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Background Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer. Methods We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors. Results Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore. Conclusions RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

Alzheimer's disease (AD) is clinically characterized by gradual onset over years with worsening of cognition. The initial and most prominent cognitive deficit is commonly memory dysfunction. However, a subset of AD cases has less hippocampal atrophy than would be expected relative to the predominance of cortical atrophy. These hippocampal-sparing cases have distinctive clinical features, including the presence of focal cortical clinical syndromes. Given that previous studies have indicated that severe hippocampal atrophy corresponds to prominent loss of episodic memory, it is likely that memory impairment is initially absent in hippocampal-sparing AD cases. Here, we report on a patient with an 8-year history of delusional jealousy with insidious onset who was clinically diagnosed as possible AD and pathologically confirmed to have AD with relatively preserved neurons in the hippocampus. This patient had delusional jealousy with a long pre-dementia stage, which initially was characterized by lack of memory impairment. Head magnetic resonance imaging findings showed preserved hippocampal volume with bilateral enlarged ventricles and mild-to-moderate cortical atrophy. Head single-photon emission computed tomography revealed severely decreased regional cerebral blood flow in the right temporal lobe. The resolution of the delusion was attributed to pharmacotherapy by an acetylcholinesterase inhibitor, suggesting that the occurrence of delusional jealousy was due to the disease process of AD. Although the neural basis of delusional jealousy remains unclear, this hippocampal-sparing AD case may be classified as an atypical presentation of AD.

Japan approved clozapine for treatment-resistant schizophrenia in June 2009. The aim of this study was to evaluate clozapine's efficacy and tolerability in Japanese patients. A twelve-week, single-arm clinical trial of clozapine in treatment-resistant schizophrenia inpatients, was conducted under real-world conditions using raters masked for type of antipsychotic. Thirty-eight patients were recruited, with 33 (86.8%) completing the trial. At week 12, clozapine was associated with significant improvement in the Positive and Negative Syndrome Scale (PANSS) total (p0.0001), PANSS positive (p0.0001), negative (p = 0.0055) and general subscale scores (p0.0001). Significant improvements occurred in all PANSS scores by week 4, the first post-baseline psychopathology rating. Altogether, 50.0% of patients showed ≥20% reduction in PANSS total score, 20.6% had ≥30% reduction and 14.7% had40% reduction. Eighteen patients (47.4%) were discharged before week 12. However, all patients experienced ≥1 adverse event. Two of 38 patients (5.2%) dropped out due to moderate leucopenia and one of them developed agranulocytosis after stopping clozapine. However, both patients recovered. Eight adverse events (hypersalivation, fatigue, sedation, constipation, insomnia, nausea/vomiting, chest pain and leucopenia) were observed in 34-79% of patients. These findings suggest that clozapine is beneficial in Japanese treatment-resistant schizophrenia patients. However, attention should be paid to patients' adverse events.

Aims and background MK615 is produced from Japanese apricot and contains several cyclic triterpenes, such as oleanolic and ursolic acids. MK615 was shown to strongly suppress cutaneous in-transit metastasis in a patient with malignant melanoma. The present investigation was undertaken to clarify the antitumor effects of MK615 in vitro and in vivo. Methods Several human cancer cell lines were exposed to MK615 for 7 days to examine its antiproliferative effects. The effect of MK615 on in vivo growth of human pancreatic cancer MIAPaCa-2 cells was also examined. Results MK615 inhibited the growth of several human cancer cell lines in a concentration-dependent way. Pancreatic cancer MIAPaCa-2 cells were highly sensitive to the growth-inhibiting effects of MK615. Treatment with MK615 preferentially induced cell death in human cancer cells while sparing normal cells such as human umbilical vein endothelial cells (HUVEC) and mouse bone marrow cells. When MIAPaCa-2 cells were incubated with MK615 in the presence of antioxidant, growth-inhibition was significantly reduced, and MK615 induced the accumulation of reactive oxygen species in cancer cells but not in HUVEC. MK615, in both the presence and absence of gemcitabine, significantly inhibited the growth of human pancreatic cancer cells as xenografts without apparent adverse effects. Conclusions MK615, a supplement produced from Japanese apricot, may have therapeutic value in treating human cancers through a reactive oxygen species-dependent mechanism.

Aims: Previous studies have reported that the incidence of obstructive sleep apnea syndrome (OSAS) in patients with depression is higher than in the general population. We examined the risk factors to predict OSAS in mood disorder patients with depressive symptoms. Method: We conducted polysomnography for patients who satisfied the following criteria: (i) diagnosis of major depressive disorder or bipolar disorder according to the Mini-International Neuropsychiatric Interview (MINI); (ii) a score of ≥10 on the Hamilton Rating Scale for Depression (HAM-D); (iii) fulfillment of either (a) or (b) below: (a) at least one of the following: severe snoring, witnessed apnea during sleep, excessive daytime sleepiness; (b) at least one of the following plus an oxygen desaturation index of 4% ≥5 times/h by pulse oximeter: mild snoring, sleep disturbance, headache, high blood pressure. The patients with apnea hypopnea index ≥5 were diagnosed with OSAS. Results: Of the 32 mood disorder patients who met the subject conditions, 59.4% had OSAS. The diagnosis rate with our criteria was significantly higher than the previously reported incidence of OSAS in patients with depression. There was no significant difference among diagnosis rates as to individual risk factors or the number of risk factors. A multiple regression test showed no significant association between apnea–hypopnea index and other clinical factors including depression severity. Conclusion: The present results showed that OSAS can be detected at a remarkably higher rate by considering appropriate OSAS risk factors in mood disorder patients, and suggested that there is a high rate of undetected and therefore untreated OSAS among mood disorder patients.

Alzheimer's disease (AD) is clinically characterized by gradual onset over years with worsening of cognition. The initial and most prominent cognitive deficit is commonly memory dysfunction. However, a subset of AD cases has less hippocampal atrophy than would be expected relative to the predominance of cortical atrophy. These hippocampal-sparing cases have distinctive clinical features, including the presence of focal cortical clinical syndromes. Given that previous studies have indicated that severe hippocampal atrophy corresponds to prominent loss of episodic memory, it is likely that memory impairment is initially absent in hippocampal-sparing AD cases. Here, we report on a patient with an 8-year history of delusional jealousy with insidious onset who was clinically diagnosed as possible AD and pathologically confirmed to have AD with relatively preserved neurons in the hippocampus. This patient had delusional jealousy with a long pre-dementia stage, which initially was characterized by lack of memory impairment. Head magnetic resonance imaging findings showed preserved hippocampal volume with bilateral enlarged ventricles and mild-to-moderate cortical atrophy. Head single-photon emission computed tomography revealed severely decreased regional cerebral blood flow in the right temporal lobe. The resolution of the delusion was attributed to pharmacotherapy by an acetylcholinesterase inhibitor, suggesting that the occurrence of delusional jealousy was due to the disease process of AD. Although the neural basis of delusional jealousy remains unclear, this hippocampal-sparing AD case may be classified as an atypical presentation of AD.

The aim of this study was to examine the effects of negative cognition on PBI score before and after treatment for depression. Forty major depressive disorder outpatients were assessed with the PBI scale and Structured Interview Guide for Hamilton Rating Scale for Depression (SIGH-D) at the time of the first medical examination (baseline) and 8 weeks later. The SIGH-D scores decreased by about 50% from baseline to 8 weeks, but there was no significant change in the PBI scores of the depressed outpatients from baseline to 8 weeks. Analysis of covariance with the SIGH-D scores as covariate was conducted for PBI scores between baseline and 8 weeks to remove effects of MDD. No significant differences were found on any of the PBI scales. Even though the therapeutic values on the SIGH-D of the depressed patients indicated that depressive symptoms were reduced by about 50%, depression level did not influence the PBI scores. This study provides evidence for the stability of parental representations throughout treatment, as measured by the PBI.

アイスクリームの脂肪球凝集,官能検査によるドライネス評価およびメルトダウン試験に及ぼすフリージング時に使用するダッシャータイプとその回転数の影響について検討した.(1) ダッシャー容積率が高い程,およびその回転数が速い程1.2μm以下の脂肪球は減少し脂肪球の凝集は進み,7-15μmの巨大な脂肪球の凝集体の特異的な増加が見られた.(2) 15・Dよりもダッシャー容積率が高い80・Dの方が脂肪球の凝集は促進された.(3) 同一ダッシャーを使用するとき回転数が110rpmよりも220rpmの方が脂肪球の凝集が促進された.(4) アイスクリームの性状としてはダッシャー容積率が高い程,およびその回転数が速い程ドライネスが増加することが判明した.(5) アイスクリームの性状としてダッシャー容積率が高い程,およびその回転数が速い程メルトダウンのスピードは遅く保型性は優れていた.

アイスクリームの脂肪球凝集に及ぼすフリーザー出口温度とオーバーランの影響について検討した.(1) フリージングする前のアイスクリームミックス中の脂肪球は1.2μm以下が主であるが,フリージングすることにより脂肪球の凝集が進み,それらがフリーザー出口温度の低下と共に成長し保型性が良くなることが判明した.(2) オーバーランが高くなり気泡が多く取り込まれるに従って,脂肪球の凝集が進み,保型性も良くなることが判明した.(3) フリーザー出口温度の低下,およびオーバーランが高くなるにつれてアイスクリームのドライネスが増加した.さらに温度の低下,およびオーバーランが高くなる程この傾向が顕著になるのが認められた.(4) フリーザー出口温度の低下,およびオーバーランが高くなるにつれて脂肪球の凝集は進むが,脂肪球の凝集体の粒径も変化し,大きな粒径に移行した.脂肪球の粒度分布は1.2μm以下のものが減少し, 3～4μm, 8～15μmの脂肪球の特異的な増加が見られた.

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy. We report here that a novel fusicoccin derivative (ISIR-042), but not its parent or related compounds such as fusicoccin A and cotylenin A, is more cytotoxic to hypoxic cells than to normoxic cells. The hypoxia-induced accumulation of hypoxia-inducible factor (HIF)-1α and the phosphorylation of Akt were effectively inhibited by treatment with ISIR-042, suggesting that the preferential cytotoxicity toward hypoxic cells is associated with a reduction of HIF-1α and Akt activation. ISIR-042 inhibited the growth of human pancreatic cancer MIAPaCa-2 cells while sparing normal endothelial cells, and significantly inhibited the growth of MIAPaCa-2 cells as xenografts without apparent adverse effects. Pancreatic cancer cells expressing CD24 and CD44 exhibited characteristics of stem cells. Treatment with gemcitabine increased this stem cell-enriched population, and this effect was significantly inhibited by ISIR-042, suggesting that ISIR- 042 preferentially inhibits stem/progenitors in pancreatic cancer cell lines compared with chemotherapeutic agents. These results suggest that ISIR-042 may be a potential therapeutic agent for hypoxic tumors such as pancreatic cancer.

Previous studies have reported that the incidence of obstructive sleep apnea syndrome (OSAS) in patients with depression is higher than in the general population. We examined the risk factors to predict OSAS in mood disorder patients with depressive symptoms.We conducted polysomnography for patients who satisfied the following criteria: (i) diagnosis of major depressive disorder or bipolar disorder according to the Mini-International Neuropsychiatric Interview (MINI); (ii) a score ofor =10 on the Hamilton Rating Scale for Depression (HAM-D); (iii) fulfillment of either (a) or (b) below: (a) at least one of the following: severe snoring, witnessed apnea during sleep, excessive daytime sleepiness; (b) at least one of the following plus an oxygen desaturation index of 4%or =5 times/h by pulse oximeter: mild snoring, sleep disturbance, headache, high blood pressure. The patients with apnea hypopnea indexor =5 were diagnosed with OSAS.Of the 32 mood disorder patients who met the subject conditions, 59.4% had OSAS. The diagnosis rate with our criteria was significantly higher than the previously reported incidence of OSAS in patients with depression. There was no significant difference among diagnosis rates as to individual risk factors or the number of risk factors. A multiple regression test showed no significant association between apnea-hypopnea index and other clinical factors including depression severity.The present results showed that OSAS can be detected at a remarkably higher rate by considering appropriate OSAS risk factors in mood disorder patients, and suggested that there is a high rate of undetected and therefore untreated OSAS among mood disorder patients.

Introduction: Clinical features and genetic backgrounds of acute myeloid Leukemia (AML) are heterogenous and the prognosis is poor. Cooperating alterations of several genes including oncogenes or tumor suppressor genes lead to development of Leukemia. AML leukemogenesis is thought to require at least two different types of genetic change: class I mutations, which confer a proliferative or survival advantage; and class II mutations, which inhibit myeloid differentiation and apoptotic activity. In hematological malignancies with 11p15 translocations, the NUP98 gene is reportedly fused to various partner genes, often including homeobox genes, such as HOXA9, A11, A13, C11, C13, D11, D13 and PMX. We previously revealed that high frequencies of FLT3 internal tandem duplication (FLT3-ITD), NRAS mutations (NRASMT), and WT1 mutations (WT1MT) in myeloid malignancies with NUP98-HOX fusions were found and their concurrent mutations with NUP98-HOX fusions were associated with clinical prognosis of the patients. However, biological outcome of the combination of NUP98-HOX fusions with FLT3-ITD, NRASMT or WT1MT remains to be investigated. To elucidate the mechanism resposible for the leukemogenesis, we examined the function of NUP98-HOX fusions and the concomitant somatic mutations in vitro. Methods: Full length of NUP98-HOXA9 (NHA9), FLT3-ITD (51 bp-duplication), NRASMT (G13V), and WT1MT (Arg250trp) was cloned into a plasmid that has a mammalian replication initiation region and a nuclear matrix attachment region, which possesses great ability to persist gene amplification for long periods. Each plasmid or the combination of the two plasmids (NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT) were transfected into IL3-dependent mouse myeloid 32D cell line. Cell proliferation was determined in medium without IL3. Cellular differentiation was examined in the medium containing G-CSF using May-Giemsa staining and fluorescence-activated cell sorting (FACS). Apoptosis was quantitated using FACS. Colony forming assay was performed in methyl cellulose medium with stem cell factor and IL3. Moreover, autophagy induced by tamoxifen was checked. Results: Compared to the empty vector, IL3-independent cellular growth was significantly increased in the cells transfected with NHA9 and FLT3-ITD alone, but not in NRASMT and WT1MT alone. In contrast, co-transfection of NRASMT or WT1MT with NHA9 significantly increased the IL3 independent proliferation of 32D cells. Similarly, cells transfected with NHA9/FLT3-ITD showed the enhanced IL3-independent proliferation. CD11b and Gr1, of which represent myeloid differentiation marker, decreased in all the transfected cells except for NRASMT. While transfection with FLT3-ITD, NRASMT and WT1MT increased the morphological nuclear segmentation of the cells, NHA9/FLT3-ITD, NHA9/NRASMT, and NHA9/WT1MT abrogated the nuclear segmentation. Both Annexin V positive/propidium iodide negative cells and active caspase 3-positive cells declined in all transfected cells. Transfection of NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT tended to increase the number of colony forming unit compared to the single gene-transfected cells. After treatment with tamoxifen, monodansylcadaverine, which was used to reveal autophagic vacuoles, was incorporated into empty vector-transfected cells, not into all transfected cells. Conclusions: NHA9 acquired proliferation capacity, blockage of differentiation, and anti-apoptosis activity, FLT3-ITD did proliferation capacity and anti-apoptosis activity, NRASMT and WT1MT did anti-apoptosis activity. By contrast, NHA9 with FLT3-ITD, NRASMT, or WT1MT conferred growth advantage, inhibited differentiation and apoptosis, augmented self-renewality. In vivo study demonstrated that NHA9 or each genetic mutation (FLT3-ITD and NRASMT) alone was insufficient to achieve myeloid leukemia. These suggested that leukemogenesis of NUP98-HOX fusion genes with simultaneous gene mutations might be induced by not only harmonization between class I and class II mutations but also self-renewality and anti-autophagic cell death. Disclosures No relevant conflicts of interest to declare.