Transcriptosome and serum cytokine profiling of an atypical case of myelodysplastic syndrome with progression to acute myelogenous leukemia

A Native American-Indian female presenting with anemia and thrombocytosis was diagnosed with myelodysplastic syndrome (MDS, refractory anemia). Over the course of 5 years she developed cytopenias and periods of leukocytosis with normal bone marrow (BM) blast counts, features of an unclassifiable MDS/MPS syndrome. The patient ultimately progressed to acute myelogenous leukemia (AML, FAB M2) and had a normal karyotype throughout her course. The episodes of leukocytosis were associated with infectious complications. Transformation to AML was characterized by a BM blast percentage of 49%. Peripheral blood and BM samples were obtained for serum protein analysis and gene expression profiling (GEP) to elucidate her disease process. An ELISA assay of the serum analyzed approximately 80 cytokines, which demonstrated that hepatocyte growth factor/scatter factor and insulin-like growth factor binding protein 1 were markedly elevated compared to normal. GEP demonstrated a unique "tumor molecular profile," which included overexpression of oncogenes (HOXA9, N-MYC, KOC1), proliferative genes (PAWR, DLG5, AKR1C3), invasion/metastatic genes (FN1, N-CAM-1, ITGB5), pro-angiogenesis genes (c-Kit), and down regulation of tumor suppressor genes (SUI1, BARD1) and anti-apoptotic genes (PGLYRP, SERPINB2, MPO). Hence, a biomics approach has provided insight into elucidating disease mechanisms, molecular prognostic factors, and discovery of novel targets for therapeutic intervention.     

Stable Response after Administration of Stem Cell Factor Combined with Granulocyte Colony-Stimulating Factor in Aplastic Anemia

We report successful treatment with 25 microg/kg of recombinant methionyl human stem cell factor (SCF) combined with 400 microg/m2 of recombinant human granulocyte colony-stimulating factor (G-CSF) in 2 patients with aplastic anemia refractory to immunosuppressive therapy. In one patient, hemoglobin levels increased from 6.4 g/dL to 11.3 g/dL after 36 weeks of SCF/G-CSF treatment. Thereafter, the platelet count (24.0 x 10(9)/L) began to improve without the therapy, and as of week 272, the platelet count was 125.0 x 10(9)/L with a leukocyte count of 8.4 x 10(9)/L and a hemoglobin level of 12.9 g/dL. In the other patient, more than 3 years of SCF/G-CSF treatment ameliorated hemoglobin levels and platelet counts from 5.8 g/dL to 15.9 g/dL and 8.0 x 10(9)/L to 50.0 x 10(9)/L, respectively. After cessation of SCF/G-CSF treatment, the positive response was sustained, and the platelet count improved further to 71.0 x 10(9)/L as of week 242. These observations suggest the clinical benefit of SCF/G-CSF administration to patients with refractory aplastic anemia.     

Diversity of human immune system multigene families and its implication in the genetic background of rheumatic diseases

A large number of molecules in the immune system are encoded by multigene families. These genes are rich in pairs of activating and inhibitory receptors that share the same ligands, thereby playing a crucial role in immunoregulation. Furthermore, multigene families tend to be highly polymorphic. Thus, multigene families are strong candidates for containing genes that enhance susceptibility to immune system-related diseases. Here, we review studies from our group, as well as other investigators, on three multigene families that belong to the immunoglobulin (Ig) - like receptor superfamily: Fcgamma receptor (FCGR), killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LILR) families. FCGR genes have been implicated in susceptibility to systemic lupus erythematosus (SLE). In FCGR2B encoding an inhibitory receptor expressed in B cells, monocytes and dendritic cells, a polymorphism within the transmembrane region, Ile232Thr, was identified and found to be associated with susceptibility to SLE in three Asian populations. Functional analyses revealed that SLE-associated FcgammaRIIb-232Thr was less efficient in entering the membrane lipid raft, and exhibited reduced inhibitory potential against B cell receptor signaling. Although the frequency of this polymorphism was low in Caucasians, another polymorphism within the promoter region was reported to be associated with SLE. KIR/HLA combinations have been shown to be associated with various autoimmune and infectious diseases. Recently, LILR families have also been found to be highly polymorphic, and association with several diseases has been identified. These results emphasize the role of multigene families in the diversity of human immune response and susceptibility to diseases.     

Age-Related Changes of Transforming Growth Factor β1 in Japanese Children

BackgroundTransforming growth factor β1 (TGFβ1) is an important factor in immunomodulation. The expression of TGFβ1 has been shown to be influenced by the C-509 T polymorphism in the TGFβ1 gene. We investigated age-related changes of plasma TGFβ1 levels in a birth-cohort study. In addition, the genotypes of the C-509 T polymorphism were investigated in allergic and non-allergic subjects.MethodsSixty-four neonates who met the following criteria were enrolled in this cohort study: 1) full-term vaginally delivery; 2) underwent DNA polymorphism analysis; and 3) questionnaire forms were filled out by parents at 0, 6 and 14 months of age. The umbilical cord blood at 0 months and peripheral blood at 6, and 14 months were collected. Plasma TGFβ1 levels were measured at 0, 6 and 14 months of age. Genomic DNA was extracted from their umbilical cord blood. The genotype of the subjects was examined for the presence of C- 509 T.ResultsThe plasma TGFβ1 level at 6 months was the highest of the 3 measurements (at 0, 6, and 14 months of age). The TGFβ1 levels at 14 months in allergic subjects were significantly higher than those in non-allergic subjects (p = 0.03). All subjects with bronchial asthma (n = 3) had the TT genotype of the C-509 T polymorphism.ConclusionsThe plasma TGFβ1 levels change with age. In addition, TGFβ1 may play a role in the pathogenesis of bronchial asthma.     

Blood flow sensing mechanism via calcium signaling in vascular endothelium

The structure and function of blood vessels adapt to environmental changes, for example, physical development and exercise. This phenomenon is based on the ability of endothelial cells (ECs) to sense and respond to blood flow. ECs are in direct contact with blood flow and exposed to shear stress. A number of recent studies have revealed that ECs recognize changes in shear stress and transmit signals to the interior of the cell, which leads to cellular responses that involve changes in cell morphology, cell function, and gene expression. Cultured human pulmonary artery ECs (HPAECs) showed Ca2(+) influx via an ATP-operated cation channel, P2X4, in response to shear stress. We have recently found that shear-induced activation of P2X4 requires endogenously released ATP and that shear stress induced HPAECs to release ATP, which was mediated by cell-surface ATP synthase located in caveolae. To gain insight into its significance, we generated a P2X4-deficient mouse. P2X4(-/-) mice do not exhibit normal EC responses to flow, such as Ca2(+) influx and subsequent production of NO, a potent vasodilator. Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2X4(-/-) mice. Furthermore, P2X4(-/-) mice have higher blood pressure than wild-type mice. Moreover, no adaptive vascular remodeling is observed in the P2X4(-/-) mice. Thus, P2X4-mediated shear stress mechanotransduction plays an important role in the vascular homeostasis, including the control of blood pressure and vascular remodeling.     

In vivo mutagenesis in the lungs of gpt-delta transgenic mice treated intratracheally with 1,6-dinitropyrene

1,6-Dinitropyrene (1,6-DNP) is a ubiquitous airborne pollutant found in diesel exhaust. In this study, mutagenesis was examined in the lungs of gpt-delta transgenic mice after intratracheal instillation of 0-0.1 mg 1,6-DNP. In addition, the 1,6-DNP-induced gpt mutation spectrum was compared with that of control mice. A single intratracheal injection of 0-0.05 mg 1,6-DNP resulted in significant dose-dependent increases in mutant frequency; the induced mutant frequency declined at the 0.1 mg dose. The average lung mutant frequencies at doses of 0.025, 0.05, and 0.1 mg 1,6-DNP were 2.9-, 4.1-, and 1.9-times higher than for control mice ((0.50+/-0.16)x10(-5)). The major mutations induced by 1,6-DNP included G:C-->A:T transitions, G:C-->T:A transversions, and 1-base deletions. Among the G:C-->A:T transitions isolated from 1,6-DNP-treated mice, five (at nucleotide positions 64, 110, 115, 116, and 418) were observed in four or more animals. These positions therefore are potential hotspots for 1,6-DNP mutation. The predominant frameshift mutations following 1,6-DNP treatment included single base pair deletions at G:C (9/13=69%). The results of this study indicate that 1,6-DNP is mutagenic for the lungs of mice.     

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.