Enhanced expression of myogenic differentiation factors and skeletal muscle proteins in human amnion-derived cells via the forced expression of MYOD1.

OBJECTIVES: Mesenchymal stem cells are expected to be an ideal cell source for cellular and gene therapy. We previously showed that cells derived from the human placenta can be induced to differentiate into myotubes in vitro and to express dystrophin in mdx/scid mice in vivo. In this study, we examined whether amnion-derived cells can be efficiently transduced and differentiated using lentiviral vectors carrying human MYOD1. METHODS: The amnion-derived cells were isolated from human preterm placentas. They were transduced with the MYOD1 vector, and mRNA levels for MYOD1, MYF5, MYOG, MYH2 and DMD were determined by quantitative-reverse transcriptase-polymerase chain reaction, and also examined immunocytochemically. RESULTS: Approximately 70% of amnion-derived cells were efficiently transduced by the lentiviral vectors. MYOD1 activates MYF5 and MYOG, MYH2 and DMD after a 7-day culture. The concerted upregulations of these myogenic regulatory factors enhanced MYH2 and DMD expressions. PAX7 was below the detectable level. Both myosin heavy chain and dystrophin were demonstrated by immunocytochemistry. CONCLUSIONS: MYOD1 activates MYF5 and MYOG, the transcription factor genes essential for myogenic differentiation, and the concerted upregulation of these myogenic regulatory factors enhanced MYH2 and DMD expressions. The amniotic membrane is an immune-privileged tissue, making MYOD1-transduced amnion-derived cells an ideal cell source for cellular and gene therapy for muscle disorders. This is the first report showing that amnion-derived cells can be modified by exogenous genes using lentiviral vectors. Furthermore, MYOD1-transduced amnion-derived cells are capable of the dystrophin expression necessary for myogenic differentiation.

An SNP in CYP39A1 is associated with severe neutropenia induced by docetaxel.

PURPOSE: Docetaxel is one of the most widely used chemotherapy drugs for gynecological cancers. A dose-limiting factor of docetaxel is severe neutropenia, and previous reports showed that grade 4 neutropenia was observed in approximately 70% of Japanese patients treated with docetaxel. In order to elucidate a valid biomarker for docetaxel-induced neutropenia, we analyzed 42 Japanese patients with gynecological cancers such as ovarian cancer and endometrial cancer of the uterus. METHODS: As a first step, AUC of docetaxel was examined in 10 patients and 1,936 SNPs of 225 genes were genotyped using DMET Plus™ genotyping systems. RESULTS: The first screening revealed that 28 SNPs were associated with the AUC (P < 0.05), and we analyzed the associations between the 28 SNPs and neutrophil counts in the other 32 patients, with the result that CYP39A1 (rs7761731) was found to be the only SNP significantly associated (P = 0.049 OR = 9.0) with the incidence of grade 4 neutropenia among 28 SNPs. CONCLUSIONS: This SNP in CYP39A1 may be a useful biomarker for predicting the risk of docetaxel-induced neutropenia.

A case of congenital dyserythropoietic anemia type 1 in a Japanese adult with a CDAN1 gene mutation and an inappropriately low serum hepcidin-25 level.

We describe the first case of genetically diagnosed congenital dyserythropoietic anemia (CDA) type 1 in a Japanese man. The patient had hemolytic anemia since he was a child, and he developed diabetes, hypogonadism, and liver dysfunction in his thirties, presumably from systemic iron overload. When he was 48 years old a diagnosis was finally made by genetic analysis that revealed a homozygous mutation of CDAN1 gene (Pro1129Leu). His serum hepcidin-25 level was inappropriately low. We conclude that physicians should be aware of the possibility of CDA in a patient with anemia and systemic iron overload at any age.

High-dose chemotherapy followed by autologous and allogeneic peripheral blood stem cell transplantation for recurrent disseminated trilateral retinoblastoma.

INTRODUCTION: Trilateral retinoblastoma (TRb) is an intracranial neurogenic tumor associated with unilateral or bilateral retinoblastoma and has very poor prognosis. Patients typically die from leptomeningeal tumor dissemination. CASE REPORT: A 3-year-old girl who had been diagnosed with TRb had a disseminated relapse after a tumorectomy, cerebrospinal irradiation, and conventional chemotherapy. The disseminated tumor disappeared after the first autologous peripheral blood stem cell transplantation (PBSCT) with high-dose melphalan and thiotepa. During the second complete remission, a second autologous PBSCT with high-dose busulfan and melphalan was performed. Seven months after the first PBSCT, the second relapse occurred, and we subsequently performed an allogeneic PBSCT with myeloablative chemotherapy consisting of melphalan, thiotepa, and cyclophosphamide. The patient showed clinical improvement after the allogeneic PBSCT. CONCLUSION: Although high-dose chemotherapies have a curative effect for some patients with TRb, the prognoses of disseminated tumors are still poor. Further examination of the high-dose chemotherapy is necessary for the time, the conditioning drugs, and the hematopoietic stem cell sources.

Shared molecular targets in pediatric gliomas and ependymomas.

BACKGROUND: Recent advances in multidisciplinary treatment approaches have improved the overall prognosis of pediatric brain tumors, but some patients remain refractory to treatment and do poorly. Several molecularly targeted therapies are under development for the treatment of brain tumors, and high-grade gliomas in adults are a particular area of study. PROCEDURE: To better understand if these new therapies can be used in pediatric populations, we examined the expression of the following seven marker genes involved in signaling pathways targeted by new therapies: ß-catenin, suppressor of fused (SUFU), erythroblastic leukemia viral oncogene homolog (ERBB) 2, platelet-derived growth factor receptorα (PDGFRα), proliferating cell nuclear antigen (PCNA), secreted protein acid and rich in cysteine (SPARC), and granulocyte colony-stimulating factor receptor (G-CSFR). Samples from 27 patients with the primitive neuroectodermal tumor (PNET)/medulloblastomas (MBs) (n = 8), ependymomas (n = 5), or gliomas (n = 14) were assessed by quantitative real-time PCR. [Correction made here after initial online publication]. We assigned an EXP score to compare across samples and determined the levels of gene expression among tumor cell types. RESULTS: Gene expression varied among the different tumors, but, within a tumor type, clear expression patterns were seen. The expression of SUFU, ERBB2, and PCNA in metastatic MBs were greater than that seen in non-metastatic MBs. Most glioma cases highly expressed PDGFRα and G-CSFR. Additionally, the expression patterns of gliomas and ependymomas were similar (r = 0.77, P = 0.04), but PNET/MBs substantially differed from gliomas (r = -0.37, P = 0.41) or ependymomas (r = 0.23, P = 0.62). CONCLUSIONS: The development of new drugs targeting up-regulated pathways may be useful for the treatment of pediatric brain tumors. As new drugs are developed, gliomas and ependymomas may be treated with similar compounds.

Double high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation for primary disseminated medulloblastoma: a report of 3 cases.

We performed double high-dose chemotherapy followed by peripheral blood stem cell transplantation (PBSCT) in 3 children with medulloblastoma and primary leptomeningial dissemination, including spinal metastasis. After resection of the main tumor mass, 30.6 Gy whole craniospinal radiation therapy and 4 or 5 courses of conventional chemotherapy with vincristine (1.5 mg/m), carboplatin (560 mg/m), ifosfamide (9000 mg/m), and etoposide (500 mg/m), and 2 courses of high-dose thiotepa (680 mg/m) and melphalan (240 mg/m) therapy with PBSCT were administered. Two patients with low erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene expression achieved long-term survival (41 mo and 40 mo) but the patient with high ERBB2 expression relapsed 9 months after the second PBSCT.

Carbamazepine-induced hemolytic and aplastic crises associated with reduced glutathione peroxidase activity of erythrocytes.

Although pure red cell aplasia is a well-known side effect of carbamazepine treatment, intravascular hemolytic anemia is rare. We describe a 5-year-old boy who developed concurrent intravascular hemolytic anemia and erythroblastopenia, probably due to carbamazepine. Carbamazepine treatment was subsequently discontinued, and the patient was treated with red blood cell transfusions, haptoglobin, and methylprednisolone. His hematologic abnormalities were almost fully recovered within 2 weeks. Examination of the patient's and mother's erythrocyte enzyme activities revealed mildly decreased erythrocyte glutathione peroxidase (GSH-Px) activity. We speculate that patients with reduced GSH-Px activity are at a high risk of developing carbamazepine-induced hemolytic crisis and/or aplastic crisis.

Glycolytic inhibition by mutation of pyruvate kinase gene increases oxidative stress and causes apoptosis of a pyruvate kinase deficient cell line.

OBJECTIVE: SLC3 is a Friend erythroleukemic cell line established from the Pk-1(slc) mouse, a mouse model of red blood cell type-pyruvate kinase (R-PK) deficiency. This study was aimed to elucidate the mechanisms attributing to apoptosis induced by R-PK deficiency. MATERIALS AND METHODS: SLC3 and a control Friend cell line, CBA2, were cultured in a condition of glucose deprivation or supplementation with 2-deoxyglucose, and apoptosis was detected by annexin V. We established two stable transfectants of SLC3 cells with human R-PK cDNA, and examined the effect of R-PK on an apoptotic feature by cell cycle analysis. Intracellular oxidation was measured with 2',7'-dichlorofluorescin diacetate. DNA microarray analysis was performed to examine gene-expression profiles between the two transfectants and parental SLC3. RESULTS: SLC3 was more susceptible than CBA2 to apoptosis induced by glycolytic inhibition. The forced expression of R-PK significantly decreased cells at the sub G0/G1 stage in an expression-level dependent manner. Microarray analysis showed that proapoptotic genes, such as Bad, Bnip3, and Bnip3l, were downregulated in the transfectants. In addition, peroxiredoxin 1 (Prdx1) and other antioxidant genes, such as Cat, Txnrd1, and Glrx1 were also downregulated. A significant decrease of dichlorofluorescein fluorescence was observed by R-PK expression. Preincubation with a glutathione precursor showed a significant decrease of apoptosis. CONCLUSION: These results indicated that glycolytic inhibition by R-PK gene mutation augmented oxidative stress in the Friend erythroleukemia cell, leading to activation of hypoxia-inducible factor-1 as well as downstream proapoptotic gene expression. Thus, R-PK plays an important role as an antioxidant during erythroid differentiation.

Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency.

BACKGROUND AND OBJECTIVES: Red blood cell pyruvate kinase (R-PK) deficiency is the most common glycolytic enzyme defect associated with hereditary non-spherocytic hemolytic anemia. Cases with the most severe deficiency die in the peri- or neonatal period and no specific therapy exists at present. To test whether the targeted overexpression of the normal R-PK gene in erythroid cells could reduce hemolysis in R-PK mutant mice, we performed a genetic rescue study using human R-PK transgenic mice. DESIGN AND METHODS: Human R-PK promoter driven with human mLCR of the human b-globin locus was used for the erythroid-specific expression of human R-PK in murine erythrocytes. The transgenic lines were mated with homozygous R-PK mutant mice and subsequently backcrossed. Mutant homozygotes with the mLCR-R-PK transgene were examined for any therapeutic effects of transgene expression. RESULTS: Two PK transgenic lines, hRPK_lo and hRPK_hi, were obtained. R-PK activity of the transgenic mice reached as high as three times that of the animals with the endogenous PK gene. Overexpression of human R-PK in the homozygous mutant mice successfully reduced hemolytic anemia. Improvements of hemolysis were evaluated by hemoglobin concentration, reticulocyte count, and spleen weight, which showed significant correlations with the levels of expression of the transgene. Recovery from metabolic disturbance in mutant red blood cells was shown as normalized concentrations of the glycolytic intermediates upstream of PK. In addition, there was a remarkable negative correlation between R-PK activity and the number of TUNEL-positive erythroid progenitors in the spleen. INTERPRETATION AND CONCLUSIONS: These results indicate that overexpression of the wild-type PK gene in mutant erythroid cells ameliorates both erythroid apoptosis and the shortened red blood cell lifespan observed in PK mutant mice. It is likely that the level of transgene expression required to achieve evident therapeutic effects should be equivalent to or more than that of the endogenous PK gene. This gene-addition strategy may be suitable for clinical application if there is a high level of transgene expression of R-PK in erythroid progenitors/red blood cells.

Splenic infarction after Epstein-Barr virus infection in a patient with hereditary spherocytosis.

We describe the first patient with hereditary spherocytosis (HS) known to have developed splenic infarction following infectious mononucleosis (IM). An 18-year-old Japanese man was referred to our hospital in November 2004 because of continuous fever and icterus. He had undergone cholecystectomy at the age of 14 years. On patient admission in November 2004, a physical examination showed marked hepatosplenomegaly, icterus, and jaundice. He had a white blood cell count of 14.9 x 10(9)/L with 9.5% atypical lymphocytes, a red blood cell count of 2.93 x 10(12)/L, and a hemoglobin concentration of 7.8 g/dL. Microspherocytes were observed in the patient's peripheral blood smear, and immunoglobulin M antibody to Epstein-Barr virus (EBV) viral capsid antigen was detected. The patient's diagnosis was HS with IM. On day 4 of admission, the patient complained of severe abdominal pain. Abdominal computed tomography scanning revealed findings of splenic infarction. Two months after the occurrence of splenic infarction, a splenectomy was performed. A pathohistologic examination of the resected spleen revealed no evidence of thrombosis or arterial occlusion. We assume that the cause of splenic infarction was insufficient blood flow to oxygenate the entire spleen during the acute enlargement of the spleen.

Ineffective erythropoiesis in mutant mice with deficient pyruvate kinase activity.

OBJECTIVE: A deficiency of pyruvate kinase (PK) is the most common cause of hereditary nonspherocytic anemia due to glycolytic enzyme defects. Red cells are poorly deformable due to adenosine triphosphate depletion in individuals with a PK deficiency and are destroyed in the microcirculation of the reticuloendothelial system, leading to extravascular hemolysis. The pathophysiology of PK deficiency has been widely studied in PK-deficient mice (PK-1(slc)). We examined the effects of a PK deficiency on erythroid progenitor maturation using these mice. MATERIALS AND METHODS: The appearance of apoptotic cells in spleen of PK-1(slc) mice was examined by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) staining. We also assayed hematopoietic stem cell colony formation in vitro in the spleen of PK-1(slc) mice, to investigate erythropoiesis, and annexin V binding, as a measure of apoptotic cells in constitutive erythroid colonies, to evaluate the maturation of erythroid progenitors. RESULTS: The number of hematopoietic progenitors including colony-forming unit erythroids, burst-forming unit erythroids (BFU-E), colony-forming unit granulocyte-macrophages, and multilineage colony-forming units in the spleens of PK-1(slc) was remarkably increased indicating hematopoiesis, and enhanced erythropoiesis in particular. TUNEL assays identified apoptotic cells in the splenic red pulp of the PK-1(slc) mice. Two-color flow cytometry detected apoptotic cells among anti-TER119-positive cells, suggesting that apoptotic cells were of erythroid lineage. Cells undergoing apoptosis were detected in cultures of BFU-E generated from bone marrow cells of PK-1(slc) mice. CONCLUSIONS: The results in this study indicate that the metabolic disturbance in PK deficiency alters not only the survival of red cells but also the maturation of erythroid progenitors, resulting in ineffective erythropoiesis.

[Survey on the threshold for platelet transfusions].

We carried out a survey on platelet transfusions performed in nine general hospitals. We evaluated 303 adults who received a total of 24455 units over 1864 platelet transfusions. The underlying diseases were hematologic disorders with chemotherapy (59.7%), hematologic disorders without chemotherapy (15.5%), hematopoietic stem cell transplantation (18.5%), and others (2.0%). The patient platelet count before transfusion (platelet trigger value) was measured in only 77.1%. The platelet trigger value differed greatly between the hospitals, with an average of 2.2 x 10(4)/microl, a minimum of 1.3 x 10(4)/microl, and maximum of 3.2 x 10(4)/microl. Only 55.3% of the platelet transfusions carried out complied with the Platelet Transfusion Guideline published by the Ministry of Health, Labour and Welfare. The hospitals surveyed could be divided into those who gave mainly around 10 units and those who gave over 10 units. The total dose of platelets transfused was more in the hospitals that used mainly 15 or more unit-PCs than in the hospitals that used mainly 10 unit-PCs. These results indicate that platelet transfusion may be greatly reduced by complying with the 2 x 10(4)/microl of platelet transfusion threshold and by selecting less than 10 units of PC per transfusion.

Molecular basis of Japanese variants of pyrimidine 5'-nucleotidase deficiency.

The type-I isoform of pyrimidine 5'-nucleotidase (P5N-I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N-I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87-bp sequence. The sequence is located 6.2-kb downstream of the exon 2 and 2.7-kb upstream of the exon 3 sequence; consequently, the P5N-I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N-I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients were homozygous for each mutation. The mutant P5N-I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos-7. The degradation of P5N with 425C (L142P) was significantly faster than a wild-type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin-proteasome pathway.

Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency.

In this study, possible adverse effects of pyruvate kinase (PK) deficiency on the maturation of erythroid progenitors were investigated. A 4-year-old Japanese girl with severe PK deficiency underwent splenectomy to reduce her need for blood transfusions. The spleen was examined a histologically, and the hematopoietic progenitors in the spleen were assayed to evaluate the extramedullary hematopoiesis of this PK-deficient subject. The number of hematopoietic progenitors including CFU-GM, BFU-E and CFU-GEMM in the spleen of the PK-deficient patient was much higher than those found in control spleens, indicating enhanced extramedullary hematopoiesis. TUNEL assay demonstrated apoptotic cells in the splenic red pulp of the PK-deficient patient. The expression of 7A6 antigen was detected in cells isolated from spleen and in cells cultured in vitro, but only in those cells that were positive for glycophorin A. These results provide evidence that the metabolic disturbances in PK deficiency affect not only the survival of red cells but also the maturation of erythroid progenitors, which results in premature cell death, i.e., apoptosis.

A novel missense mutation (1060G --> C) in the phosphoglycerate kinase gene in a Japanese boy with chronic haemolytic anaemia, developmental delay and rhabdomyolysis.

We report the case of a 3-year-old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8-9 g/dl, his reticulocyte counts were 300-500 x 109/l, and his total bilirubin level was 25.65-42.75 micro mol/l. The patient suffered two episodes of respiratory infection-associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3.0 years, his developmental milestones (developmental quotients measured using the Tsumori-Inage methods) score was 49% (normal 74-131%), and his height was below average by -2.0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (< 10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G-->C) PGK1 gene mutation. This mutation results in the Ala-353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.

Maturation of adult peripheral blood CD38(+)CD4(+) T cells demonstrated by cytokine production in response to a superantigen, TSST-1.

This study looks at immunoincompetent CD4(+) T cells in adult peripheral blood (APB) using cytokine production in response to a superantigen as a measure of function. We compared the function of APB CD38(+)CD4(+) and CD38(-/low)CD4(+) T cells to that of cord blood (CB) CD4(+) T cells. APB CD4(+) T cell blasts produce substantial amounts of IL-2 in response to TSST-1 restimulation, while CB CD4(+) T cell blasts produce less. APB CD38(+)CD4(+) T cells produce low levels of IL-4 and IFN-gamma in response to TSST-1, even after activation, while APB CD38(-/low)CD4(+) T cells retain their ability to produce high levels of these cytokines despite high CD38 expression. These results suggest that the developmental stage of APB CD38(+)CD4(+) T cells lies between that of CB CD4(+) T cells and APB CD38(-/low)CD4(+) T cells and that APB CD38(+)CD45RO(-)CD4(+) T cells gradually cease to express CD38 as they acquire full function. We reconsider CD4(+) cell maturation and response to TSST-1 and discuss the implications of T cell maturity on infectious diseases.

Gene expression and biological significance of hexokinase in erythroid cells.

Red blood cells (RBCs) express two hexokinase (HK) isoforms, HK-I and HK-R. Both isozymes are generated from the HK-I gene by use of an alternate promoter. Gene structure and exon-intron organization of the HK-I gene have been elucidated from a sequence of three contiguous genomic clones localized at human chromosome 10. The sequence spans about 131 kb, and consists of 25 exons, which include 6 testis- and 1 erythroid-specific exons. HK-R has been shown as an erythroid-specific isozyme whose expression is turned on in the early erythroid-progenitors and is significantly induced during their differentiation. HK-R unfolds major HK activity in immature RBCs and is rapidly degraded during the maturation process. HK-I has a porin-binding domain in its N-terminus. Recent studies have shown that HK isozymes with a porin-binding domain play a role in mitochondrial integrity, suggesting that HK-I-deficient erythroid cells might be eliminated by apoptosis. It is most likely that RBCs are most labile as a result of HK-I/R deficiency since the HK-I gene but not the other isozyme genes are expressed in fetal and adult RBCs.