Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.

Gene targeting studies in mice have shown that the transcription factor Ikaros plays an essential role in lymphoid development and as a tumor suppressor in T cells, whereas the related gene Aiolos functions as a tumor suppressor in B cells. We analyzed the expression levels of the Ikaros gene family, Ikaros and Aiolos, in human bone marrow samples from patients with adult acute lymphoblastic leukemia [ALL (n = 46; B-cell ALL = 41; T-cell ALL = 5)]. Overexpression of the dominant negative isoform of Ikaros gene Ik-6 was observed in 14 of 41 B-cell ALL patients by reverse transcription-PCR, and the results were confirmed by sequencing analysis and immunoblotting. None of the other dominant negative isoforms of the Ikaros gene were detected by reverse transcription-PCR analysis. Southern blotting analysis with PstI digestion revealed that those patients with the dominant negative isoform Ik-6 might have small mutations in the Ikaros locus. We did not detect any overexpression of dominant negative isoforms of Aiolos in adult ALL patients. These results suggest that Ikaros plays a key role in human B-cell malignancies through the dominant negative isoform Ik-6.

Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Phorbol ester-mediated regulation of CD10/neutral endopeptidase transcripts in acute lymphoblastic leukemias.

The cell-surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (CD10/NEP) hydrolyzes a variety of peptide substrates and regulates related peptide-mediated cellular responses. Because the enzyme functions as part of a peptide regulatory loop, the fact that CD10/NEP itself varies with cellular activation is of considerable interest. In hematopoietic and nonhematopoietic cell types, the levels of CD10/NEP protein and enzymatic activity correlate with transcript abundance. For these reasons, we investigated the regulation of CD10/NEP transcripts in the phorbol ester-treated acute lymphoblastic leukemia cell line, REH. When REH cells are treated with phorbol myristate acetate (PMA), CD10/NEP transcripts rapidly decrease in a labile protein-dependent manner. PMA has a modest effect on CD10/NEP transcription and significantly reduces CD10/NEP mRNA stability. Of note, the predicted secondary structure of the CD10/NEP 3' untranslated region includes several stem loop structures that may affect the stability of CD10/NEP transcripts.

Ikaros expression in human hematopoietic lineages.

The Ikaros gene has been implicated in lymphoid development and proliferation from the results of gene targeting studies in mice. Recently we reported that the Ikaros gene may be involved in the disease progression of chronic myelogenous leukemia (CML). In this report, we investigated Ikaros isoforms in human non-lymphoid leukemia cell lines and normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E)-derived colonies. We evaluated Ikaros gene expression by RT-PCR, Southern blotting, sequencing analysis, Northern blotting, and immunoblotting.Ikaros isoforms Ik-1 and Ik-2, 3 were predominantly expressed in human non-lymphoid leukemia cell lines. Ik-4 and Ik-8 were also detectable as a minor population. In contrast to the previous report in mice, multiple Ikaros isoforms were expressed in human CFU-GM and BFU-E-derived colonies, and the dominant-negative isoform Ik-6 was not detectable. We also showed that human Ikaros isoforms contained an additional coding sequence in the N-terminal region, which was highly homologous to the sequence reported in mice. These observations suggest that the Ikaros gene may play some role in the development of human non-lymphoid lineage hematopoiesis. Moreover, the finding that the dominant-negative isoform Ik-6, which was overexpressed in patients with blast crisis of CML, was rarely detectable in non-lymphoid lineages supports its pathogenetic role in human hematologic malignancies.

Administration of granulocyte colony-stimulating factor induces hyporesponsiveness to lipopolysaccharide and impairs antigen-presenting function of peripheral blood monocytes.

OBJECTIVE: The incidence and severity of acute graft-vs-host disease after allogeneic transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) are not greater than those after conventional bone marrow transplantation despite infusion of more than one log greater number of donor T cells in PBSC. It has been postulated that monocytes from G-CSF-mobilized donors suppress alloreactivity of donor T cells. MATERIALS AND METHODS: We investigated the phenotype and function of monocytes in normal individuals receiving 10 microg/kg of G-CSF for 4 days. RESULTS: Monocytes were phenotypically and functionally different after G-CSF administration from steady-state monocytes. They were characterized by an increased CD14(+)CD16(+) subpopulation, reduced expression of HLA-DR, and diminished ability to produce tumor necrosis factor-alpha and interleukin-10 to lipopolysaccharide, compared with steady-state monocytes. These alterations were not replicated by culturing monocytes with G-CSF in vitro, suggesting an indirect effect of G-CSF. In addition, the antigen-presenting function of G-CSF-mobilized monocytes was impaired. CONCLUSION: Hyporesponsiveness of G-CSF-treated monocytes to lipopolysaccharide with regard to tumor necrosis factor-alpha production, together with impaired antigen-presenting function, may be responsible for the unexpectedly low incidence of graft-vs-host disease after G-CSF-mobilized PBSC transplantation.

Establishment and characterization of a novel ALL-L3 cell line (BALM-18): induction of apoptosis by anti-IgM and inhibition of apoptosis by bone marrow stroma cells.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-18, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) at diagnosis using bone marrow stroma cells (BST) as feeder cells. The primary leukemia cells did not grow without feeder cells. As with the primary leukemia cells, BALM-18 showed an immunophenotype of Burkitt's lymphoma group I [CD10+, CD20+, CD23-, CD39-, CD77+] and carried the t(8;14)(q24;q32) chromosomal abnormality which is highly associated with ALL-L3 and Burkitt's lymphoma. It also revealed a significantly low level of bcl-2 protein. Strikingly, anti-human IgM antibody did induce apoptosis in induction experiments. However, it was reversed by the addition of anti-CD40 antibody or BST cells, whereas the culture supernatant of the stroma cells did not show any effect on the inhibition of apoptosis. BALM-18 may be useful for analyzing both the mechanisms of anti-IgM induced apoptosis and signaling during the inhibition of apoptosis by CD40 or BST cells.

Synergistic effects of pegylated recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor on mobilization of hematopoietic progenitor and stem cells with long-term repopulating ability into peripheral blood in mice.

We investigated the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization and the combined effect of PEG-rHuMGDF plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) in C57BL/6 mice. Treatment of mice with PEG-rHuMGDF increased the numbers of day 8 and day 12 spleen colony-forming units (CFU-S), and pre-CFU-S in the PB. Ten days administration of PEG-rHuMGDF could mobilize higher numbers of days 8 and day 12 CFU-S than 5 days administration. An optimal dose of PEG-rHuMGDF mobilized a higher number of committed progenitor cells (day 8 CFU-S) and a lower number of immature progenitor cells (pre-CFU-S) into PB than rhG-CSF. The combined administration of optimal or suboptimal doses of PEG-rHuMGDF and rhG-CSF induced synergistic effects on mobilization of CFU-S and pre-CFU-S into PB compared to either factor alone. Four months after sex-mismatched PBPC transplantation, long-term donor-derived engraftment was observed in all recipients that had been transplanted with PBPCs mobilized by rhG-CSF and/or PEG-rHuMGDF, as determined by Y-chromosome polymerase chain reaction (PCR) analysis. Our data suggest that cytokine-induced pathways for PBPC mobilization may be different between PEG-rHuMGDF and rhG-CSF and indicate that PEG-rHuMGDF alone or the combination with rhG-CSF may be useful in effective PBPC mobilization.

G-CSF reduces IFN-gamma and IL-4 production by T cells after allogeneic stimulation by indirectly modulating monocyte function.

Despite a 10-fold increase of T cell dose, the incidence and severity of acute GVHD following allogeneic transplantation of G-CSF-mobilized PBSC is not increased compared to BMT. Experimental murine studies demonstrate that G-CSF polarizes donor T cells toward a type 2 cytokine response. To determine whether G-CSF alters T cell cytokine responses, we investigated the effects of G-CSF administration on T cell proliferative and cytokine responses to alloantigen and Con A in nonadherent PBMC (NAC) and CD3+ T cells obtained from normal individuals before and after G-CSF administration (10 microg/kg x 4 days). Although T cell proliferative and cytokine (IFN-gamma and IL-4) responses to alloantigen stimulation and Con A were significantly reduced in post-G-CSF NAC, they were restored by the removal of non-T cells from post-G-CSF NAC. Furthermore, there was less T cell alloreactivity in MLR in the presence of autologous post-G-CSF monocytes than in the presence of pre-G-CSF monocytes. This alteration was not replicated in vitro by culturing PBMC with G-CSF. These results suggest that G-CSF administration suppresses T cell proliferative and cytokine (IFN-gamma and IL-4) responses to allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplantation (2000).

Allogeneic peripheral blood stem cell transplantation for the treatment of chronic active Epstein-Barr virus infection.

The prognosis of chronic active Epstein-Barr virus infection (CAEBV) is very poor. We describe a 24-year-old male with severe CAEBV who was treated with allogeneic peripheral blood stem cell transplantation (allo-PBSCT). On admission, EBER-1 in lymphocytes infiltrating the liver, EBV-DNA in peripheral blood mononuclear cells (PBMC) and monoclonal NK cell proliferation were confirmed. After unsuccessful chemotherapy, he received an allo-PBSCT from his HLA-identical sister. Although he died of pulmonary hemorrhage on day +19, EBV-DNA was undetectable by PCR in PBMC, and the post-mortem liver showed no EBER-1-positive lymphocytes. This experience suggests that EBV-positive lymphocytes in CAEBV may be eradicated by allo-PBSCT, thereby raising the possibility of a new treatment modality. Bone Marrow Transplantation (2000) 26, 805-808.

Plasma stromal cell-derived factor-1 during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization.

In this report, we examined plasma stromal cell-derived factor-1 levels in normal healthy donors for allogeneic peripheral blood stem cell transplantation (PBSCT) and in patients for autologous PBSCT using an enzyme-linked immunosorbent assay. The average level of plasma stromal cell-derived factor-1 was 2197 pg/ml before granulocyte colony-stimulating factor administration and 1899 pg/ml on day 4, demonstrating a significant decrease in the peripheral blood of healthy donors (P=0.0003). In patients for autologous PBSCT, a significant decrease of plasma stromal cell-derived factor-1 in the peripheral blood was also observed (P=0.0464). However, the physiologic gradient of stromal cell-derived factor-1 between peripheral blood and bone marrow was never inverted in normal healthy donors or in autologous PBSCT patients. Our results suggest that stromal cell-derived factor-1 may not be involved in the granulocyte colony-stimulating factor-induced release of CD34(+) cells to the peripheral blood. Further studies of a possible additive effect of granulocyte colony-stimulating factor and stromal cell-derived factor-1 are warranted.

Hepatic graft-versus-host disease presenting as an acute hepatitis after allogeneic peripheral blood stem cell transplantation.

Hepatic graft-versus-host disease (GVHD) generally presents as cholestatic jaundice, and increased serum alkaline phosphatase (ALP) is followed by hyperbilirubinemia and clinical jaundice. Currently accepted standards for evaluating the clinical severity of GVHD are based not on serum aminotransferase levels but on the serum bilirubin level. We describe a 17-year-old Japanese female who had increased aminotransferases without cholestasis on day 23 after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Liver biopsy revealed lymphocytic infiltration of the portal tracts and pericentral necrosis of the lobuli. The limiting plates were not clearly defined due to cellular infiltrates. There was periductal lymphocytic infiltration and vacuolization of the biliary epithelial cells with exocytosis, compatible with GVHD of cholangiohepatitic type. These findings indicate that acute hepatic GVHD may present as acute hepatitis and this should be included in the differential diagnosis for patients with increased aminotransferases after allogeneic stem cell transplantation.

Hematopoietic progenitor cells from allogeneic bone marrow transplant donors circulate in the very early post-transplant period.

Despite the therapeutic efficacy of allogeneic bone marrow transplantation (allo-BMT), circulating hematopoietic progenitor cells after bone marrow transplantation have not been well characterized. In the present study, we focused on these 'post-transplant circulating progenitor cells (PTCPC)' which may be on their way to bone marrow. We analyzed the number of myeloid progenitor cells (CFU-GM) per 10 ml of peripheral blood (PB) on days 0 (just before transplantation), 1 (8-15 h after the completion of transplantation), 2, 3, 5, 7, 10, 14, 17, 21, 28 and 35 after allo-BMT in five transplant patients using a standard methylcellulose assay. In addition, high proliferative potential colony-forming cells (HPP-CFC) of the harvested donor bone marrow (BM) and day 1 PB of recipients were assayed in five patients. The origin of HPP-CFC from day 1 PB was analyzed by polymerase chain reaction of a DNA region containing a variable number of tandem repeats. The replating potential of these HPP-CFC was evaluated by a secondary colony assay. The proportion of CD38negative cells among CD34+ cells in the harvested BM and day 1 PB was evaluated by two-color flow cytometric analysis. The number of CFU-GM on day 1 ranged from 6 to 73/10 ml PB, and became undetectable on day 5. The reappearance of PTCPC was observed on day 14, along with hematopoietic recovery. The proportion of HPP-CFC among myeloid colonies from day 1 PB was significantly higher than that from harvested BM (44.3+/-10.4% vs 11.3+/-2.1%, respectively, n=5, P=0.0030). These HPP-CFC from day 1 PB were confirmed to be of donor origin. More than 90% of these HPP-CFC had replating potential. Two-color flow cytometric analysis revealed that the proportion of CD34+CD38negative cells was significantly higher in day 1 PB than in the harvested BM (61.0+/-16.5% vs 9.3+/-3.5%, respectively, n=7, P=0.0002). These observations suggest that both primitive and committed transplanted myeloid progenitor cells may circulate in the very early period following allo-BMT.

Granulocyte colony-stimulating factor and lineage-independent modulation of VLA-4 expression on circulating CD34+ cells.

Although the use of allogeneic transplants of peripheral blood stem/progenitor cells (PBSCs) is increasing, the precise mechanism of PBSC mobilization has not yet been fully clarified. We examined the expression of some adhesion molecules on CD34+ cells from steady-state bone marrow (BM), granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs, and cytotoxic drugs plus G-CSF-mobilized PBSCs. Irrespective of mobilization method, very late antigen (VLA)-4 expression on circulating CD34+ cells was significantly lower than on steady-state BM CD34+ cells. To elucidate the influence of lineage commitment on VLA-4 expression of circulating CD34+ cells, we analyzed VLA-4 expression on different subsets of CD34+ cells with or without CD33, CD38, CD5, or CD10 antigens, or Glycophorin A in G-CSF-mobilized PBSCs and steady-state BM from related donors, using 3-color flow cytometry. VLA-4 on circulating CD34+ subsets was less expressed than on each corresponding subset of steady-state BM CD34+ cells. Furthermore, VLA-4 positive rates showed no significant difference among the CD34+ subsets. Finally, the data comparing CD34+ cells from steady-state and G-CSF-mobilized PBSCs revealed no differences in terms of VLA-4 expression. These data suggest that reduced expression of VLA-4 may be a result of peripheralization of CD34+ cells from bone marrow, which occurs in a G-CSF- and lineage-independent fashion.

Successful engraftment of allogeneic peripheral blood stem cell transplant after nonmyeloablative preparative regimen with cytarabine and cyclophosphamide: report of 2 cases.

We report our experience with allogeneic peripheral blood stem cell transplantation (allo-PBSCT) following a nonmyeloablative conditioning regimen consisting of cytarabine (8 g/m2) and cyclophosphamide (120 mg/kg) in the treatment of 2 patients aged 50 and 55 years with refractory chronic myelomonocytic leukemia and chronic myeloid leukemia in accelerated phase, respectively. Our nonmyeloablative regimen was well tolerated by older patients at high risk of regimen-related toxicity by the conventional conditioning regimen but was immunosuppressive enough to achieve mixed chimerism. After allo-PBSCT, we monitored chimerism in these patients by fluorescence in situ hybridization using X- and Y-specific probes and polymerase chain reaction-based analysis of a variable number of tandem repeats. We found that full chimerism and graft-versus-leukemia (GVL) effects could be induced in these patients by donor lymphocyte infusions and withdrawal of posttransplantation immunosuppressive therapy. Our observations suggest that a nonmyeloablative conditioning regimen can establish mixed chimerism and that donor lymphocyte infusion may induce GVL effects in older patients at high risk of regimen-related toxicity.

Transient factor VIII inhibitor in a hemophilia patient after staphylococcal septic shock syndrome.

We report a transient type I factor VIII inhibitor that arose in a 30-year-old hemophilia patient just after staphylococcal septicemia. This situation usually occurs early in the course of substitution therapy with factor VIII concentrate in hemophilia patients. Although disseminated intravascular coagulation and acute respiratory distress syndrome developed after septic shock, the patient recovered following intravenous administration of antibiotics (meropenem and gentamycin), an antithrombin preparation, high-dose methylprednisolone, and recombinant factor VIII concentrate (rFVIII). During this therapy, however, activated partial thromboplastin time gradually lengthened. On the seventh day of hospitalization, intracranial hemorrhage occurred with right hemiplegia, even though the substitution therapy had continued at the same dosage (30 U/kg per day) of rFVIII. At that point, 4 Bethesda units of the type I inhibitor against factor VIII were detected in the plasma. Increased amounts (46 U/kg per day) of rFVIII and prednisolone were administered, and hypothermic therapy was initiated. Following these treatments, the patient's general condition gradually improved, and within 25 days the inhibitor titer dropped to undetectable levels and did not recur during treatment. These clinical findings suggest that the staphylococcal septic shock may have acted as a trigger in the development of transient factor VIII inhibitor in this patient.

Analysis of circulating hematopoietic progenitor cells after peripheral blood stem cell transplantation.

We investigated the kinetics of posttransplant circulating progenitor cells (PTCPC) in the early phase after autologous (auto-) and allogeneic (allo-) peripheral blood stem cell transplantation (PBSCT). We analyzed the number of myeloid progenitor cells (CFU-GM) per 10 ml of peripheral blood (PB) on days 0 (just prior to transplantation), 1 (12-15 hours after completion of first transplantation), 2, 3, 5, 7, 10, 14, 17, 21 and 28 (after auto-PBSCT), and also additionally on day 35 after allo-PBSCT. A standard methylcellulose colony assay was used for analysing the number of CFU-GGM and BFU-E on all of the days. In addition, high proliferative potential-colony forming cells (HPP-CFC) of the harvested PBSC from donors and day 1 PB from recipients were assayed in 5 allo-PBSCT patients. Furthermore, a proportion of CD38- cells among CD34+ cells in the harvested PBSC and day 1 PB was evaluated by two-color flow cytometric analysis in 5 allo-PBSCT patients. The number of CFU-GM on day 1 ranged from 7 to 119 per 10 ml PB after auto-PBSCT, and from 15 to 61 per 10 ml PB after allo-PBSCT. After these transient increases, PTCPC diminished rapidly. Then, PTCPC emerged again on day 7 after auto-PBSCT and on day 10 or 14 after allo-PBSCT along with neutrophil recovery. A proportion of HPP-CFC among myeloid colonies from day 1 PB of recipients was significantly higher than that from the harvested PBSC from donors (65.6 +/- 12.7% vs. 17.4 +/- 13.0%, respectively, n = 5, P = 0.0013). In addition, two-color flow cytometric analysis revealed that the proportion of CD34+CD38- cells was significantly higher in day 1 PB of recipients than in the harvested PBSC from donors (57.5 +/- 17.6% vs. 11.7 +/- 4.9%, n = 5, P = 0.005). These observations suggest that both primitive and committed transplanted myeloid progenitor cells may circulate in the very early period following PBSCT.

Severe bleeding tendency caused by leukemic infiltration and destruction of vascular walls in chronic neutrophilic leukemia.

Bleeding is reportedly one of the major causes of death in patients with chronic neutrophilic leukemia (CNL), but thrombocytopenia, abnormal platelet functions, or coagulopathy has been confirmed to be the cause of the bleeding tendency in only a small proportion of the patients. We report the case of a 49-year-old woman with CNL who experienced episodes of cutaneous and recurrent multiple cerebral hemorrhages without severe thrombocytopenia, detectable abnormal platelet functions, or coagulating dysfunction. Histological examination of specimens obtained at autopsy showed extensive infiltration and destruction of vascular walls by leukemic cells, which could explain her severe bleeding tendency. This study is the first to clearly show that the infiltration and destruction of vascular walls by leukemic cells can cause fatal bleeding episodes without warning from laboratory findings. Further studies are needed to elucidate the mechanism of the infiltration and destruction of blood vessels by CNL cells and to develop effective measures to control the growth and infiltration of CNL cells.

Allogeneic peripheral blood stem cell transplantation in 23 adult patients with hematologic malignancies: a single-center experience.

We describe a single-center experience of 23 consecutive patients (median age, 35 years) with hematologic malignancies who received allogeneic peripheral blood stem cell transplants (alloPBSCTs) from HLA-identical siblings. Ten patients had standard-risk disease and 13 had high-risk disease. Twenty-one patients received alloPBSCT as a primary transplant, and the remaining 2, with high-risk disease, as a second transplant after posttransplantation relapse. All donors received daily subcutaneous injections of granulocyte colony-stimulating factor at a dose of 10 microg/kg, and peripheral blood stem cells were collected by 1 to 3 aphereses. Median numbers of CD34+ and CD3+ cells infused were 5.8 x 10(6)/kg (range, 1.3-19.7 x 10(6)/kg) and 4.9 x 10(8)/kg (range, 1.9-8.6 x 10(8)/kg), respectively. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and methotrexate (18 patients) or CyA and methylprednisolone (5 patients). Rapid hematologic engraftment was observed in 20 of the 23 patients. Median days to absolute neutrophil counts >0.5 x 10(9)/L and platelet counts >20 x 10(9)/L were 12 (range, 9-18 days) and 14 (range, 10-128 days), respectively. Acute GVHD of grade 2-4 was observed in 6 of 20 evaluable patients (30%) and extensive chronic GVHD in 8 of 15 evaluable patients (53%). Ten of the 23 patients (44%) were surviving in continuous complete remission 191 to 1492 days (median, 643 days) posttransplantation. Treatment-related death within 100 days posttransplantation was observed in 6 of the 23 patients (26%). Six of the 23 patients (26%) developed relapse at a median 81 days (range, 38-160 days) posttransplantation. Further study is needed to assess the precise benefits of alloPB-SCT compared with allogeneic bone marrow transplantation.

G-CSF-induced mobilization of peripheral blood stem cells for allografting: comparative study of daily single versus divided dose of G-CSF.

We conducted a comparative study on a daily single versus a divided dose of G-CSF for G-CSF-induced mobilization of peripheral blood stem cells (PBSC) in eleven HLA-identical sibling donors of allogeneic PBSC transplantation (PBSCT). Six donors received double subcutaneous injections of G-CSF at a dose of 5 micrograms/kg x 2/day for 5 days (Group A), while the remaining five received single subcutaneous injection at a dose of 10 micrograms/kg/day for 5 days (Group B). The numbers of circulating CD34+ cells, myeloid progenitors (CFU-GM) and erythroid progenitors (BFU-E) reached peak values at day 5 of G-CSF administration in both groups. The mean number of CD34+ cells harvested per apheresis was 4.4 x 10(6)/kg (cells/body weight of each donor, range: 0.8-7.9 x 10(6)/kg) in Group A and 5.1 x 10(6)/kg (range: 3.0-9.0 x 10(6)/kg) in Group B. There were no significant differences between these two groups in total numbers of CFU-GM, BFU-E, or T-lymphocytes harvested. Adverse effects including mild to moderate bone pain and thrombocytopenia were transient and well tolerated. No difference was observed in the incidence of adverse effects between the two groups. These observations suggest that there is no difference in G-CSF-induced mobilization of PBSC between daily single and divided dose of G-CSF to collect a sufficient number of PBSC for engraftment after allo-PBSCT.

Molecular characterization of total kininogen deficiency in Japanese patients.

Kininogens are multifunctional plasma glycoproteins. There are two forms of human kininogen: low molecular weight kininogen (LK) and high molecular weight kininogen (HK). Both are derived from the same gene by alternative splicing. Some patients with kininogen deficiency have been reported to be deficient only in HK while others are deficient in both HK and LK (total kininogen deficiency). We analyzed three Japanese patients with total kininogen deficiency by the Csp45I digestion study of exon 5 as previously reported in Williams trait and found that two had the same point mutation of C to T at base 22 of exon 5, resulting in a transition of CGA (Arg) codon to TGA (Stop) codon. This is the first report of molecular characterization of total kininogen deficiency in the Japanese population.