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The Polycomb group protein Bmi1 is a constituent of the Polycomb repressive complex 1, and it is an important molecule for the regulation of the self-renewal of hematopoietic stem cells. In the field of clinical hematology, there are reports that the level of Bmi1 expression in blast cells is related to the prognosis of acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. We investigated whether the level of Bmi1 expression in leukemic cells is related to the prognosis and the characteristics of childhood acute lymphoblastic leukemia. In all the leukemic blast cells, Bmi1 gene expression was lower value than that in normal B cells. There were no correlations between the level of Bmi1 gene expression in leukemic blast cells and other parameters, including prognosis. Here, we report that the level of Bmi1 expression in blast cells is not related to the prognosis of pediatric acute lymphoblastic leukemia.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3′ acceptor splice site (3′-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5′ donor splice site (5′-dss) of intron 8 (Int8/5′-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3′-consensus acceptor splice site, resulted in a substitution of Gly154 to Val (G154V; G G 716C→G T C). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5′-dss/t(+2)c mutation inactivated the authentic 5′ splice site of intron 8 and led to the utilization of a cryptic 5′-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3′ and 5′ splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo. —Ikeda, Y., A. Takagi, Y. Nakata, Y. Sera, S. Hyoudou, K. Hamamoto, Y. Nishi, and A. Yamamoto. Novel compound heterozygous mutations for lipoprotein lipase deficiency: a G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5′ splice site mutation of intron 8. J. Lipid Res. 2001. 42: 1072–1081.

For difficult to treat neuropathic pain from cancer, adjuvant analgesics are often used with opioids. We present the case of a 5-year-old girl who was diagnosed with meningitis caused by malignant T-cell lymphoma. She had severe neuropathic pain not relieved by increasing doses of a fentanyl infusion. Intravenous administration of ketamine and lidocaine in combination with fentanyl provided excellent analgesia without significant side effects. Ketamine and lidocaine can be safely infused together with concomitant opioids for the treatment of refractory neuropathic pain caused by cancer.

Although the occurrence of posttransfusion urticaria in the immunosuppressive period is believed to be rare, from our experiences, this disease develops regardless of the immune status of the patients. Therefore, we performed a retrospective study to determine whether posttransfusion urticaria develops during the period of bone marrow suppression.This study included 20 patients who developed urticaria as a complication of blood transfusion from January 2010 to January 2011 at the Department of Pediatrics, Hiroshima University Hospital. We retrospectively analyzed the patients' the blood examination data obtained before blood transfusion to elucidate the mechanism underlying posttransfusion urticaria.White blood cell counts were low before the patients developed urticaria; particularly, neutrophil counts were significantly low. Furthermore, the monocyte, eosinophil, and basophil counts were significantly lower before the development of urticaria.Urticaria tends to develop in a condition of neutropenia. Thus, care must be taken to prevent the development of this disease during the neutropenia period.

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD–protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1–transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1–transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

A 26-year-old man with chronic granulomatous disease complicated by multiple liver abscess was admitted to our hospital for hepatic resection and allogeneic bone marrow transplantation (BMT) from an HLA-matched sibling. We diagnosed the patient with Aspergillus liver abscesses based on computed tomographic findings, elevated serum levels of beta-D-glucan, positive test for galactomannan antigen, and the findings of laboratory cultures. Since the liver abscess could not be treated by drainage and administration of antifungals, we resected the posterior segments of the liver, which contained the abscess (S1, S6). However, abscess recurred in the remaining part of the liver 1 month later. The patient received allogeneic BMT from an HLA-matched sibling. During BMT, we continuously administered liposomal amphotericin B (L-AMB) via the hepatic artery (25 mg/day) to treat the liver abscess. There were no adverse effects during hepatic arterial infusion of L-AMB, and the liver abscess disappeared after BMT. These results suggest that hepatic arterial infusion of L-AMB is effective in treating fungal abscess in the liver.

summary Chronic granulomatous disease (CGD) is an inherited immunodeficiency disorder caused by defects in NADPH oxidase and characterized by recurrent life-threatening bacterial and fungal infections. Although CGD has been considered to be a target for gene therapy, bone marrow transplantation (BMT) is now selected as the radical treatment in most cases. We performed BMT in a patient with CGD with severe infections and experienced respiratory complications of diffuse alveolar haemorrhage and/or infection-associated alveolar haemorrhage. We suggest that attention be paid to signs of onset of alveolar haemorrhage during BMT in CGD patients.

Human umbilical cord blood (CB) has been used successfully in stem cell transplantation. A subpopulation of CD34(+) cells expresses chemokine receptor CXCR4 which is critical for bone marrow engraftment in human hematopoietic stem cells. Here, we demonstrate the effect of short-term culture on CXCR4 expression on umbilical CB-derived CD34(+) cells and subsequent engraftment capability in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Surface CXCR4 expression on CD34(+) cells increased after incubating the cells in medium alone for 2 h; this effect was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34(+) cells in culture for 2 h, although these cells showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. Furthermore, cultured human CD34(+) cells showed improved engraftment into the bone marrow of NOD/SCID mice compared to noncultured or AMD3100-treated CD34(+) cells. These observations suggest that increased cell surface expression of CXCR4 on CD34(+) cells improved the engraftment of human umbilical CB cells into bone marrow through enhanced homing activity.

Objective The Polycomb -group ( PcG ) genes regulate global gene expression in many biological processes, including hematopoiesis, by manipulating specific target genes. It is known that various PcG genes regulate self-renewal of hematopoietic stem cells (HSCs). Here we have shown that the reciprocal expression of PcG proteins regulates self-renewal and differentiation of HSCs. Methods We used murine and human bone marrow cells and evaluated the reciprocal expression of PcG proteins on the basis of their respective intranuclear distributions. PcG- gene expression in HSCs was knocked down by small interfering RNAs. The function of each gene in HSCs was analyzed in vitro and in vivo. Results Cells were either Bmi1-positive or Mel-18−positive. The Bmi1-positive cells contained very little amounts of Mel-18 and vice versa. The bmi1 -knockdown marrow cells did not show HSC function, while the mel-18 −knockdown marrow cells showed increased stem cell function. Results of the analysis on human cells were similar to those observed in case of murine cells. In a clinical investigation, transplantation using sources with a low Bmi1 to Mel-18 ratio was associated with early hematopoietic recovery. Conclusion Reciprocal expression of Bmi1 and Mel-18 regulated HSC function. Here, we observed that expression of the PcG genes— bmi1 and mel-18 —is correlated with self-renewal and differentiation of HSCs. Thus, it was suggested that the balance between Bmi1 and Mel-18 regulates self-renewal of HSCs.

Cyclic neutropenia (CyN) in childhood and severe congenital neutropenia (SCN) are congenital disorders that cause chronic neutropenia. Mutations in the neutrophil elastase gene, ELA2, have been reported in patients with CyN and in those with SCN. We examined granulopoietic defects in CyN patients with those in SCN patients.Three patients with CyN and four with SCN were enrolled in this study. Bone marrow cells were enriched based on the expression of CD34, Kit, and granulocyte colony-stimulating factor receptor (G-CSFR). The purified cells were assayed for colony formation, proliferation, and mRNA expression of granular enzymes.All patients showed heterozygous mutations of ELA2. Flow cytometric analysis demonstrated no differences in the frequency of CD34, Kit, and G-CSFR expression between CyN patients and normal subjects. Significant differences in granulocyte/macrophage (GM)-colony formation of CD34(+)/Kit(+) cells were observed among CyN patients, SCN patients, and normal subjects in response to hematopoietic factors. Impaired granulopoiesis was found in both CD34(+)/Kit(+)/G-CSFR(+) and CD34(+)/Kit(+)/G-CSFR- cells in patients with CyN, whereas this impairment was observed only in CD34(+)/Kit(+)/G-CSFR(+) cells in SCN patients, as previously reported. The mRNA expression of granular enzymes in myeloid precursors and the transcription levels during myeloid cell differentiation in CyN patients were comparable to those in normal subjects, in contrast to the abnormal transcription of granular enzymes in SCN patients.These results suggest that the underlying granulopoietic abnormalities differ between CyN and SCN, and emphasize the presence of additional genetic pathophysiology specific to each disease.

A case is presented of predisposing a patient's father with obligate heterozygous lipoprotein lipase (LPL) deficiency to mild hypertriglyceridemia in Japanese I-family members (n=8) with patient DI, who was a compound heterozygote for a novel missense mutation of G154V (GG(716)C--GTC/Gly(154) Val) in exon 5 and a novel splice mutation (Int8/5'-dss/t(+2)c; a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (dss)) in intron 8 of the LPL gene.The patient's father and paternal grandmother were heterozygotes for the Int8/5'-dss/t(+2)c allele, while the patient's mother and maternal grandmother were heterozygotes for the G154V allele. These four heterozygous carriers with one defective LPL allele showed 45-57% of the mean LPL activity and mass in the post-heparin plasma (PHP) observed in normal individuals. Among the four heterozygous carriers, the patient's father, who was40 years old, nonobese and hyperinsulinemia, manifested mild hypertriglyceridemia (type IV hyperlipoproteinemia). The remaining three healthy heterozygous carriers (two were40 years old and the other was40 years old) were all normolipidemic state.In this family, hyperinsulinemia as a marker of insulin resistance may be a strong determinant of hypertriglyceridemia in the carrier with heterozygous LPL deficiency.

Abstract 4520 The impact of ABO major incompatibility in hematopoietic stem cell transplantation (SCT) using conventional myeloablative conditioning has been reported to be negligible in clinical settings. To date, there are scant studies about its clinical significance using reduced-intensity nonmyeloablative SCT (RISCT). In this study we analyzed 32 cases performed HSCT using fludarabine-based RISCT (median age was 15 years old ranged from 7 months to 35 years old, 10 cases with and 22 cases without ABO major incompatibility) and 19 cases using conventional myeloablative SCT (median age was 8 years old ranged from 1 to 16 years old, 6 cases with and 13 cases without ABO major incompatibility) in our institution from January, 2005 to April, 2010. All patients using RISCT were non-malignant disorders including 13 patients with chronic granulomatous disease, 11 with aplastic anemia, and 5 with severe congenital neutropenia. On the other hand, all patients with conventional myeloablative SCT were pediatric malignant disorders including 8 patients with acute lymphoblastic leukemia, and 7 with acute myelogenic leukemia. As shown in Table, in RISCT, the number of red blood cell (RBC) transfusion after SCT was significantly higher and the last day of RBC transfusion was also significantly longer in patients with ABO major incompatibility than those in patients with ABO compatibility. Notably, two patients with ABO major incompatibility had prolonged delay of RBC engraftment and pure red cell aplasia due to the presence of high titer of anti-A antibody. Furthermore, the prolonged recovery of platelet and neutrophil were also observed in patients with ABO major incompatibility (Table). On the other hand, in HSCT using conventional myeloablative conditioning, no differences in the recovery of RBC and platelet were noted irrespective of ABO incompatibility. These data suggest that RISCT with ABO major incompatibility affects the engraftment and recovery of multi-lineage blood cells. Thus, the alternatives of donors may be an important role in transplant-related morbidity of RISCT with ABO incompatibility.TablePatients using RISCTABO major incompatibilityCompatibleIncompatibleP valuesPatients NumbersN=22N=10The number of RBC transfusion* (time)2.1 ± 3.113.6 ± 26.7 Disclosures: No relevant conflicts of interest to declare.

Objective It has generally been believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs). In the present study, we tested the hypothesis that HSCs also give rise to adipocytes. Materials and Methods Using transplantation of a single enhanced green fluorescent protein−positive (EGFP + ) HSC and primary culture, we examined generation of adipocytes from HSCs. Results Adipose tissues from clonally engrafted mice showed EGFP + adipocytes that stained positive for leptin, perilipin, and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator−activated receptor-γ agonist, significantly enhanced the number of EGFP + adipocytes. When EGFP + bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with Oil Red O and Sudan Black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture- and sorting-based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. Conclusion Together, these studies clarify the current controversy regarding the ability of HSCs to give rise to adipocytes. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.