Small Maf Compound Mutants Display Central Nervous System Neuronal Degeneration, Aberrant Transcription, and Bach Protein Mislocalization Coincident with Myoclonus and Abnormal Startle Response

The small Maf proteins form heterodimers with CNC and Bach family proteins to elicit transcriptional responses from Maf recognition elements (MAREs). We previously reported germ line-targeted deficiencies in mafG plus mafK compound mutant mice. The most prominent mutant phenotype was a progressive maf dosage-dependent neuromuscular dysfunction. However, there has been no previous report regarding the effects of altered small-maf gene expression on neurological dysfunction. We show here that MafG and MafK are expressed in discrete central nervous system (CNS) neurons and that mafG::mafK compound mutants display neuronal degeneration coincident with surprisingly selective MARE-dependent transcriptional abnormalities. The CNS morphological changes are concurrent with the onset of a neurological disorder in the mutants, and the behavioral changes are accompanied by reduced glycine receptor subunit accumulation. Bach/small Maf heterodimers, which normally generate transcriptional repressors, were significantly underrepresented in nuclear extracts prepared from maf mutant brains, and Bach proteins fail to accumulate normally in nuclei. Thus compound mafG::mafK mutants develop age- and maf gene dosage-dependent cell-autonomous neuronal deficiencies that lead to profound neurological defects

Clinicopathological analysis of primary adrenal diffuse large B-cell lymphoma: effectiveness of rituximab-containing chemotherapy including central nervous system prophylaxis

BACKGROUND: Primary adrenal lymphoma (PAL) is an extremely rare subtype of extranodal non-Hodgkin’s lymphoma. Some researchers have reported some of the characteristics of PAL and its association with poor prognosis; however, the clinicopathological features of PAL remain to be elucidated. METHODS: From 2008 to 2011 we experienced seven cases of PAL in our institutions. We retrospectively analyzed the clinical and pathological features of these patients. RESULTS: The patients ranged in age from 50 to 85 years, with a median of 71 years. The overall male:female ratio was 6:1. All seven patients were diagnosed with diffuse large B-cell lymphoma (DLBCL) pathologically. Bilateral adrenal involvement was confirmed in five patients. The median largest tumor diameter at diagnosis was 58 mm. The Ki-67 index was generally high (>70%). All patients were treated with rituximab-containing chemotherapy, and central nervous system (CNS) prophylaxis was conducted for three patients. One patient with CNS involvement at the time of the diagnosis also received whole-brain radiation. The overall survival rate at two years was 57% (median follow-up; 24.8 months). It is noteworthy that the three patients who received a full course of the rituximab-containing regimen and CNS prophylaxis are currently alive without disease relapse, and that none of the seven patients died due to progression of lymphoma. CONCLUSIONS: Primary adrenal DLBCL can be a clinically aggressive disease entity. Rituximab-containing chemotherapy combined with CNS prophylaxis could be a reasonable option for the treatment of PAL; however, analyses of more PAL cases are needed for the establishment of this strategy

A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy against Anemia of Chronic Disease

<div><p>Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, <i>HAMP</i>, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice <i>in vivo</i>. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased <i>HAMP</i> expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits <i>HAMP</i> expression. Transcriptional profiling confirmed the downregulation of <i>HAMP</i>. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of <i>HAMP</i> expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated <i>HAMP</i> suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of <i>Gdf15</i> and downregulation of <i>Hamp</i> expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.</p> </div

K7174 treatment suppresses hepcidin production <i>in vivo</i>.

<p>(A) Quantitative RT-PCR analysis of <i>Gdf15</i> and <i>Hamp</i> expression in liver samples from control (PBS) or K7174-treated mice (<i>n</i> = 8, mean ± SE, * <i>P</i> < 0.05). The expression levels of PBS-treated liver samples were set to 1. (B) Serum hepcidin (hepcidin1) levels in control (PBS) or K7174-treated mice (<i>n</i> = 8, mean ± SE, * <i>P</i> < 0.05).</p

<i>GDF15 is responsible for K7174-mediated</i> HAMP <i>gene downregulation in HepG2 cells.</i>

<p>(A) Retrovirus-mediated GDF15 overexpression in HepG2 cells. Anti-GDF15 antibody was used to detect endogenous GDF15 protein. Alpha-Tubulin was used as a loading control. (B) Quantitative RT-PCR analysis of <i>HAMP</i> expression in GDF15 overexpressed HepG2 cells. The expression level of each target gene relative to that of <i>GAPDH</i> was calculated (<i>n</i> = 3, mean ± SE, * <i>P</i> < 0.05). The expression level of DMSO-treated control cells was set to 1. (C) Experimental strategy for siRNA-mediated GDF15 knockdown after K7174 treatment in HepG2 cells. (D) Western blotting analysis of whole-cell extracts from GDF15-silenced HepG2 cells, treated with K7174 or DMSO. Anti-GDF15 antibody was used to detect endogenous GDF15 protein. Alpha-Tubulin was used as a loading control. (E) Quantitative RT-PCR analysis of <i>GDF15</i> and <i>HAMP</i> expression in GDF15-silenced HepG2 cells, treated with K7174 or DMSO. The expression level of each target gene relative to that of <i>GAPDH</i> was calculated (<i>n</i> = 3, mean ± SE, * <i>P</i> < 0.05). The expression levels of DMSO- and control siRNA-treated control cells were set to 1.</p

Expression profiling identified GDF15 among K7174-regulated genes.

<p>(A) Expression profiling results based on HepG2 cells treated with K7174 (20 µM, 24 h) or 0.1% DMSO as a control. The heat map depicts the fold change resulting from K7174 treatment. The top 50 upregulated (left) and downregulated (right) genes after K7174 treatment are shown. Arrows indicate the positions of <i>DDIT3</i> (CHOP), <i>CEBPB</i> and <i>BMP4</i>. (B,C) Quantitative RT-PCR validation of array results for upregulated (B) and downregulated (C) genes. The expression level of each target gene relative to that of <i>GAPDH</i> was calculated (<i>n</i> = 5, mean ± SE, * <i>P</i> < 0.05). The expression levels of DMSO-treated control cells were set to 1. (D) Western blotting analysis of whole-cell extracts from control (DMSO) or K7174-treated HepG2 cells. Anti-GDF15 antibody was used to detect endogenous GDF15 protein. Alpha-Tubulin was used as a loading control. (E) ELISA to measure GDF15 concentration in culture media from control (DMSO) or K7174-treated HepG2 cells (<i>n</i> = 5, mean ± SE, * <i>P</i> < 0.05).</p

K7174 induced GDF15 in the K562 hematopoietic cell line.

<p>(A) Quantitative RT-PCR analysis of GDF15 in control (DMSO) or K7174-treated K562 cells (<i>n</i> = 3, mean ± SE, * <i>P</i> < 0.05). The expression level of DMSO-treated control cells was set to 1. (E) ELISA to measure GDF15 concentration in culture media from control (DMSO) or K7174-treated K562 cells (<i>n</i> = 3, mean ± SE, * <i>P</i> < 0.05).</p

K7174 suppresses HAMP expression in HepG2 cells.

<p>Quantitative RT-PCR analysis of <i>HAMP</i> expression in control (DMSO) or K7174-treated HepG2 cells. The expression level of each target gene relative to that of <i>GAPDH</i> was calculated (<i>n</i> = 5, mean ± SE, * <i>P</i> < 0.05). The expression level of DMSO-treated control cells was set to 1.</p

Advantages of Higher Busulfan Dose Intensity in Fludarabine-Combined Conditioning for Patients with Acute Myeloid Leukemia Undergoing Cord Blood Transplantation

The alkylating agent busulfan is commonly used as conditioning in allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia (AML). However, a consensus has not yet been reached regarding the optimal busulfan dose in cord blood transplantation (CBT). Therefore, we conducted this large nationwide cohort study to retrospectively analyze the outcomes of CBT in patients with AML receiving busulfan at intermediate (6.4 mg/kg i.v.; BU2) or higher (12.8 mg/kg i.v.; BU4) doses within a fludarabine/i.v. busulfan (FLU/BU) regimen. Among 475 patients who underwent their first CBT following FLU/BU conditioning between 2007 and 2018, 162 received BU2 and 313 received BU4. Multivariate analysis identified BU4 as a significant factor for longer disease-free survival (hazard ratio [HR], .85; 95% confidence interval [CI], .75 to .97; P = .014) and a lower relapse rate (HR, .84; 95% CI, .72 to .98; P = .030). No significant differences were observed in non-relapse mortality between BU4 and BU2 (HR, 1.05; 95% CI, .88-1.26; P = .57). Subgroup analyses showed that BU4 provided significant benefits for patients who underwent transplantation while not in complete remission (CR) and those age <60 years. Our present results suggest that higher busulfan doses are preferable in patients undergoing CBT, particularly those not in CR and younger patients

Outcomes of poor peripheral blood stem cell mobilizers with multiple myeloma at the first mobilization: A multicenter retrospective study in Japan

Abstract Autologous stem cell transplantation (ASCT) remains an important therapeutic strategy for multiple myeloma; however, a proportion of patients fail to mobilize a sufficient number of peripheral blood stem cells (PBSCs) to proceed to ASCT. In the present study, we aimed to clarify the characteristics and outcomes of poor mobilizers. Clinical data on poorly mobilized patients who underwent PBSC harvest for almost 10 years were retrospectively collected from 44 institutions in the Japanese Society of Myeloma (JSM). Poor mobilizers were defined as patients with less than 2 × 106/kg of CD34+ cells harvested at the first mobilization. The proportion of poor mobilization was 15.1%. A sufficient dataset including overall survival (OS) was evaluable in 258 poor mobilizers. Overall, 92 out of 258 (35.7%) poor mobilizers did not subsequently undergo ASCT, mainly due to an insufficient number of PBSCs. Median OS from apheresis was longer for poor mobilizers who underwent ASCT than for those who did not (86.0 vs. 61.9 mon., p = 0.02). OS from the diagnosis of poor mobilizers who underwent ASCT in our cohort was similar to those who underwent ASCT in the JSM database (3y OS rate, 86.8% vs. 85.9%). In this cohort, one‐third of poor mobilizers who did not undergo ASCT had relatively poor survival. In contrast, the OS improved in poor mobilizers who underwent ASCT. However, the OS of extremely poor mobilizers was short irrespective of ASCT