KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.

Comparison of graft-versus-host-disease and survival after HLA-identical sibling bone marrow transplantation in ethnic populations.

The association of ethnicity with the incidence of graft-versus-host disease (GVHD) and other clinical outcomes after transplantation is controversial. We compared the results of HLA-identical sibling bone marrow transplantations for leukemia, performed between 1990 and 1999, among different ethnic populations, including 562 Japanese, 829 white Americans, 71 African Americans, 195 Scandinavians, and 95 Irish. Results for adults and children were analyzed separately. Multivariate analyses of adult patients showed that white Americans, African Americans, and Irish cohorts were at significantly higher risk for acute GVHD than Japanese or Scandinavian cohorts (relative risk [RR] = 1.77, P < .001; RR = 1.84, P < .006; RR = 2.22, P < .001, respectively). White Americans, African Americans, and Irish, but not Scandinavians, were at significantly higher risk for early (within 3 months of transplantation) transplant-related mortality (TRM) compared with Japanese (RR = 2.99, P < .001; RR = 5.88, P < .001; RR = 2.66, P < .009, respectively). No differences in the risk for chronic GVHD, relapse, and overall survival were noted. In the pediatric cohort (limited to Japanese and white Americans), white Americans were at significantly higher risk for acute (RR = 1.93; P = .04) and chronic (RR = 3.16; P = .002) GVHD. No differences in other clinical outcomes were noted. Our findings suggest that ethnicity may influence the risk for GVHD, though overall survival rates after transplantation remain similar.

Modulation of polymorphonuclear leukocyte apoptosis in the critically ill by removal of cytokines with continuous hemodiafiltration.

Delay of polymorphonuclear leukocyte (PMN) apoptosis caused by hypercytokinemia is considered to be a potential cause of tissue damage and resultant organ failure. We evaluated whether continuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter (PMMA-CHDF), which can remove cytokines in the circulating blood, can modulate apoptosis in peripheral blood neutrophils and thereby reduce tissue damage and organ dysfunction in 25 critically ill patients. Following the completion of a 3-day PMMA-CHDF session, serum cytokine levels were significantly decreased and the percentage of apoptotic PMNs was significantly increased. A significant correlation was observed between the PMMA-CHDF-induced increase in the percentage of apoptotic PMNs and the degree of decrease in the serum interleukin-6 level. A significant correlation was also found between the increase in the percentage of apoptotic PMNs and improvement in sequential organ failure assessment score following PMMA-CHDF. These results suggest that PMMA-CHDF in critically ill patients with hypercytokinemia and concomitant delay in apoptosis of PMNs can alleviate the delay of PMN apoptosis through the removal of serum cytokines and thus may result in avoidance of organ dysfunction.

Effect of filgrastim administration for steady-state mobilization of peripheral blood stem cells.

To obtain a better (optimal) schedule of peripheral blood stem cell (PBSC) collection by steady-state granulocyte colony-stimulating factor administrations for autologous or allogeneic transplantations, we compared the effect of doses of filgrastim (8 microg/kg/day versus 16 microg/kg/day) for the steady-state mobilization of PBSCs. The effects of a filgrastim dose of 8 microg/kg/day were not significantly different from those of a dose of 16 microg/kg/day. In the group of patients receiving 8 microg/kg/day, the CD34+ cells over 3 x 10(6)/kg donor body weight were harvested in 3 patients who did not have a long history of receiving combination chemotherapy. The administration of 8 microg/kg filgrastim was adopted also for allogeneic PBSC mobilization for 24 healthy donors. All healthy donors donated an adequate number of PBSCs (CD34+ cells over 4 x 10(6)/kg of recipient body weight) and tolerated this mobilization well with no serious complications. In PBSC mobilization with healthy donors, the maximal yields of CD34+ cells from Day 4 to Day 6 were seen on the fifth day in most cases.

Board certification of medical doctors and medical technologists in transfusion medicine in Japan.

The Japan Society of Blood Transfusion (JSBT) organized a Board for certification of medical doctors in Transfusion Medicine (Board certified medical doctors by the JSBT) and a Board for certification of medical technologists for transfusion medicine (qualified medical technologists in Transfusion Medicine). For certified medical doctors the JSBT co-ordinated with the Japanese Association of Medical Science and with the Japan Medical Association, and for qualified medical technologists the JSBT co-organized with the Japanese Society of Laboratory Medicine, the Japanese Association of Medical Technologist and the College of Clinical Pathology of Japan. By May 2001, 259 of the certified medical doctors and 875 of the qualified medical technologists have been certified and are participating actively to increase the level of transfusion practice at individual facilities.