High-dose cytosine arabinoside and etoposide with total body irradiation as a preparatory regimen for allogeneic hematopoietic stem-cell transplantation in patients with acute lymphoblastic leukemia.

One approach to improving the outcome of allogeneic hematopoietic stem-cell transplantation for acute lymphoblastic leukemia (ALL) is to intensify the pretransplant conditioning regimen without increasing toxicity. We used an intensified conditioning regimen consisting of high-dose cytosine arabinoside (3 g/m(2) twice daily i.v. for 3 consecutive days, total six doses), high-dose etoposide (1 g/m(2) once daily i.v. during the first 2 days) and total body irradiation (TBI) (HDACE-TBI) in ALL patients. We retrospectively analyzed 21 patients treated with HDACE-TBI, of whom 18 were in complete remission (CR) and three were in non-CR at transplantation. Although gastrointestinal toxicities were common, critical regimen-related toxicities were not seen in any patients. One patient demonstrated veno-occlusive disease, which could be controlled conservatively. The disease-free survival rate of 18 patients in CR at transplantation was 61%. These results demonstrate that the HDACE-TBI combination regimen is a feasible alternative to other preparatory regimens and does not increase the regimen-related toxicity.

GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies.

GATA-1 germline mutation in mice results in embryonic lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether the obligate presence of GATA-1 in erythroid cells is due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor complementary DNAs (cDNAs) placed under the transcriptional control of the GATA-1 gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1-deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryonic lethal phenotype of the GATA-1 mutation. However, adult mice rescued with the latter transgenes developed anemia, while GATA-1 transgenic mice did not. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis. The results also show that there are biochemical distinctions among the hematopoietic GATA proteins and that during adult hematopoiesis the hematopoietic GATA factors are not functionally equivalent.

Cyclosporine A-associated fatal central nervous system angiopathy in a bone marrow transplant recipient: an autopsy case.

We report here the case of a 32-year-old woman who suffered from a unique angiopathy in the central nervous system (CNS). She died of multiple infarcts in the brain stem and cerebellum during treatment with cyclosporine A after bone marrow transplantation for refractory anemia with excess of blasts. The autopsy findings showed segmental narrowing of the basilar artery, in which circumferential dissection of the internal elastic lamina had occurred. The distal portion of the basilar artery was obstructed by upward dislocation of the dissected intima. Similar angiopathy was also observed at multiple sites along the basilar artery branches. These findings suggest endothelial damage, including vasoconstriction and dissection of the CNS arteries.

Contribution of TNF-alpha and IL-10 gene polymorphisms to graft-versus-host disease following allo-hematopoietic stem cell transplantation.

Some cytokines are believed to play a role in the development of acute and chronic GVHD after allo-hematopoietic stem cell transplantation. It has been reported that TNF-alpha and IL-10 gene polymorphisms are associated with the production of those cytokines and the development of graft failure after organ transplantation and systemic lupus erythematosus. We examined whether TNF-alpha and IL-10 gene polymorphisms affect the severity of acute GVHD (aGVHD) and chronic GVHD (cGVHD). Sixty-two and 54 patients were available for the analysis of aGVHD and cGVHD, respectively. We analyzed the gene polymorphisms derived from pre- and post-transplant blood cells. Donor-derived TNF2 allele (A) was more frequently detected in patients with aGVHD III/IV than those aGVHD 0-II (2/6 vs 2/56) (P = 0.04). The donors of the patients with cGVHD more frequently possessed a greater number of alleles (allele 13 or more which contain 26 or more CA repeats) in IL-10.G than those without (13/26 vs 5/28) (P = 0.02), and the patients with cGVHD had more CA repeats in donor-derived IL-10.G than those without (mean = 25.2 vs 23.4) (P = 0.01). Donor-derived TNF-308 and IL-10.G alleles may contribute to severe aGVHD and cGVHD, respectively, and will help us distinguish those patients at high risk for GVHD.

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene.

A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.

Expression of eosinophil peroxidase in the immature basophil cell line KU812-F.

Although peroxidase activity in basophils can be detected by optical and ultrastructural cytochemistry, its characteristics remain to be determined. We have demonstrated the characteristics of peroxidase activity induced in the immature basophil cell line, KU812-F. Ultrastructurally, peroxidase activity was detected in granules as well as in the perinuclear space and endoplasmic reticulum. Immunocytochemistry revealed that KU812-F cells were stained by anti-eosinophil peroxidase antibodies, and eosinophil peroxidase mRNA, not myeloperoxidase, was detected in the cells using Northern hybridization and reverse transcription-polymerase chain reaction. Eosinophil peroxidase can be one of the molecules shared with eosinophils and basophils. The biological function of eosinophil peroxidase detected in basophils remains uncertain.

Role of GATA-1 in proliferation and differentiation of definitive erythroid and megakaryocytic cells in vivo.

To elucidate the contributions of GATA-1 to definitive hematopoiesis in vivo, we have examined adult mice that were rendered genetically defective in GATA-1 synthesis (Takahashi et al, J Biol Chem 272:12611, 1997). Because the GATA-1 gene is located on the X chromosome, which is randomly inactivated in every cell, heterozygous females can bear either an active wild-type or mutant (referred to as GATA-1.05) GATA-1 allele, consequently leading to variable anemic severity. These heterozygous mutant mice usually developed normally, but they began to die after 5 months. These affected animals displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells) was expressed profusely in the abnormally abundant cells generated in the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally differentiate, this leads to progenitor proliferation in the spleen and consequently death. Thus, GATA-1 plays important in vivo roles for directing definitive hematopoietic progenitors to differentiate along both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human myelodysplastic syndrome and thus may serve as a useful model for this disorder.

Ablation of Nrf2 function does not increase the erythroid or megakaryocytic cell lineage dysfunction caused by p45 NF-E2 gene disruption.

Maf recognition elements (MAREs or NF-E2 binding sites) have been shown to be vital for erythroid- and megakaryocyte-specific gene expression. Transcription factor NF-E2 is composed of p45, a large subunit belonging to the CNC family proteins, and a small Maf subunit, and is thought to activate transcription through its binding to MAREs in both the erythroid and megakaryocytic cell lineages. While p45 gene knockout mice exhibit thrombocytopenia due to abnormal terminal differentiation of megakaryocytes, and the mutant mice die of massive bleeding within a week after birth, anemia is not apparent in these animals. Disruption of the nrf2 gene, encoding another CNC family protein, results in no hematological abnormalities. We have therefore tested the hypothesis that Nrf2 might compensate for the p45 deficiency in erythroid lineage cells of p45-knockout mice, thereby masking the anticipated anemia. However, we failed to detect any greater failure in either erythroid or megakaryocytic cell development in Nrf2 plus p45 compound mutant mice as compared to with either individual homozygous mutation. These data suggest that p45 and Nrf2 may both be dispensable for hematopoietic cell development, and that other factors regulate erythroid- and megakaryocyte-specific gene expression through their required MAREs.

Primary familial polycythaemia associated with a novel point mutation in the erythropoietin receptor.

Primary familial and congenital polycythaemia (PFCP) is a rare disease characterized by congenital erythrocytosis inherited in an autosomal dominant fashion. Recently, mutations in the erythropoietin receptor (EpoR) have been identified in PFCP families. We describe a Japanese family with an autosomal dominant inheritance of PFCP. An in vitro colony assay demonstrated hypersensitivity of erythroid progenitors to erythropoietin (Epo) in affected family members. Sequence analysis of RT-PCR products amplified from the C-terminal region of EpoR transcripts in affected family members revealed that they were all heterozygous for C and T bases at position 5986, which suggested a genetic mutation (C to T) on one allele of EpoR. This mutation gave rise to a translation termination codon TAG at amino acid 435. Thus, the resulting EpoR is a truncated protein product lacking all 74 amino acids downstream of the mutation. To date, all genetic mutations affecting a family with PFCP, including this one, have been located in the cytoplasmic negative regulatory region of the EpoR. All mutations gave rise to truncated Epo receptors between Tyrosine 427 and Tyrosine 455. The phosphotyrosines in this region of EpoR have been demonstrated to be binding sites for SHP-1 phosphatase. Therefore PFCP is presumably brought about as a result of genetic mutations which cause the loss of the SHP-1 binding site in the cytoplasmic region of EpoR.

Profile of cell cycle in hematopoietic malignancy by DNA/RNA quantitation using 7AAD/PY.

Using 7-amino-actinomycin-D/pyronin Y (7AAD/PY), we analyzed the surface phenotypes and cell cycle of 22 hematopoietic cell lines based on their cellular DNA/RNA content. Populations of G1a, G1b, S, and G2M, the DNA index (DI), and the RNA index of S phase (SRI) were calculated by means of DNA/RNA dot plots. Two new parameters were extracted from the cell-cycle profiles: the nucleic acid index of S phase (NI) and the coefficient of variations in the RNA at S phase (SVC). DNA/RNA dot plots of cell lines revealed four characteristic profiles of the cell cycle, defined with the calculated NI and SCV. These were type 0 (small NI, large SCV), type I (small NI, small SCV), type II (large NI, small SCV), and type III (large NI, large SCV). Type O included four stem cell lines: one t(1;19) leukemia, two Ph1+ acute lymphocytic leukemia (ALL), and one biphenotypic crisis of chronic granulocytic leukemia (CGL). Type I included five ALL cell lines: three T-ALL and two common B-ALL. Type II contained 10 myeloid cell lines: five AML and five myeloid crisis of CGL. Type III contained three relatively immature lymphoma cell lines: two Burkitt's lymphoma and one follicular center lymphoma. Calculated NI/SCV (%) were as follows: type 0, 2.27 +/- 0.19/16.7 +/- 3.7; type I, 2.20 +/- 0.30/11.1 +/- 0.7; type II, 3.64 +/- 0.52/11.8 +/- 1.0; and type III, 3.60 +/- 0.53/17.5 +/- 1.9. Cell-cycle analysis of blasts using 7AAD/PY combined with surface phenotyping may yield important information for classifying hematopoietic malignancy within 2 hours of patient admission.

Gadolinium DTPA-enhanced magnetic resonance imaging of cerebral contusions.

The morphological characteristics of cerebral contusions in head trauma patients suggest that an increase in cerebrovascular permeability is responsible for the contusion edema which develops within 1-3 days posttrauma. In the present study, 10 patients with cerebral contusions (mean age, 38 years old; 8 males and 2 females) were examined by gadolinium (Gd)-DTPA enhanced magnetic resonance imaging (MRI) at 1-2 days after trauma. Gd-DTPA (0.3 mmol/kg) was infused intravenously over a period of 30 min. MRIs were taken before, and at 2 and 4 hours after initiation of the Gd-DTPA administration. It was found that contusion edema areas were frequently enhanced by Gd-DTPA at 2 hours. The enhancement diminished at 4 hours. These findings appear to be inconsistent with the results of previously reported similar studies in which enhancement was detected at 6-9 days posttrauma but not during the period earlier than 6 days. This discrepancy may be attributable to the presence of a high blood concentration of Gd-DTPA for a longer period of time and a delay in the time at which MRIs were taken in the present study. The present data indicate that an increased cerebrovascular permeability occurs at as early as 1-2 days posttrauma, and suggest that contusion edema which progresses during the initial 1-3 days may be at least partially vasogenic in nature.