was no statistical significance. Three patients (18.8%) had a clinical acute rejection (AR) episode 4 to 6 weeks after transplantation, and AUC0–12 at nighttime was significantly lower (18.4% on average) in patients with AR in comparison to those without AR. There was no statistical significance in maximum concentration (Cmax) or morning/night trough levels between patients with and without AR. In regard to the correlation between tacrolimus concentrations in each sampling time and AUC0–12, the morning trough concentrations were less predictable for daytime AUC0–12 (r2 = 0.125), but there was a weak correlation to nighttime AUC0–12 (r2 = 0.424). Tacrolimus concentrations at 2, 3, and 6 hours after the morning dose (C2, C3, and C6) had a good correlation against daytime AUC. The results of this study indicate that the variance on the clinical pharmacokinetics of tacrolimus between daytime and nighttime in renal transplant patients is not significant, while the lower nighttime AUC corresponded to the occurrence of AR.

Circadian pharmacokinetics of mycophenolic Acid and implication of genetic polymorphisms for early clinical events in renal transplant recipients.

Background. We investigated the mycophenolic acid (MPA) chronopharmacokinetics and the relation between MPA circadian exposure and the incidence of acute rejection (AR). The association between selected genetic polymorphisms and clinical events or MPA circadian exposure was also studied. Methods. Thirty recipients were studied one month after renal transplantation. Mycophenolate mofetil (MMF) was administered twice a day at a single dose of 0.5 g in four patients, 0.75 g in eight patients, and 1 g in 18 patients. Results. The daytime area under the concentration-time curve (AUC0–12) was larger than the nighttime AUC0–12 (55.09 vs. 50.54 &mgr;g·hr/ml, P=0.049). The Cmax and tmax of MPA after the morning dose were respectively higher and shorter than those after the night dose. Seven patients (23.3%) had AR episodes. The MMF single dose per body weight (12.46 mg/kg in patients with AR vs. 16.99 in patients without AR), daytime and nighttime AUC0–12 (32.41 vs. 62.00 and 24.44 vs. 57.88 &mgr;g·hr/ml) and morning trough level of MPA (1.03 vs. 3.83 &mgr;g/ml) were significantly lower in patients with AR than in those without AR. The percentage of patients requiring diminished dose of MMF due to diarrhea was higher among patients with the multidrug resistance 1 (MDR1) C3435T T allele than among those with the CC genotype (P=0.049). Conclusion. MPA pharmacokinetics showed circadian variations, and a lower MPA AUC in both daytime and nighttime was associated with the occurrence of AR in the early stage after renal transplantation. The MDR1 C3435T polymorphism might be associated with diarrhea due to MPA.

Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture

Long-term severe thrombocytopenia following human placental and umbilical cord blood (CB) transplantation is a significant clinical problem. We studied the ex vivo expansion of megakaryocytic progenitor cells (CFU-Meg) from cryopreserved/thawed leukocyte concentrates (LC) of CB prepared by the Tokyo Cord Blood Bank protocol. The LC cells were cultured in serum-free culture medium supplemented with a combination of early-acting cytokines including thrombopoietin (TPO), flt3-ligand (FL), and stem cell factor (SCF). Combination of TPO plus FL, TPO plus SCF, and all of these cytokines together resulted in 8.9-, 7.7-, and 8.4-fold increases in CFU-Meg, respectively, by Day 5 of culture. Our results showed that this simple expansion strategy has the potential for expanding CFU-Meg from cryopreserved/thawed LC cells from CB.

Effects of amifostine on the proliferation and differentiation of megakaryocytic progenitor cells

This study investigated the effects of amifostine, a clinically usable radioprotector or chemoprotector, on the proliferation and differentiation of normal and X-irradiated cluster of differentiation 34 positive (CD34+) megakaryocytic progenitor cells (colony-forming unit in megakaryocytes, CFU-Meg) from human placental and umbilical cord blood (CB) in vitro. Amifostine significantly accelerated megakaryocyte colony formation in a plasma clot culture supplemented with recombinant human thrombopoietin because of an increase in immature CFU-Meg-derived large megakaryocyte colony formation. An analysis of the cells that were harvested from the culture showed that amifostine induced a 70- and an 83-fold increase in the total cell and CFU-Meg numbers, respectively, and produced hyperploid megakaryocytes of more than 8 N ploidy. The radioprotective effect of amifostine on the clonal growth of X-irradiated CD34+ CFU-Meg was observed by treatment before or after irradiation. These findings suggest that the action of amifostine extends from immature CFU-Meg to the terminal differentiation of megakaryopoiesis, and its radioprotective effect is shown in megakaryopoiesis and thrombopoiesis.

電子カルテ上への「添付文書情報提供システム」の構築とその運用に関する研究

To promote the appropriate use of pharmaceuticals and to prevent side effects, physicians need package inserts on medicinal drugs as soon as possible. A medicinal drug information service system was established for electronic medical records to speed up and increase the efficiency of package insert communications within a medical institution. Development of this system facilitates access to package inserts by, for example, physicians. The time required to maintain files of package inserts was shortened, and the efficiency of the drug information service increased. As a source of package inserts for this system, package inserts using a standard generalized markup language (SGML) form were used, which are accessible to the public on the homepage of the Organization for Pharmaceutical Safety and Research (OPSR). This study found that a delay occurred in communicating revised package inserts from pharmaceutical companies to the OPSR. Therefore a pharmaceutical department page was set up as part of the homepage of the medical institution for electronic medical records to shorten the delay in the revision of package inserts posted on the medicinal drug information service homepage of the OPSR. The usefulness of this package insert service system for electronic medical records is clear. For more effective use of this system based on the OPSR homepage pharmaceutical companies have been requested to provide quicker updating of package inserts.

Investigation of cost and medical service fee for pharmaceutical management in home medical care

Due to the evolvement of the aged society and the steep rise in medical costs, the environment encircling the medical care industry has been changing remarkably. For this reason, it has become both necessary and fundamental for a community pharmacist to participate in home medical care through the pharmaceutical management service. We have studied the associated costs and medical service fees for pharmaceutical management in home medical care. The costs and medical service fees were calculated based on the pharmaceutical management service data collected during the three years from November 1998 to October 2001. As a result, the medical service fees were calculated using the old system which lasted until March 2002. Calculations using this system took into account 550 points per visit, up to two visits per month. Under the new system which started in April 2002, the number of visits taken into account is four times a month, 500 points for the first visit, 300 points from the second through to the forth visit. Then, we simulated a break-even point (BEP). It is clear that it is difficult for any community pharmacy to be specialized in home medical care. In order for the pharmacist to actively participate in home medical care in the future, it is necessary to further improve the system.

INHIBITORY EFFECT OF MURINE KIDNEY EXTRACTS ON MAST CELLS DERIVED FROM HUMAN UMBILICAL CORD BLOOD

We examined the effect of aqueous murine kidney extract (MKE) on the growth of mast cells prepared from the liquid culture of human umbilical cord blood CD34+ cells in the presence of a combination of recombinant human stem cell factor (SCF) and interleukin-6 (IL-6). Cultured cells were mature mast cells that expressed CD117 antigen on their surface, a specific marker for human mast cell, and they contained 6.53 pg histamine per cell. Adding MKE resulted in a 53% inhibition of mast cell growth and a 40% decrease in histamine content in mast cells in a serum-free liquid culture stimulated by SCF and IL-6. The inhibitory molecule for the growth of human mature mast cells was estimated at about 30 kDa of protein from gel-filtration HPLC. This fraction also inhibited the growth of murine peritoneal cells-derived mast cells. These results suggest that MKE contains regulator(s) that suppress the growth of mast cells and histamine synthesis, and that act beyond species specificity.

Colony-Promoting Activity in Mice Kidneys with Phenylhydrazine Hemolytic Anemia

Aqueous anemic mice kidney extracts (MKE) were assessed colony-promoting activity (CPA) of hematopoietic progenitor cells in serum-free cultures stimulated by interleukin-3 and erythropoietin (Epo). Mice with hemolytic anemia followed by phenylhydorazine (PHZ) injection for 3 days showed a decrease in the hematocrit (25.4%) and an increase in serum Epo by 14-fold of the control on day 3 after the treatment. At 3 days, the total number of hematopoietic progenitor cells in the bone marrow of PHZ mice decreased by 67% of the control, while these cells in the spleen increased to 22-fold of the control on day 3 and 55-fold on day 6. A significant increase in CPA was observed in MKE prepared from PHZ mice kidneys. Additionally, bone marrow suppressive anemia induced by 5-fluorouracil resulted in enhanced CPA the same as for PHZ mice, but in contrast, anemia with suppression of Epo-production due to nephrotoxicity induced by cisplatin caused a decrease in CPA. These results suggest that CPA in MKE correlates with hematopoietic conditions, and may have a definite role in hematopoiesis through the function of the kidney.

Partial Purification and Characterization of a Factor for the Enhancement of Colony Formation in Vitro by Myeloid Progenitor Cells

We have purified a factor, hematopoietic promoting factor (HPF), from porcine kidney extract (PKE), which exhibits a promoting activity on granulocyte/macrophage (GM) colony and burst-forming-unit-erythroid (BFU-E)-derived colony formation by progenitors from murine bone marrow cells in vitro. The addition of HPF resulted in an enhancement of the GM colonies as well as BFU-E-derived colonies, but did not enhance the colony-forming-unit-erythroid (CFU-E)-derived colony formation. HPF was added to the BFU-E cultures together with cytokines, such as recombinant murine interleukin-3 (IL-3), recombinant murine GM colony-stimulating-factor (GM-CSF) and recombinant human G-CSF, which have all been shown to enhance BFU-E growth. The combination of HPF plus these cytokines resulted in an enhancement of benzidine negative colony formation in comparison to the case of each cytokine alone; however, no increase was found on BFU-E colony formation. HPF is able to enhance the granulopoiesis and erythropoiesis in vitro. And the synergistic activity of HPF is significantly affected by the presence of cytokines in the cultures.