Prevalence of osteoporosis and osteopenia assessed by densitometry in Japanese puerperal women.

AIM: The aim is to examine the prevalence of low bone mineral density (BMD) (osteoporosis and osteopenia) of lumbar and femoral bones in puerperal women for the prenatal and postnatal care to ensure their optimal bone health. METHODS: We analyzed the first scan data of 1079 Japanese puerperal women without bone fracture (mean age 33.5 ± 4.5 years) who had undergone deliveries at Niigata City General Hospital for 10 years. We measured BMDs of the lumbar spine [LS], femoral neck [FN], and total hip [TH] with dual-energy X-ray absorptiometry (DXA) within 30 days after delivery. RESULTS: The rates of osteoporosis of LS, FN, TH, and comprehensive diagnosis (CD) were 0.6%, 4.8%, 1.5%, and 5.4%, respectively, and osteopenia were 20.2%, 39.5%, 29.3%, and 44.3%, respectively. The multivariable-adjusted odds ratio (OR) for body mass index (BMI) before pregnancy reflected significant decreases in risk for low BMD with an OR of 0.78 (95% CI: 0.74-0.82) with the 1 kg/m2 increases. In lean women (BMI < 18.5), 71% of them had low BMD. CONCLUSIONS: Approximately 50% of puerperal women had low BMD without bone fracture. BMD measurements at puerperium, especially for lean women, may be very useful for identifying at risk of osteoporosis in future.

Taurine supplementation for prevention of stroke-like episodes in MELAS: a multicentre, open-label, 52-week phase III trial.

OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of high-dose taurine supplementation for prevention of stroke-like episodes of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), a rare genetic disorder caused by point mutations in the mitochondrial DNA that lead to a taurine modification defect at the first anticodon nucleotide of mitochondrial tRNALeu(UUR), resulting in failure to decode codons accurately. METHODS: After the nationwide survey of MELAS, we conducted a multicentre, open-label, phase III trial in which 10 patients with recurrent stroke-like episodes received high-dose taurine (9 g or 12 g per day) for 52 weeks. The primary endpoint was the complete prevention of stroke-like episodes during the evaluation period. The taurine modification rate of mitochondrial tRNALeu(UUR) was measured before and after the trial. RESULTS: The proportion of patients who reached the primary endpoint (100% responder rate) was 60% (95% CI 26.2% to 87.8%). The 50% responder rate, that is, the number of patients achieving a 50% or greater reduction in frequency of stroke-like episodes, was 80% (95% CI 44.4% to 97.5%). Taurine reduced the annual relapse rate of stroke-like episodes from 2.22 to 0.72 (P=0.001). Five patients showed a significant increase in the taurine modification of mitochondrial tRNALeu(UUR) from peripheral blood leukocytes (P<0.05). No severe adverse events were associated with taurine. CONCLUSIONS: The current study demonstrates that oral taurine supplementation can effectively reduce the recurrence of stroke-like episodes and increase taurine modification in mitochondrial tRNALeu(UUR) in MELAS. TRIAL REGISTRATION NUMBER: UMIN000011908.

Molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction.

Molecular hydrogen (H2) was believed to be an inert and nonfunctional molecule in mammalian cells; however, we overturned the concept by reporting the therapeutic effects of H2 against oxidative stress. Subsequently, extensive studies revealed multiple functions of H2 by exhibiting the efficacies of H2 in various animal models and clinical studies. Here, we investigated the effect of H2 on free-radical-induced cytotoxicity using tert-butyl hydroperoxide in a human acute monocytic leukemia cell line, THP-1. Cell membrane permeability was determined using lactate dehydrogenase release assay and Hoechst 33342 and propidium iodide staining. Fatty acid peroxidation and mitochondrial viability were measured using 2 kinds of fluorescent dyes, Liperfluo and C11-BODIPY, and using the alamarBlue assay based on the reduction of resazurin to resorufin by mainly mitochondrial succinate dehydrogenase, respectively. Mitochondrial membrane potential was evaluated using tetramethylrhodamine methyl ester. As a result, H2 protected the cultured cells against the cytotoxic effects induced by tert-butyl hydroperoxide; H2 suppressed cellular fatty acid peroxidation and cell membrane permeability, mitigated the decline in mitochondrial oxidoreductase activity and mitochondrial membrane potential, and protected cells against cell death evaluated using propidium iodide staining. These results suggested that H2 suppresses free-radical-induced cell death through protection against fatty acid peroxidation and mitochondrial dysfunction.

Hydrogen inhalation during normoxic resuscitation improves neurological outcome in a rat model of cardiac arrest independently of targeted temperature management.

BACKGROUND: We have previously shown that hydrogen (H2) inhalation, begun at the start of hyperoxic cardiopulmonary resuscitation, significantly improves brain and cardiac function in a rat model of cardiac arrest. Here, we examine the effectiveness of this therapeutic approach when H2 inhalation is begun on the return of spontaneous circulation (ROSC) under normoxic conditions, either alone or in combination with targeted temperature management (TTM). METHODS AND RESULTS: Rats were subjected to 6 minutes of ventricular fibrillation cardiac arrest followed by cardiopulmonary resuscitation. Five minutes after achieving ROSC, post-cardiac arrest rats were randomized into 4 groups: mechanically ventilated with 26% O2 and normothermia (control); mechanically ventilated with 26% O2, 1.3% H2, and normothermia (H2); mechanically ventilated with 26% O2 and TTM (TTM); and mechanically ventilated with 26% O2, 1.3% H2, and TTM (TTM+H2). Animal survival rate at 7 days after ROSC was 38.4% in the control group, 71.4% in the H2 and TTM groups, and 85.7% in the TTM+H2 group. Combined therapy of TTM and H2 inhalation was superior to TTM alone in terms of neurological deficit scores at 24, 48, and 72 hours after ROSC, and motor activity at 7 days after ROSC. Neuronal degeneration and microglial activation in a vulnerable brain region was suppressed by both TTM alone and H2 inhalation alone, with the combined therapy of TTM and H2 inhalation being most effective. CONCLUSIONS: H2 inhalation was beneficial when begun after ROSC, even when delivered in the absence of hyperoxia. Combined TTM and H2 inhalation was more effective than TTM alone.

Protective effect of molecular hydrogen against oxidative stress caused by peroxynitrite derived from nitric oxide in rat retina.

BACKGROUND: Oxidative and nitrative processes have an important role in the pathogenesis of glaucomatous neurodegeneration. Oxidative stress occurs when cellular production of reactive oxygen species outweighs the protective capacity of antioxidant defences. Reactive oxygen species are generated as by-products of cellular metabolism, primarily in the mitochondria. Herein, we present a novel investigation of the effects of molecular hydrogen (H2 ) on retinal cells exposed to oxidative stress. METHODS: We cultured adult rat retinal tissues in an organotypic culture system with a nitric oxide donor, S-nitroso-N-acetylpenicillamine, in the presence or absence of H2 . Loss of mitochondrial membrane potential and apoptosis of retinal cells were analysed using a MitoTMRE detection kit and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. Tyrosine nitration levels and oxidative stress damage in the retina were evaluated using immunohistochemical staining. Retinal damage was quantified by measuring the numbers of cells in the ganglion cell and inner nuclear layers and the thickness of the retina. RESULTS: H2 suppressed loss of mitochondrial membrane potential and apoptosis in retinal cells. Moreover, H2 decreased the tyrosine nitration level and suppressed oxidative stress damage in retinal cells. S-nitroso-N-acetylpenicillamine treatment decreased the cell numbers in the ganglion cell layer and inner nuclear layer, but the presence of H2 inhibited this reduction. These findings suggest that H2 has a neuroprotective effect against retinal cell oxidative damage, presumably by scavenging peroxynitrite. CONCLUSIONS: H2 reduces cellular peroxynitrite, a highly toxic reactive nitrogen species. Thus, H2 may be an effective and novel clinical tool for treating glaucoma and other oxidative stress-related diseases.

Successful management of severe intrahepatic cholestasis of pregnancy: report of a first Japanese case.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a cholestasis condition caused by elevated levels of serum bile acids that mainly occurs in the third trimester of pregnancy. Maternal symptoms include pruritus; elevation of transaminases, biliary enzymes, and bilirubin levels; and abnormal liver function tests. Fetal symptoms include spontaneous preterm labor, fetal distress, and intrauterine death. It is more prevalent in the Caucasians and is rarely found in Asian countries, including Japan. The etiology of ICP has been reported as involving various factors such as, environmental factors, hormone balance, and genetic components. The genetic factors include single-nucleotide polymorphisms (SNPs) in the genes of canalicular transporters, including ABCB4 and ABCB11. It has also been reported that the combination of these SNPs induces severe cholestasis and liver dysfunction. CASE PRESENTATION: Here, we report for the first time a 24-year Japanese case of severe ICP diagnosed by typical symptoms, serum biochemical analysis, and treated with the administration of ursodeoxycholic acid which improved cholestasis and liver injury and prevented fetal death. The sequence analysis showed SNPs reported their association with ICP in the ABCB11 (rs2287622, V444A) and ABCB4 (rs1202283, N168N) loci. CONCLUSION: The risk of ICP has been reported to be population-specific, and it is rare in the Japanese population. Our case was successfully treated with ursodeoxycholic acid and the genetic sequence analysis has supported the diagnosis. Because genetic variation in ABCB4 and ABCB11 has also been reported in the Japanese population, we need to be aware of potential ICP cases in pregnant Japanese women although further studies are necessary.

Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

Caloric restriction primes mitochondria for ischemic stress by deacetylating specific mitochondrial proteins of the electron transport chain.

RATIONALE: Caloric restriction (CR) confers cardioprotection against ischemia/reperfusion injury. However, the exact mechanism(s) underlying CR-induced cardioprotection remain(s) unknown. Recent evidence indicates that Sirtuins, NAD(+)-dependent deacetylases, regulate various favorable aspects of the CR response. Thus, we hypothesized that deacetylation of specific mitochondrial proteins during CR preserves mitochondrial function and attenuates production of reactive oxygen species during ischemia/reperfusion. OBJECTIVE: The objectives of the present study were (1) to investigate the effect of CR on mitochondrial function and mitochondrial proteome and (2) to investigate what molecular mechanisms mediate CR-induced cardioprotection. METHODS AND RESULTS: Male 26-week-old Fischer344 rats were randomly divided into ad libitum-fed and CR (40% reduction) groups for 6 months. No change was observed in basal mitochondrial function, but CR preserved postischemic mitochondrial respiration and attenuated postischemic mitochondrial H(2)O(2) production. CR decreased the level of acetylated mitochondrial proteins that were associated with enhanced Sirtuin activity in the mitochondrial fraction. We confirmed a significant decrease in the acetylated forms of NDUFS1 and cytochrome bc1 complex Rieske subunit in the CR heart. Low-dose resveratrol treatment mimicked the effect of CR on deacetylating them and attenuated reactive oxygen species production during anoxia/reoxygenation in cultured cardiomyocytes without changing the expression levels of manganese superoxide dismutase. Treatment with nicotinamide completely abrogated the effect of low-dose resveratrol. CONCLUSIONS: These results strongly suggest that CR primes mitochondria for stress resistance by deacetylating specific mitochondrial proteins of the electron transport chain. Targeted deacetylation of NDUFS1 and/or Rieske subunit might have potential as a novel therapeutic approach for cardioprotection against ischemia/reperfusion.

Transgenic type2 diabetes mouse models for in vivo redox measurement of hepatic mitochondrial oxidative stress.

BACKGROUND: Oxidative stress is involved in the progression of diabetes and its associated complications. However, it is unclear whether increased oxidative stress plays a primary role in the onset of diabetes or is a secondary indicator caused by tissue damage. Previous methods of analyzing oxidative stress have involved measuring the changes in oxidative stress biomarkers. Our aim is to identify a novel approach to clarify whether oxidative stress plays a primary role in the onset of diabetes. METHODS: We constructed transgenic type 2 diabetes mouse models expressing redox-sensitive green fluorescent proteins (roGFPs) that distinguished between mitochondria and whole cells. Pancreas, liver, skeletal muscle, and kidney redox states were measured in vivo. RESULTS: Hepatic mitochondrial oxidation increased when the mice were 4 weeks old and continued to increase in an age-dependent manner. The increase in hepatic mitochondrial oxidation occurred simultaneously with weight gain and increased blood insulin levels before the blood glucose levels increased. Administering the oxidative stress inducer acetaminophen increased the vulnerability of the liver mitochondria to oxidative stress. CONCLUSIONS: This study demonstrates that oxidative stress in liver mitochondria in mice begins at the onset of diabetes rather than after the disease has progressed. GENERAL SIGNIFICANCE: RoGFP-expressing transgenic type 2 diabetes mouse models are effective and convenient tools for measuring hepatic mitochondrial redox statuses in vivo. These models may be used to assess mitochondria-targeting antioxidants and establish the role of oxidative stress in type 2 diabetes.

Overexpression of KLF15 transcription factor in adipocytes of mice results in down-regulation of SCD1 protein expression in adipocytes and consequent enhancement of glucose-induced insulin secretion.

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic ß cells mediated through modulation of oxidative stress.

Multiple paraovarian cysts requiring emergency surgery: A rare clinical finding.

This report presents an unusual case of multiple paraovarian cysts that required emergency surgery due to a paraovarian cyst being entrapped by another paraovarian cyst. Laparoscopic surgery is considered useful for diagnostic and therapeutic purposes and is, therefore, recommended owing to difficulty in differentiating paraovarian cysts from ovarian cysts.

Laparoscopic surgery for a hydrocele of the canal of Nuck with an ovarian tumor: An extremely rare clinical finding.

This clinical image presents an unusual report of simultaneous laparoscopic resection of a hydrocele of the canal of Nuck and an ovarian tumor. Laparoscopic treatment with a proper approach is a useful technique in some cases.

Acute psychiatric symptoms in a young woman with anti-N-methyl D-aspartate receptor encephalitis: A case of successful early diagnosis and therapeutic intervention.

This clinical image presents a report on the diagnosis and treatment of anti-NMDAR encephalitis, a rare disease. This report emphasizes the importance of a differential diagnosis for acute psychiatric symptoms. Accurate and timely diagnosis is critical for the selection and implementation of treatment and for optimal patient outcomes.

Hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress.

Molecular hydrogen (H(2)) is an efficient antioxidant that diffuses rapidly across cell membranes, reduces reactive oxygen species (ROS), such as hydroxyl radicals and peroxynitrite, and suppresses oxidative stress-induced injury in several organs. ROS have been implicated in radiation-induced damage to lungs. Because prompt elimination of irradiation-induced ROS should protect lung tissue from damaging effects of irradiation, we investigated the possibility that H(2) could serve as a radioprotector in the lung. Cells of the human lung epithelial cell line A549 received 10 Gy irradiation with or without H(2) treatment via H(2)-rich PBS or medium. We studied the possible radioprotective effects of H(2) by analyzing ROS and cell damage. Also, C57BL/6J female mice received 15 Gy irradiation to the thorax. Treatment groups inhaled 3% H(2) gas and drank H(2)-enriched water. We evaluated acute and late-irradiation lung damage after H(2) treatment. H(2) reduced the amount of irradiation-induced ROS in A549 cells, as shown by electron spin resonance and fluorescent indicator signals. H(2) also reduced cell damage, measured as levels of oxidative stress and apoptotic markers, and improved cell viability. Within 1 wk after whole thorax irradiation, immunohistochemistry and immunoblotting showed that H(2) treatment reduced oxidative stress and apoptosis, measures of acute damage, in the lungs of mice. At 5 mo after irradiation, chest computed tomography, Ashcroft scores, and type III collagen deposition demonstrated that H(2) treatment reduced lung fibrosis (late damage). This study thus demonstrated that H(2) treatment is valuable for protection against irradiation lung damage with no known toxicity.

Development of Cancer Immunotherapy Targeting the PD-1 Pathway.

Immune checkpoint inhibitors are causing a paradigm shift in cancer treatment. Immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) dampen T cell activation to avoid autoimmunity and the destructive effects of an excessive inflammatory response. Immune checkpoint signaling can be exploited by tumors to escape host immune surveillance, and immune checkpoint inhibitors enhance antitumor immunity by releasing the brakes on the immune system. PD-1 was identified in 1992 by Honjo and colleagues at Kyoto University. Studies in animal models revealed that PD-1 blockade can inhibit tumorigenesis and tumor metastasis. In addition, PD-1 blockade showed fewer adverse effects than CTLA-4 blockade. Based on these findings, a humanized monoclonal antibody against human PD-1 called nivolumab was developed. Since PD-1 blockade targets lymphocytes rather than tumor cells, the therapeutic effects last longer, even if mutations occur during tumorigenesis. Furthermore, because it does not depend on specific tumor antigens, PD-1 blockade can be applied to various kinds of tumors.

Rap1b Promotes Notch-Signal-Mediated Hematopoietic Stem Cell Development by Enhancing Integrin-Mediated Cell Adhesion.

Hematopoietic stem cells (HSCs) emerge from hemogenic endothelium (HE) within the ventral portion of the dorsal aorta during vertebrate development. In zebrafish, Notch signaling induces HE specification from posterior lateral plate mesoderm (PLPM) cells as they migrate over the ventral surface of the somite. During migration, PLPM cells make close contact with Notch-ligand-expressing somitic cells to acquire HE identity. Herein, we show in zebrafish that the small GTPase Rap1b regulates HSC development by potentiating Notch-mediated HE specification. PLPM cells migrate toward the midline along the somite boundary where fibronectin accumulates. Rap1b stimulates integrin ß1 to enhance PLPM cell adhesion to fibronectin localized at the somite boundary. Rap1b-induced integrin-ß1-mediated adhesion to fibronectin leads to the spreading of PLPM cells to facilitate their physical contact with the Notch-ligand-expressing somitic cells, thereby promoting Notch-mediated HE specification. Thus, we have revealed an unexpected role of Rap1-induced integrin-mediated cell adhesion in HSC development.

Prevention of chemotherapy-induced alopecia by the anti-death FNK protein.

Many anticancer drugs attack rapidly dividing cells, including not only malignant cells but also hair follicle cells, and induce alopecia. Chemotherapy-induced alopecia (CIA) is an emotionally distressing side effect of cancer chemotherapy. There is currently no useful preventive therapy for CIA. We have previously constructed anti-death rFNK protein from rat Bcl-x(L) by site-directed mutagenesis to strengthen cytoprotective activity. When fused to the protein transduction domain (PTD) of HIV/Tat, the fusion protein PTD (TAT)-rFNK successfully entered cells from the outside in vitro and in vivo to exhibit anti-death activity against apoptosis and necrosis. Here, we show that topical application of FNK protected against CIA in a newborn rat model. The protective activity against hair-loss was observed in 30-1000 nM TAT-rFNK administrative groups in a dose-dependent manner. Furthermore, a human version of FNK (hFNK) fused to other PTD peptides exhibited a protective ability. These results suggest that PTD-FNK possesses protective activity against CIA and is not restricted to a sequence of PTD peptides or species of FNK. Thus, PTD-FNK represents potential to develop a useful method for preventing CIA in cancer patients.

Effects of Molecular Hydrogen Assessed by an Animal Model and a Randomized Clinical Study on Mild Cognitive Impairment.

BACKGROUND: Oxidative stress is one of the causative factors in the pathogenesis of neurodegenerative diseases including mild cognitive impairment (MCI) and dementia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive antioxidant. OBJECTIVE: We assess the effects of drinking H2-water (water infused with H2) on oxidative stress model mice and subjects with MCI. METHODS: Transgenic mice expressing a dominant-negative form of aldehyde dehydrogenase 2 were used as a dementia model. The mice with enhanced oxidative stress were allowed to drink H2-water. For a randomized double-blind placebo-controlled clinical study, 73 subjects with MCI drank ~300 mL of H2-water (H2-group) or placebo water (control group) per day, and the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) scores were determined after 1 year. RESULTS: In mice, drinking H2-water decreased oxidative stress markers and suppressed the decline of memory impairment and neurodegeneration. Moreover, the mean lifespan in the H2-water group was longer than that of the control group. In MCI subjects, although there was no significant difference between the H2- and control groups in ADAS-cog score after 1 year, carriers of the apolipoprotein E4 (APOE4) genotype in the H2-group were improved significantly on total ADAS-cog score and word recall task score (one of the sub-scores in the ADAS-cog score). CONCLUSION: H2-water may have a potential for suppressing dementia in an oxidative stress model and in the APOE4 carriers with MCI.

Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184-16193 poly-cytosine tract), which confer resistance to cisplatin.

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 µg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184-16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.