Masahiro Kobayashi

Reliable Application Layer Multicast Over Combined Wired and Wireless Networks

During the last several years, the Internet has evolved from a wired infrastructure to a hybrid of wired and wireless domains by spreading worldwide interoperability for microwave access (WiMAX), Wi-Fi, and cellular networks. Therefore, there is a growing need to facilitate reliable content delivery over such heterogeneous networks. On the other hand, application layer multicast (ALM) has become a promising approach for streaming media content from a server to a large number of interested nodes. ALM nodes construct a multicast tree and deliver the stream through this tree. However, if a node leaves, it cannot deliver the stream to its descendant nodes. In this case, quality-of-service (QoS) is compromised dramatically. Especially, this problem is exacerbated in wireless networks because of packet errors and handovers. In order to cope with this problem, multiple-tree multicasts have been proposed. However, existing methods fail to deliver contents reliably in combined wired and wireless networks. In this paper, we propose a method to ensure the robustness of node departure, while meeting various bandwidth constraints by using layered multiple description coding (LMDC). Finally, we evaluate the proposed method via extensive simulations by using the network simulator (ns-2). By comparing our proposed method with the existing ones, we demonstrate that our method provides better performance in terms of total throughput, relative delay penalty (RDP), and relative delay variation (RDV). The results indicate that our approach is a more reliable content delivery system when compared with contemporary methods in the context of heterogeneous networks containing wired and wireless environments.

Phase I study of glasdegib (PF‐04449913), an oral smoothened inhibitor, in Japanese patients with select hematologic malignancies

The hedgehog signaling pathway regulates multiple morphogenetic processes during embryogenesis. Aberrant activation of the hedgehog pathway signal transduction in adult tissues is associated with the pathogenesis of hematologic malignancies and solid tumors. We report findings from an open‐label, multicenter phase I trial of the selective, small‐molecule hedgehog signaling inhibitor glasdegib (PF‐04449913) in Japanese patients with select advanced hematologic malignancies. Glasdegib was administered as once‐daily oral doses (25, 50 and 100 mg) in 28‐day cycles after a lead‐in dose on Day −5. The primary objectives were to determine first‐cycle dose‐limiting toxicities, safety, vital signs and laboratory test abnormalities. Secondary objectives included evaluation of pharmacokinetics, pharmacodynamics and preliminary evidence of clinical activity of glasdegib. No dose‐limiting toxicities were noted in the 13 patients in the present study. All patients experienced at least one treatment‐emergent, all‐causality adverse event. The most frequent treatment‐related adverse events (observed in ≥3 patients) were dysgeusia (n = 9), muscle spasms (n = 5), alopecia, decreased appetite (n = 4 each), and increased blood creatinine phosphokinase, constipation and diarrhea (n = 3 each). Two deaths occurred during the study and were deemed not to be treatment‐related due to disease progression. Glasdegib demonstrated dose‐proportional pharmacokinetics, marked downregulation of the glioma‐associated transcriptional regulator GLI1 expression in normal skin, and evidence of preliminary clinical activity, although data are limited. Glasdegib was safe and well tolerated across the dose levels tested. It is confirmed that the 100‐mg dose is safe and tolerable in Japanese patients, and this dose level will be examined in the future clinical trial.

A novel heterozygous ALAS2 mutation in a female with macrocytic sideroblastic anemia resembling myelodysplastic syndrome with ring sideroblasts: a case report and literature review

Dear Editor, Sideroblastic anemias are a group of disorders with the common feature of bone marrow ring sideroblasts (RS), reflecting excess mitochondrial iron deposition [1]. In adults, these syndromes are commonly associated with myelodysplastic syndrome (MDS), and a significant portion of cases result from a mutation in the RNA splicing machinery component splicing factor 3b, subunit 1 (SF3B1) [2]. Congenital sideroblastic anemia is rare and represented by X-linked sideroblastic anemia (XLSA), which is attributed to mutations in the X-linked gene erythroid-specific 5-aminolevulinate synthase (ALAS2) [1, 3]. ALAS2 encodes the enzyme catalyzing the first and rate-limiting steps in the heme biosynthesis pathway in erythroid cells. XLSA is predominantly observed in hemizygous males and manifests as microcytic anemia with systemic iron overload, whereas most heterozygous female carriers are asymptomatic and may exhibit only minor red cell abnormalities; however, some cases develop into microcytic sideroblastic anemia due to unbalanced lyonization, skewed X chromosome inactivation, or age-related clonal hematopoiesis [4–6]. Here, we review recently recognized adult female cases of macrocytic sideroblastic anemia caused by heterozygous ALAS2 mutation. A 61-year-old woman had macrocytic anemia since adulthood and was receiving iron chelation therapy. Her son is healthy, but her mother had anemia of unclear etiology. At her first visit to our hospital, the white blood cell count was 2400/μL; red blood cell count, 1.96 × 10/μL; hemoglobin, 7.3 g/dL; hematocrit, 22.1%; mean corpuscular volume, 112.8 fL; and platelet count, 8.6 × 10/μL. Serum biochemical analysis demonstrated increased ferritin (913.2 ng/mL). Bone marrow analysis revealed RS (60% of all erythroblasts) (Fig. 1a), with normal karyotype and mild megaloblastoid changes in the erythroid lineage (Fig. 1b), implying MDS with RS (MDS-RS). After obtaining written informed consent, we conducted Sanger sequencing to identify the genetic alteration associated with RS formation. Surprisingly, genomic DNA sequencing of whole blood revealed a novel 488 G > A heterozygous ALAS2 mutation (Fig. 1c). The mutation was germline because it involved the buccal cells (Fig. 1d), and it resulted in an amino acid substitution of arginine for histidine at residue 163. No mutation was identified in SF3B1. In vitro enzymatic analysis with bacterially expressed recombinant ALAS2 protein [7] confirmed that the mutation severely abrogated its enzymatic activity (Fig. 1e), supporting the diagnosis of congenital sideroblastic anemia rather thanMDSRS. Similar to previous reports [8, 9], we observed complete skewing toward the expression of wild-type ALAS2mRNA in reticulocytes (Fig. 1f, g). Thus, the severe loss-of-function mutation of ALAS2 might perturb terminal erythroid maturation, leading to sideroblastic anemia in heterozygous females and embryonic lethality in hemizygous males, which is supported by the observation that anemia was detected exclusively in females (Fig. 1g). Regarding the mechanisms of macrocytic anemia occurrence, the compensatory stimulation of erythropoiesis within wild-type ALAS2-expressing erythroblasts might play a role. Nevertheless, further accumulation of cases and the establishment of animal models are required. * Hideo Harigae harigae@med.tohoku.ac.jp

Iron-heme-Bach1 axis is involved in erythroblast adaptation to iron deficiency

Iron plays the central role in oxygen transport by erythrocytes as a constituent of heme and hemoglobin. The importance of iron and heme is also to be found in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is deactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1−/− mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses these genes in erythroblasts under iron deficiency to balance the levels of heme and globin. Moreover, an increase in genome-wide DNA methylation was observed in erythroblasts of Bach1−/− mice under iron deficiency. These findings reveal the principle role of iron as a regulator of gene expression in erythroblast maturation and suggest that the iron-heme-Bach1 axis is important for a proper adaptation of erythroblast to iron deficiency to avoid toxic aggregates of non-heme globin.

Dynamics of absorption, metabolism, and excretion of 5-aminolevulinic acid in human intestinal Caco-2 cells

5-Aminolevulinic acid (ALA) is a precursor for the biosynthesis of porphyrins and heme. Although the oral administration of ALA has been widely applied in clinical settings, the dynamics of its absorption, metabolism, and excretion within enterocytes remain unknown. In this study, after enterocytic differentiation, Caco-2 cells were incubated with 200 µM ALA and/or 100 µM sodium ferrous citrate (SFC) for up to 72 h. Both ALA and the combination of ALA and SFC promoted the synthesis of heme, without affecting the expression of genes involved in intestinal iron transport, such as DMT1 and FPN. The enhanced heme synthesis in Caco-2 cells was more pronounced under the effect of the combination of ALA and SFC than under the effect of ALA alone, as reflected by the induced expression of heme oxygenase 1 (HO-1), as well as a reduced protein level of the transcriptional corepressor Bach1. Chromatin immunoprecipitation analysis confirmed Bach1 chromatin occupancy at the enhancer regions of HO-1, which were significantly decreased by the addition of ALA and SFC. Finally, Transwell culture of Caco-2 cells suggested that the administered ALA to the intestinal lumen was partially transported into vasolateral space. These findings enhance our understanding of the absorption and metabolism of ALA in enterocytes, which could aid in the development of a treatment strategy for various conditions such as anemia.

Forced FOG1 expression in erythroleukemia cells: Induction of erythroid genes and repression of myelo-lymphoid transcription factor PU.1

The transcription factor GATA-1-interacting protein Friend of GATA-1 (FOG1) is essential for proper transcriptional activation and repression of GATA-1 target genes; yet, the mechanisms by which FOG1 exerts its activating and repressing functions remain unknown. Forced FOG1 expression in human K562 erythroleukemia cells induced the expression of erythroid genes (SLC4A1, globins) but repressed that of GATA-2 and PU.1. A quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated increased GATA-1 chromatin occupancy at both FOG1-activated as well as FOG1-repressed gene loci. However, while TAL1 chromatin occupancy was significantly increased at FOG1-activated gene loci, it was significantly decreased at FOG1-repressed gene loci. When FOG1 was overexpressed in TAL1-knocked down K562xa0cells, FOG1-mediated activation of HBA, HBG, and SLC4A1 was significantly compromised by TAL1 knockdown, suggesting that FOG1 may require TAL1 to activate GATA-1 target genes. Promoter analysis and quantitative ChIP analysis demonstrated that FOG1-mediated transcriptional repression of PU.1 would be mediated through a GATA-binding element located at its promoter, accompanied by significantly decreased H3 acetylation at lysine 4 and 9 (K4 and K9) as well as H3K4 trimethylation. Our results provide important mechanistic insight into the role of FOG1 in the regulation of GATA-1-regulated genes and suggest that FOG1 has an important role in inducing cells to differentiate toward the erythroid lineage rather than the myelo-lymphoid one by repressing the expression of PU.1.

Anaplastic multiple myeloma: possible limitations of conventional chemotherapy for long-term remission

Anaplastic multiple myeloma (AMM) is a very rare morphological subtype of multiple myeloma, which has been reported only sporadically, mostly in case reports (1-3). The clinical course of AMM is highly aggressive and the disease has an extremely poor prognosis, which is considerably different from that of conventional myeloma. Some patients are diagnosed with AMM at disease onset, whereas others can develop anaplastic transformation during the course of conventional plasma cell myeloma. AMM can present as multiple extramedullary tumors (3). Pathologically, the pleomorphic multinucleated morphology of AMM can mimic multinucleated carcinoma (4). Due to its rarity, however, a therapeutic strategy for AMM remains to be established. A 63-year-old woman with severe lumbago and right upper limb pain for longer than 1 week was referred to us because of thrombocytopenia, high serum lactic dehydrogenase (LDH) level, and a mass on the right brachial plexus found by magnetic resonance imaging (MRI). The patient’s general condition was very poor, with severe pain and easy fatigability. She was afebrile and her vital signs were normal. There was no lymphadenopathy or hepatosplenomegaly. The complete blood count indicated thrombocytopenia (38000/μL) without anemia (hemoglobin, 12.2 g/dL). The white blood cell count (8600/μL) was normal, with a normal differentiation count (69% neutrophils, 18% lymphocytes, 9% monocytes, 3% eosinophils, and 1% metamyelocytes); there were no atypical lymphocytes, plasma cells, or blasts. Biochemical analysis revealed an extremely high serum LDH level (16200 IU/L) and mild elevation of the serum transaminase level (asparagine transaminase, 226 IU/L; alanine transaminase, 88 IU/L). The alkaline phosphatase level was within the normal range. LDH subclass analysis demonstrated the predominance of LDH2 (39%) and LDH3 (44%). Renal function was normal (serum creatinine, 0.74 mg/dL) with normal findings on urinalysis. Serum levels of uric acid (8.8 mg/dL) and inorganic phosphate (6.3 mg/dL) were elevated, but there were no abnormalities in other electrolytes. Serum total protein (6.6 g/dL) and albumin (4.5 g/dL) levels were normal. Coagulation tests revealed only slight elevation of fibrin degenerative products. Elevated levels of serum ferritin (3338 ng/mL), soluble interleukin-2 receptor (625 U/mL), and beta-2 microglobulin (2.5 mg/L) were also observed. Immunoelectrophoresis of serum and urine detected monoclonal IgD-lambda protein and Bence-Jones protein (BJP) subtypes. Serum IgG, IgA, IgM, and IgD levels were 395, 16, 7, and 197.3 mg/dL, respectively. Serum free light chain analysis indicated deviation of the kappa/ lambda ratio (kappa-chain, 1.4 mg/L; lambda-chain, 2150 mg/L). The patient was negative for anti-human immunodeficiency virus (HIV) antibody. Elevation of the Epstein–Barr virus DNA titer in peripheral blood was not observed. Bone marrow aspiration resulted in dry tap. However, biopsy revealed nodular aggregation of atypical large cells that had basophilic cytoplasm and euchromatic nuclei (Figure 1); on flow cytometry and immunohistochemical analysis, they were CD3–, CD4–, CD7+, CD10–, CD13+, CD20–, CD30–, CD33+, CD56–, CD79a–, IgG–, IgM–, IgA–, Igκ–, Igλ+, c-myc+, MPO+, and MUM1+ (Figure 1). CD38 was weakly positive and CD138 was negative. The Ki-67 labeling index was very high (95%). Epstein–Barr virus-encoded RNA was not detected by in situ hybridization. G-banding analysis revealed a complex karyotype, including duplication of the 14q32 locus (Table 1). Fluorescence in situ hybridization demonstrated no fusion signals of IgG/Myc, IgH/MAF, IgH/ FGFR3, or IgH/CCND1, and no split signal of Myc. Strong uptake of fluorodeoxyglucose (FDG) in systemic bones without bone destruction and around the right brachial plexus was observed on positron emission tomography combined with computed tomography (PET/CT) (Figure 2). There was no lymphadenopathy or hepatosplenomegaly. Magnetic resonance imaging detected infiltrating lesions around the right brachial plexus. The clinicopathological findings described above indicated atypical plasma cell dyscrasia with extreme clinical aggressiveness, features markedly different from those of conventional plasma cell myeloma, and considered to be included within the concept of AMM. We initially administered high-dose dexamethasone, which resulted in partial relief of pain, improvement of general status, and reduction of serum LDH to some extent. Thereafter, the anti-lymphoma EPOCH regimen (etoposide, doxorubicin hydrochloride, vincristine, prednisolone, and cyclophosphamide) was started. Further transient elevation of LDH was observed, but there were no signs of tumor lysis syndrome or disseminated intravascular coagulopathy. After 3 weeks, serum LDH and IgD levels had significantly decreased and abnormal cells were not detected in the bone marrow, suggesting that the EPOCH regimen was highly effective. After a total of four courses of EPOCH, a significant reduction in systemic bone FDG uptake was noted on PET/CT. Thereafter, Anaplastic multiple myeloma: possible limitations of conventional chemotherapy for long-term remission

Successful Treatment of Aggressive Mature B-cell Lymphoma Mimicking Immune Thrombocytopenic Purpura

A 55-year-old woman suffered from hemorrhagic tendency. She had severe thrombocytopenia without any hematological or coagulatory abnormalities, and a bone marrow examination revealed an increased number of megakaryocytes without any abnormal cells or blasts. No lymphadenopathy or hepatosplenomegaly was observed on computed tomography. She was initially diagnosed with immune thrombocytopenic purpura (ITP). None of the treatments administered for ITP produced a response. However, abnormal cells were eventually found during the third bone marrow examination. The pathological diagnosis was mature B-cell lymphoma. Rituximab-containing chemotherapy produced a marked increase in the patients platelet count, and her lymphoma went into complete remission.

γδ T cell clonal proliferation early after PD-1 blockade

Dear Editor, A recent study showed that programmed death 1 (PD-1) suppresses not only T cell-mediated immune responses but also overactivated T cell receptor (TCR) signaling, and its blockade can potentially promote T cell lymphomagenesis [1]. However, T cell lymphoma triggered by PD-1 inhibitors has not been reported previously. Recently, we encountered a case of secondary hepatosplenic T cell lymphoma (HSTCL) that developed early after PD-1 blockade therapy. A 73-year-old man had been diagnosed with classical Hodgkin lymphoma (HL), mixed cellularity type, 2 years previously. He received nivolumab treatment (3 mg/kg every 2 weeks) for HL that relapsed after standard chemotherapy and brentuximab vedotin treatment. After two doses of nivolumab, the patient presented with high-grade fever, general fatigue, and bleeding tendency. Laboratory investigation showed acute-onset pancytopenia (white blood cell count, 1.0 × 10/μL; hemoglobin level, 9.2 g/dL; reticulocyte count, 0.5%; and platelet count, 47 × 10/μL) with increased serum levels of lactic dehydrogenase (12,313 IU/L) and ferritin ( 2 6 6 , 8 0 0 μ g / L ) s u g g e s t i n g h emop h a g o c y t i c lymphohistiocytosis. Disseminated intravascular coagulation was also noted. Epstein–Barr virus (EBV) DNA levels in the peripheral blood were not elevated, and the patient showed neither infections nor autoimmune diseases. Whole-body computed tomography showed that the HL lesions had decreased in size compared to before nivolumab treatment. Bone marrow examination revealed clonal proliferation of atypical lymphocytes (Fig. 1a) that exhibited TCR-γδ restriction on flow cytometric analysis. Moreover, monoclonal rearrangements of the TCR-γ and TCR-δ genes were detected (Fig. 1b). Chromosomal examination showed abnormalities o f 46 ,XY,add (17 ) (p13 ) [19 /20 ] /46 , idem ,de r (1 ) t (1:3)(p34:p13)[1/20]. Bone marrow biopsy specimen showed massive infiltration of neoplastic lymphoid cells, which were immunohistochemically positive for CD3, TIA-1, and granzyme B, but negative for CD4, CD5, CD8, CD10, CD20, CD30, CD79a, and EBV-encoded RNA on in situ hybridization. Despite intensive treatment, the patient’s condition deteriorated rapidly, and he died 2 months after initiation of nivolumab. Autopsy showed massive infiltration of T cell lymphoma into the bone marrow, liver, and spleen. These findings were consistent with HSTCL, a rare subtype of extranodal T cell lymphoma. HSTCL has been reported in patients undergoing kidney transplantation or with a prior history of Crohn’s disease, systemic lupus, rheumatoid arthritis, or malaria [2–4], suggesting that HSTCL is associated with immune dysregulation and chronic antigen stimulation. These reports also included two cases of HSTCL with a history of HL [2, 3]. An experimental model suggested that CD30 molecules, characteristic of Hodgkin–Reed–Sternberg cells, can potentially trigger clonal proliferation of γδ T cells [5]. In our case, blockade of PD-1 may have accelerated γδ T cell clonal proliferation occurring during prolonged HL-associated inflammation. Another point of note in this case was that the neoplastic cells atypically showed expression of granzyme B with TIA1, although HSTCLs usually have a non-activated cytotoxic phenotype without granzyme B expression [3]. We assumed that overactivated T cell-mediated immune responses triggered by PD-1 inhibitor led to the patient’s severe inflammatory condition and aggressive clinical course. PD-1 blockade is a promising therapeutic approach for refractory * Yasushi Onishi yonishi@med.tohoku.ac.jp