Interplay of adipocyte and hepatocyte: Leptin upregulates hepcidin.

Conflicting evidence concerning leptin, an adipocyte-derived hormone, in atherogenesis and non-alcoholic fatty liver disease (NAFLD) has been reported. Iron metabolism and iron-mediated oxidative stress should be taken into consideration for the clarification of the pathogenesis of these diseases. In this study, we demonstrate that leptin receptor activation directly affects iron metabolism by the finding that serum levels of hepcidin, the master regulator of iron in the whole body, were significantly lower in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice. The administration of recombinant leptin to ob/ob mice for two weeks showed a significant increase in serum hepcidin and hepatic Hamp mRNA levels. Hamp mRNA levels were significantly correlated with hepatic iron content and BMP6 mRNA levels. Hepatic iron content was associated with the increase in mRNA levels of divalent metal transporter 1 and transferrin receptor. Our data provide evidence that the interplay of these two hormones could help improve the understanding of the pathogenesis of atherosclerosis and NAFLD.

[Sudden death associated with myocardial damage caused by microthrombi in a patient with thrombotic thrombocytopenic purpura].

We describe a 35-year-old woman with Down's syndrome who was admitted to a clinic with anorexia and vomiting. Since laboratory findings showed anemia (Hb 7.4 g/dl) and thrombocytopenia (0.5 × 104/µl), she was transferred to our hospital for treatment. Further laboratory examinations revealed schistocytes, LDH elevation, and a negative Coombs' test. Thrombotic thrombocytopenic purpura (TTP) was suspected. Plasma exchange (PEX) and prednisolone administration were thus immediately initiated. Prior to these treatments, ADAMTS13 activity was less than 5% and inhibitors were detected at a level of 0.8 Bethesda U/ml. Although her platelet count had risen to 13.0 × 104/µl by day 6 (post 4 sessions of PEX), it had decreased to 1.8 × 104/µl on day 7. Despite ongoing PEX, thrombocytopenia persisted. On day 21, she suddenly died. Autopsy findings revealed no evidence of myocardial necrosis or coronary artery thrombosis. Extensive microthrombi were, however, detected in precapillary arterioles, capillaries, and post-capillary venules of the heart. Therefore, this patient's sudden death was clinically suspected to have been caused by cardiomyopathy, which had produced cardiogenic shock.

Clonal heterogeneity of mantle cell lymphoma revealed by array comparative genomic hybridization.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) characterized by the translocation t(11;14)(q13;q32). This lymphoma exhibits a poor prognosis and remains incurable with standard chemotherapy approaches. Recently, we have shown that a majority of patients with acute-type adult T-cell leukemia/lymphoma (ATLL) have multiple subclones that were likely produced in lymph nodes. We investigated whether MCL has multiple subclones as identified in ATLL by high-resolution oligo-array comparative genomic hybridization (CGH). Eleven of 20 (55%) evaluable MCL cases had a log2 ratio imbalance, suggesting the existence of multiple subclones in MCL. Based on the proportion of every subclone relative to the main clone, we were able to speculate clonal evolution in each MCL case with multiple subclones. Our analysis gave new insights into the clonal heterogeneity quantitatively and accurately. Furthermore, genomic copy number alterations are not hierarchical events and not necessarily the initial or later events for cells to become MCL.

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling.

FMS-related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70-100% of cases of AML and in virtually all cases of B-lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small-molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off-target toxicities and drug resistance. The development of anti-FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)-induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor-dependent cell line independently of FL addition. In addition, A2 showed complement-dependent cytotoxicity activity, but was devoid of Ab-dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell-based immunotherapies.

Deregulated iron metabolism in bone marrow from adenine-induced mouse model of chronic kidney disease.

Although the primary cause of anemia in chronic kidney disease (CKD) is lack of sufficient erythropoietin (EPO), other factors may be involved, including the deregulation of iron metabolism. To clarify the mechanism of deranged erythropoiesis in CKD, we evaluated bone marrow (BM) cells in adenine-induced CKD mice. They showed even higher EPO expression in the kidney. Hepatic hepcidin mRNA and plasma hepcidin and ferritin levels were increased. Flow cytometry revealed a decrease in the number of cells expressing transferrin receptor (TfR), or late erythroid progenitors in BM; these cells correspond to proerythroblasts, and basophilic and polychromatic erythroblasts. In CKD mice, levels of erythroferrone mRNA in BM and splenic cells were significantly decreased, and MafB protein levels in BM cells were significantly increased. These results suggest that, in BM, the decrease in TfR, which may be associated with increased MafB levels, and the decrease in erythroferrone increase hepatic hepcidin expression, which may perturb iron recycling and erythropoiesis.

Rearrangement of VPS13B, a causative gene of Cohen syndrome, in a case of RUNX1-RUNX1T1 leukemia with t(8;12;21).

Variant chromosomal translocations associated with t(8;21) are observed in 3-4% of acute myeloid leukemia (AML) cases with a RUNX1-RUNX1T1 fusion gene. However, the molecular events that occur in variants of t(8;21) are not well characterized. In the present study, we report genetic features of a variant three-way translocation of t(8;12;21)(q22;p11;q22) in a patient with AML. In this patient, leukemia cells lacked azurophilic granules, which does not correspond with the classic features of t(8;21). RNA-seq analysis revealed that TM7SF3 at 12p11 was fused to VPS13B at 8q22 and VPS13B to RUNX1, in addition to RUNX1-RUNX1T1. VPS13B was located near RUNX1T1 and both were localized at the same chromosomal bands. The reading frames of TM7SF3 and VPS13B did not match to those of VPS13B and RUNX1, respectively. Disruption of VPS13B causes Cohen syndrome, which presents intermittent neutropenia with a left-shifted granulopoiesis in the bone marrow. Disruption of VPS13B may thus cause the unusual features of RUNX1-RUNX1T1 leukemia. Our case indicates that rearrangement of VPS13B may be additional genetic events in variant t(8;21).

A Relevant Method for Estimating Cell Cycle of Developing Cell: The Conspicuous Lengthening of Ts during the Early Neural Development of Cynops Embryo: cell cycle analysis/Ts lengthening/early neural development/amphibian embryo).

A new method for estimating cell cycle was proposed and the cell cycles of the presumptive neural cells of Cynops embryo from the gastrula to neurula stages were estimated. Up to the onset of gastrulation, Tg2 and Tg1 became recognizable and Ts lengthened more than 10 times of that in the morula stage. The respective phases of cell cycle, espetially Ts became prominently longer as gastrulation and neurulation proceeded. However, the Ts retained a correlation with Tgc as expressed in the following regression equation, Ts=0.795Tgc-0.090, through the early development of presumptive neural cells.

CHANGES OF CHROMOSOMES DURING THE EARLY NEURAL DEVELOPMENT OF A JAPANESE NEWT, CYNOPS PYRRHOGASTER.

The karyotype of Cynops pyrrhogaster was determined on the mitotic chromosomes in the presumptive neural area of an early gastrula. 24 chromosomes of a diploid set consisted of 8 metacentric and 4 submetacentric pairs. Individual chromosomes were identified on the basis of their morphology and characteristic C-binding patterns. Sex chromosomes were not identified. Total length of the haploid chromosome set in the presumptive neural area decreased remarkably from morulae to gastrulae, further continued to decrease up to neurulae and thereafter remained unchanged till tail-buds. Chromosome shortening occurring from morulae to gastrulae was accompanied with a prominent decrease in chromosome volume, keeping chromosome width constant. Shortening took place evenly along the longitudinal axis of a chromosome. When gastrulae and neurulae were compared concerning their positions of the appearance of the C-bands, the basic pattern remained unchanged. In certain chromosomes, the number of C-bands decreased as the result of their fusion, as gastrulae proceeded to neurulae.

Changes of Chromosome Length and Constitutive Heterochromatin in Association with Cell Division during Early Development of Cynops pyrrhogaster embryo: (chromatin condensation/constitutive heterochromatin/cell division/ neural differentiation/amphibian embryo).

The length of chromosomes in the presumptive ectoderms of Cynops embryos was measured at nine successive cell divisions from the 6th (cleavage stage) to the 14th (gastrula stage). Up to the 10th cell division (cleavage stage) the chromosome length remained constant. At the 11th cell division the chromosomes began to shorten and continued to shorten at every cell division up to the 14th cell division. The degree of shortening and the mode of variation in length corresponded to the respective developmental stages of cleavage, blastula and gastrula. During those periods when chromosomes became shortened, some fine C-bands of the paracentromeric region found in earlier stages fused together. The chromatin of interphase nuclei also showed considerable changes during chromosome shortening. Besides the size reduction of interphase nuclei, the number of C-band granules in an interphase nucleus decreased in parallel with chromosome shortening and fusion of C-bands in mitotic chromosome.

Calcification of the thoracic aorta determined by three-dimensional computed tomography predicts cardiovascular complications in patients undergoing hemodialysis.

PURPOSE: In patients on dialysis, the most common cause of death is cardiovascular disease. This is caused, at least in part, by excessive vascular calcification. Studies that have examined coronary calcification have been published, but these measurements require expensive equipment. Here, we used computed tomography to determine aortic calcification and evaluated these data as prognostic markers for cardiovascular disease. METHODS: Computed tomography with contrast medium was performed on 49 patients undergoing hemodialysis (29 males and 20 females; average age, 68.9 ± 11.0 years). A calcification score (CS) was defined as the ratio of the volume of vascular calcification to the volume of the thoracic aorta. All patients were monitored for cardiovascular end points, which included cerebral infarction or hemorrhage, myocardial infarction, electrocardiographic, or echocardiographic abnormalities that suggested myocardial ischemia, cardiac surgery, leg amputation, and hospitalization or death due to heart failure. RESULTS: Patients were followed for 3 years, with 12 patients reaching the end point. Both high CS (p = 0.007) and male gender (p = 0.009) were significantly associated with cardiovascular events. In contrast, events were not related to age, dialysis duration, diabetes mellitus, smoking status, low-density lipoprotein cholesterol level, pulse-wave velocity, maximum intima-media thickness of the carotid artery wall, systolic blood pressure, or left ventricular hypertrophy. Multiple logistic regression analysis revealed that a high baseline CS was a significant predictor for cardiovascular events (p < 0.05). CONCLUSIONS: Calcification of the thoracic aorta determined by three-dimensional computed tomography predicts cardiovascular complications in patients on hemodialysis.

Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice.

Patients in the chronic phase (CP) of chronic myelogenous leukemia (CML) have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC) phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt.) AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.

Mutation analysis of NF-κB signal pathway-related genes in ocular MALT lymphoma.

Constitutive nuclear factor-kappa B (NF-κB) activation has been reported in ocular adnexal lymphoma (OAL). TNFAIP3/A20 is a "global" inhibitor of NF-κB pathway. We have shown that OAL has preferential loss of the 6q23.3 region where TNFAIP3/A20 exist, which is suggested to involve in lymphomagenesis of OAL. The mechanisms causing NF-κB activity in OAL remain elusive. Recently, NF-κB canonical pathway genes including CARD11, CD79B and MYD88 were shown to be frequently mutated in diffuse large B-cell lymphomas. In this study, we analyzed the mutation status of these genes by direct sequencing in 24 OAL cases including 9 cases with loss of 6q23.3 previously identified by array comparative genomic hybridization. We showed that genetic alterations of these genes were not found in OAL, a finding differing from that of most B-cell lymphomas. Genetic or epigenetic alterations in other genes are likely to be relevant in pathogenesis of OAL case without A20 loss.

Chronic myeloid leukemia following chemotherapy with 5'-deoxy-5-fluorouridine for gastric cancer.

We report here a very rare case of chronic myeloid leukemia (CML) following long-term chemotherapy with 5'-deoxy-5-fluorouridine (5'-DFUR) for gastric cancer. A 69-year-old man was diagnosed with the chronic phase of CML. Six years previously, he underwent radical subtotal gastrectomy for gastric cancer, and was subsequently treated with oral anti-metabolite 5'-DFUR as adjuvant chemotherapy for 6 years. He was placed on imatinib therapy, and achieved a major molecular response 10 months after the initiation of therapy. This is the first reported case of therapy-related CML following 5'-DFUR treatment.

Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A.

Scanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.

Use of lectins as probes for analyzing embryonic induction.

Lectins were used as probes to investigate the mechanism of embryonic induction. Concanavalin (Con A) and gorse agglutinin out of 7 species of lectins tested were found to have strong neural-inducing effect on the presumptive ectoderm of newt gastrulae. Their effects were abolished by the addition of α-methyl-D-mannoside and α-L-fucose, respectively. Succinyl-Con A had a weak inducing activity in comparison to Con A. Autoradiography of3H-Con A-treated explants revealed that Con A bound to the inner surface, but not to the outer surface of ectoderm and was successively incorporated into cytoplasm.3H-Thymidine incorporation was lower in the first half and higher in the second half of the 60 h cultivation period in Con A-treated explants as compared to controls.Con A-Sepharose had a strong inductive effect. This suggests that neural induction is caused through Con A binding to the plasma membrane, but not through incorporation into the cytoplasm of the ectoderm cells.