A glucocorticoid-receptor agonist ameliorates bleomycin-induced alveolar simplification in newborn rats.

BACKGROUND: Glucocorticoids (GCs) are highly effective yet problematic agents against bronchopulmonary dysplasia (BPD). The dimeric trans-activation of GCs induces unfavorable effects, while monomeric trans-repression suppresses inflammation-related genes. Recently, non-steroidal-selective glucocorticoid-receptor agonists and modulators (SEGRAMs) with only the trans-repressive action have been designed. METHODS: Using a bleomycin (Bleo)-induced alveolar simplification newborn rat model (recapitulating arrested alveolarization during BPD), we evaluated the therapeutic effects of compound-A (CpdA), a SEGRAM. Sprague-Dawley rats were administered Bleo from postnatal day (PD) 0 to 10 and treated with dexamethasone (Dex) or CpdA from PD 0 to 13. The morphological changes and mRNA expression of inflammatory mediators, including interleukin (IL)-1ß, C-X-C motif chemokine ligand 1 (CXCL1), and C-C motif chemokine 2 (CCL2) were investigated. RESULTS: Similar to the effects of Dex, CpdA exerted protective effects on morphological derangements and inhibited macrophage infiltration and production of pro-inflammatory mediators in Bleo-treated animals. The effects of CpdA were probably mediated by GC receptor (GR)-dependent trans-repression, because unlike the Dex-treated group, anti-inflammatory genes specifically induced by GR-dependent trans-activation (such as "glucocorticoid-induced leucine zipper, GILZ") were not upregulated. CONCLUSIONS: CpdA improved lung inflammation, inhibited the arrest of alveolar maturation, and restored histological and biochemical changes in a Bleo-induced alveolar simplification model. IMPACT: SEGRAMs have attracted widespread attention because they are expected to not exhibit unfavorable effects of GCs. Compound A, one of the SEGRAMs, improved lung morphometric changes and decreased lung inflammation in a bleomycin-induced arrested alveolarization, a newborn rat model representing one of the main features of BPD pathology. Compound A did not elicit bleomycin-induced poor weight gain, in contrast to dexamethasone treatment. SEGRAMs, including compound A, may be promising candidates for the therapy of BPD with less adverse effects compared with GCs.

Long-term alpha-tocopherol supplements may improve mental development in extremely low birthweight infants.

AIM: Methods to improve the mental development of extremely low birthweight (ELBW) children are currently lacking. We assessed the effects of long-term supplementation of alpha-tocopherol on the neurological development of 259 school-aged ELBW children. METHODS: Extremely low birthweight participants were divided into three groups: group A with no alpha-tocopherol supplementation (n = 121); group B with the supplementation for <6 months (n = 104) and group C with the supplementation for more than 6 months (n = 34). We analysed the participants' data at birth and between the ages of one-and-a-half to 8 years and evaluated potential factors associated with intellectual disabilities. RESULTS: Children from group C had the best outcome. The groups' mean gestational weeks and mean ventilator days were as follows: 27.5 weeks, 16.1 days (group A); 25.7 weeks, 41.7 days (group B); and 25.1 weeks, 75.5 days (group C). Multivariate regression analysis revealed that the odds ratios for impaired mental development at 8 years were 1.5 in group B and 0.19 (p = 0.017) in group C, compared with 1.0 in group A. The association between the duration of alpha-tocopherol administration and performance intelligence quotient (IQ) was dose dependent (p = 0.03). CONCLUSION: Long-term supplementation of alpha-tocopherol appeared to improve mental development, in particular, performance IQ, in school-aged ELBW children.

Antenatal exposure to Ureaplasma species exacerbates bronchopulmonary dysplasia synergistically with subsequent prolonged mechanical ventilation in preterm infants.

INTRODUCTION: The presence of microorganisms in gastric fluid in neonates at birth is postulated to reflect antenatal infection and also to be associated with the development of bronchopulmonary dysplasia (BPD). RESULTS: A logistic regression analysis, after controlling for other risk factors, indicated that Ureaplasma-positive infants were not at increased risk for moderate/severe BPD (adjusted odds ratio (OR): 2.58, 95% confidence interval (CI): 0.57-6.89, P = 0.12). However, the association between the presence of Ureaplasma species and the risk for moderate/severe BPD increased significantly in infants on mechanical ventilation (MV) ≥2 wk (adjusted OR: 4.17, 95% CI: 1.62-44.1, P = 0.009). An analysis using a lung injury marker indicated that Ureaplasma-positive infants with MV ≥2 wk, but not other infants, showed higher serum KL-6 levels in samples taken from cord blood, and that KL-6 levels increased time-dependently up to 4 wk of age. DISCUSSION: Antenatal exposure to Ureaplasma species induces lung injury prior to birth and synergistically contributes to the development of BPD in infants requiring prolonged MV (≥2 wk). METHODS: We recovered gastric fluid specimens from 122 infants with gestational age (GA) <29 wk or birth weight <1,000 g to investigate whether these microorganisms influence respiratory outcome of BPD. A PCR analysis was used to detect urease and 16S ribosomal RNA (rRNA) genes to classify neonates into Ureaplasma-positive or Ureaplasma-negative infants.

Vitamin E and preterm infants.

In evaluating vitamin E (VE) nutritional status of preterm infants, it is essential that any data should be compared with those of healthy term infants, and never with those of adults. Moreover, it should be evaluated in terms of gestational age (GA), not birth weight (BW), because placental transfer of most nutrients from mother to fetus is dependent on GA, not BW. Judging from the limited data during the last 75 years, there was no significant correlation between GA and VE concentrations in circulation or in the red blood cells (RBCs), leukocytes, and buccal mucosal cells. In addition, the oxidizability of polyunsaturated fatty acids (PUFAs) in plasma or RBCs, as targets for protection by VE chain-breaking ability, was lower in preterm infants. However, because of the minimal information available about hepatic VE levels, which is considered a key determinant of whole body VE status, the decision on whether VE status of preterm infants is comparable with that of term infants should be postponed. Clinical trials of VE supplementation in preterm infants were repeatedly undertaken to investigate whether VE reduces severity or inhibits development of several diseases specific to preterm infants, namely retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), and germinal matrix hemorrhage - intraventricular hemorrhage (GMH-IVH). Most of these trials resulted in a misfire, with a few exceptions for IVH prevention. However, almost all these studies were performed from 1980s to early 1990s, in the pre-surfactant era, and the study populations were composed of mid-preterm infants with GAs of approximately 30 weeks (wks). There is considerable difference in 'preterm infants' between the pre- and post-surfactant eras; modern neonatal medicine mainly treats preterm infants of 28 wks GA or less. Therefore, these results are difficult to apply in modern neonatal care. Before considering new trials of VE supplementation, we should fully understand modern neonatal medicine, especially the recent method of oxygen supplementation. Additionally, a deeper understanding of recent progress in pathophysiology and therapies for possible target diseases is necessary to decide whether VE administration is still worth re-challenging in modern neonatal intensive care units (NICUs). In this review, we present recent concepts and therapeutic trends in ROP, BPD, and GMH-IVH for those unfamiliar with neonatal medicine. Numerous studies have reported the possible involvement of reactive oxygen species (ROS)-induced damage in relation to supplemental oxygen use, inflammation, and immature antioxidant defense in the development of both BPD and ROP. Various antioxidants effectively prevented the exacerbation of BPD and ROP in animal models. In the future, VE should be re-attempted as a complementary factor in combination with various therapies for BPD, ROP, and GMH-IVH. Because VE is a natural and safe supplement, we are certain that it will attract attention again in preterm medicine.

Efficacy of a mitochondrion-targeting agent for reducing the level of urinary protein in rats with puromycin aminonucleoside-induced minimal-change nephrotic syndrome.

BACKGROUND: Oxidative stress is a major factor responsible for minimal-change nephrotic syndrome (MCNS), which occurs most commonly in children. However, the influence of oxidative stress localized to mitochondria remains unclear. We examined the effect of a mitochondrion-targeting antioxidant, MitoTEMPO, in rats with puromycin aminonucleoside (PAN)-induced MCNS to clarify the degree to which mitochondrial oxidative stress affects MCNS. MATERIALS AND METHODS: Thirty Wistar rats were divided into three groups: normal saline group (n = 7), PAN group (n = 12), and PAN + MitoTEMPO group (n = 11). Rats in the PAN and PAN + MitoTEMPO groups received PAN on day 1, and those in the PAN + MitoTEMPO group received MitoTEMPO on days 0 to 9. Whole-day urine samples were collected on days 3 and 9, and samples of glomeruli and blood were taken for measurement of lipid peroxidation products. We also estimated the mitochondrial damage score in podocytes in all 3 groups using electron microscopy. RESULTS: Urinary protein excretion on day 9 and the levels of lipid peroxidation products in urine, glomeruli, and blood were significantly lower in the PAN　+　MitoTEMPO group than in the PAN group (p = 0.0019, p = 0.011, p = 0.039, p = 0.030). The mitochondrial damage score in podocytes was significantly lower in the PAN　+　MitoTEMPO group than in the PAN group (p <0.0001). CONCLUSIONS: This mitochondrion-targeting agent was shown to reduce oxidative stress and mitochondrial damage in a MCNS model. A radical scavenger targeting mitochondria could be a promising drug for treatment of MCNS.

Edaravone prevents kainic acid-induced neuronal death.

There is growing evidence that free radical generation may play a key role in the neuronal damage induced by prolonged convulsions. Free radical scavengers are known to inhibit neuronal death induced by exposure to excitotoxins. However, this neuroprotective effect has not been demonstrated with treatment after seizures had been stopped. We investigated whether 3-methyl-1-phenyl-2-pyrazolin-5-one, edaravone (Ed), a newly developed free radical scavenger that has been used clinically to treat cerebral infarction, could prevent neuronal loss when administered after the occurrence of seizures in a kainic acid (KA)-induced seizure model. Compared with KA alone, cell loss was significantly reduced when animals received Ed (10 mg/kg i.v.) just after seizures, and when Ed was administered both 60 min before (30 mg/kg i.p.) and after KA injection. Combined before-and-after treatment with Ed significantly ameliorated the KA-induced decrease of glutathione and blocked the KA-induced increase of 4-hydroxy-2-nonenal (HNE). Because before-and-after treatment with Ed significantly lessened the KA-induced increase of HNE, Ed may exert its neuroprotective effect by inhibiting lipid peroxidation. However, post-treatment with Ed prevented neuronal cell loss, while HNE and glutathione levels did not differ from those in animals without Ed, so a mechanism other than free radical scavenging must be involved in the prevention of cell loss. Patients who develop status epilepticus are unlikely to receive adequate antioxidant therapy before the onset, so it is an advantage that Ed can prevent neuronal death even when administered after seizures.

A Case of Fundus Oculi Albinoticus Diagnosed as Angelman Syndrome by Genetic Testing.

PURPOSE: To report a case of fundus oculi albinoticus diagnosed as Angelman syndrome (AS) via genetic testing. CASE REPORT: This study reports on a 4-year-old boy. Since he had been having respiratory disturbance since birth, he underwent a complete physical examination to investigate the cause. The results indicated that he had various brain congenital abnormalities, such as a thin corpus callosum, as well as hydronephrosis, an atrial septal defect, and skin similar to patients with fundus oculi albinoticus. Examination revealed bilateral fundus oculi albinoticus, mild iridic hypopigmentation, optic atrophy, and poor visual tracking. Genetic testing revealed a deletion in the Prader-Willi syndrome/AS region on chromosome 15, and together with the results of methylation analysis, his condition was diagnosed as AS. Follow-up examinations revealed no change in the fundus oculi albinoticus and optic atrophy, nor did they indicate poor visual tracking. CONCLUSIONS: When fundus oculi albinoticus and optic atrophy are observed in patients with multiple malformations, AS should be considered as a differential diagnosis.

A Case of Hydranencephaly in Which Ophthalmic Examinations Were Performed.

PURPOSE: We performed ophthalmic examinations, including optical coherence tomography (OCT), on a case diagnosed with hydranencephaly. CASE REPORT: This case involved a female infant born at the gestational age of 35 weeks and 4 days, with the birth weight of 2,152 g, who was one of monochorionic diamniotic twins, and the identical twin died in utero at the gestational age of 24 weeks. After that, examination by fetal echo indicated that she had microcephaly and ventriculomegaly. Postnatal magnetic resonance imaging (MRI) of her head indicated microcephaly and significant enlargement of the lateral ventricle on both sides, with no obvious signs of elevated intracranial pressure. The brain parenchyma of both sides of the frontal lobe, parietal lobe, and occipital lobe had marked thinning, yet that of the temporal lobe, basal ganglia, thalamus, brain stem, and cerebellum had been maintained. Moreover, no obvious hematoma or neoplastic lesions were observed. Ophthalmic examinations indicated that both of her eyes had slight light reflex, attributed to optic nerve atrophy. Examination by use of a hand-held OCT system indicated a layered structure of the retina and thinning of the ganglion cell layer. Flicker electroretinogram (ERG) examination by use of a hand-held ERG system indicated an almost normal wave. However, no clear visual reaction was observed when she was 10 months old. CONCLUSION: Our findings in this case of hydranencephaly revealed that even though the outer layer functions of the patient's retina were maintained, extensive damage to her cerebral cortex resulted in poor visual function.

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid.

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.

Identification of apo- and holo-forms of ceruloplasmin in patients with Wilson's disease using native polyacrylamide gel electrophoresis.

OBJECTIVES: To determine the serum ratio of holo-ceruloplasmin to total ceruloplasmin in patients with Wilson's disease (WD), we identified apo- and holo-forms of serum ceruloplasmin with native-PAGE electrophoresis. DESIGN AND METHODS: Serum obtained from nine patients with Wilson's disease was subjected to native-PAGE analysis. We also determined the ceruloplasmin level and ferroxidase activity in all of the samples. RESULTS: Among the nine patients, three had both forms of ceruloplasmin and six had only apo-ceruloplasmin. In the patients who had only apo-ceruloplasmin, the serum ceruloplasmin level was significantly lower than in the patients with both forms. CONCLUSIONS: Differences in the ability to incorporate copper into ceruloplasmin may have a role in ceruloplasmin concentrations in patients with Wilson's disease.

alpha-Tocopherol transfer protein expression in rat liver exposed to hyperoxia.

alpha-Tochopherol transfer protein (alpha TTP), a 32 kDa protein exclusively expressed in liver cytosol, has a high binding affinity for alpha-tochopherol. The factors that regulate the expression of hepatic alpha TTP are not clearly understood. In this study, we investigated whether or not exposure to hyperoxia (> 95% O2 for 48 h) could alter the expression of hepatic alpha TTP. We also examined the association between the expression of antioxidant enzymes (hepatic glutathione peroxidase (GPX) and Mn-superoxide dismutase (Mn-SOD)) and the expression of hepatic alpha TTP. The levels of thiobarbituric acid-reactive substances (TBARS) in both plasma and liver were significantly higher after rats were exposed to hyperoxia for 48 h when compared with the levels in control rats. Northern blotting showed a decrease in the expression of alpha TTP messenger RNA (mRNA) after hyperoxia, although the alpha TTP protein level remained constant. Expression of Mn-SOD mRNA and protein, as well as the expression of GPX mRNA, were stable after hyperoxia. These findings indicate that mRNA for hepatic alpha TTP, rather than Mn-SOD or GPX, may be highly responsive to oxidative stress.

Alpha-tocopherol content and alpha-tocopherol transfer protein expression in leukocytes of children with acute leukemia.

Alpha-tocopherol (a form of vitamin E) is a fat-soluble vitamin that can prevent lipid peroxidation of cell membranes. This antioxidant activity of alpha-tocopherol can help to prevent cardiovascular disease, atherosclerosis and cancer. We investigated the alpha-tocopherol level and the expression of alpha-tocopherol transfer protein (alpha-TTP) in the leukocytes of children with leukemia. The plasma and erythrocyte alpha-tocopherol levels did not differ between children with leukemia and the control group. However, lymphocytes from children with leukemia had significantly lower alpha-tocopherol levels than lymphocytes from the controls (58.4 +/- 39.0 ng/mg protein versus 188.9 +/- 133.6, respectively; p < 0.05), despite the higher plasma alpha-tocopherol/cholesterol ratio in the leukemia group (5.83 +/- 1.64 micromol/mmol versus 4.34 +/- 0.96, respectively; p < 0.05). No significant differences in the plasma and leukocyte levels of isoprostanes (the oxidative metabolites of arachidonic acid) were seen between the leukemia patients and controls. The plasma level of acrolein, a marker of oxidative stress, was also similar in the two groups. Investigation of alpha-TTP expression by leukocytes using real-time PCR showed no difference between the two groups. These findings suggest that there may be comparable levels of lipid peroxidation in children with untreated leukemia and controls, despite the reduced alpha-tocopherol level in leukemic leukocytes.

Two cases in which an abnormal hemoglobin (Hb Kamakura) was identified on the basis of an abnormally low glycohemoglobin levels.

We encountered two patients with abnormally low glycohemoglobin levels in spite of normal plasma glucose levels. Since the presence of an abnormal hemoglobin was suggested by high-performance liquid chromatography (HPLC), gene analysis of these two patients was conducted by single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing, and Hb Kamakura with one base substitution (beta3 Leu --> Val) in the beta globin gene was detected in both patients. There was been only one report of Hb Kamakura in Kanagawa Prefecture, and our patients represent the second and third cases, respectively.

Meconium aspiration delays normal decline of pulmonary vascular resistance shortly after birth through lung parenchymal injury.

BACKGROUND: Persistent pulmonary hypertension of the newborn is often associated with meconium aspiration syndrome (MAS) or perinatal asphyxia. OBJECTIVE: To determine the effect of meconium or asphyxia on pulmonary arterial pressure and circulating levels of vasoactive substances, we conducted a prospective study of 54 term infants, including infants with meconium-stained amniotic fluid with normal (MSAF) or abnormal (MAS) chest X-ray findings, infants with perinatal asphyxia, and controls. The purpose of this study was to determine the group most likely to have elevated pulmonary arterial pressure and a disturbed balance between vasoactive substances. METHODS: To estimate the pulmonary arterial pressure by echocardiography, we used the ratio of the right to left systolic ventricular pressure (RVP/LVP ratio). We measured the plasma concentrations of endothelin-1 (ET-1), cyclic guanosine monophosphate (cGMP) as an indicator of nitric oxide (NO) production, and 6-keto-prostaglandin F(1)α (6-keto-PGF(1)α) for the estimation of prostacyclin concentration. We also measured KL-6 as a marker of lung injury. RESULTS: The RVP/LVP ratio was significantly higher in the MAS group than the other groups on day 0. Although ET-1 and 6-keto-PGF(1)α levels were comparable among all groups, the cGMP level on days 3-5 and the KL-6 level throughout the first postnatal week were significantly higher in the MAS group. CONCLUSIONS: It is possible that meconium aspiration delays normal decline of pulmonary vascular resistance shortly after birth through lung parenchymal injury. The subsequent increase of cGMP in MAS may be an adaptive response to prevent further elevation of pulmonary arterial pressure by inducing NO.

Neonatal streptococcal toxic shock syndrome caused by Streptococcus dysgalactiae subsp. equisimilis.

We report a neonatal infection with Streptococcus dysgalactiae subsp. equisimilis occurring through maternal transmission and presenting as streptococcal toxic shock syndrome 12 hours after birth. Pediatricians and obstetricians should be aware of the possibility of this infectious disease when examining newborns with fever. These observations suggest that antenatal maternal screening for S. dysgalactiae subsp. equisimilis should be considered.

Hypothermic inhibition of apoptotic pathways for combined neurotoxicity of iron and ascorbic acid in differentiated PC12 cells: reduction of oxidative stress and maintenance of the glutathione redox state.

Recent clinical trials have demonstrated the efficacy and safety of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy (HIE). We previously reported that the levels of non-protein-bound iron and ascorbic acid (AA) are increased in the CSF of infants with HIE. In this study, we investigated the effect of hypothermia on the combined cytotoxicity of Fe and AA for differentiated PC12 cells. The optimal settings for hypothermic treatment were a temperature of 30-32 degrees C, rescue time window of less than 6 h, and minimum duration of at least 24 h. Hypothermia effectively prevented the loss of the mitochondrial transmembrane potential from 6 h to 72 h (end of the study period) and attenuated the release of apoptotic proteins (cytochrome c and apoptosis-inducing factor) at 6 h of exposure to Fe-AA. Activation of caspase-3 was also delayed until 24 h. Akt was transiently activated, although no influence of temperature was observed. Elevation of oxidative stress markers, including ortho-, meta-, and di-tyrosine (markers of protein oxidation) and 4-hydroxynonenal (lipid peroxidation) was significantly attenuated when the temperature was reduced by 5 degrees C. The half-cell reduction potential (Ehc) of GSSG/2GSH redox couple ranged from -220 to -180 mV in unstressed differentiated PC12 cells, and apoptosis was triggered when Ehc exceeded -180 mV. Hypothermia prevented Ehc from rising above -180 mV within 24 h of exposure to Fe-AA. In conclusion, hypothermia prevented cell death due to Fe-AA toxicity by inhibiting apoptotic pathways through maintenance of a reduced cellular environment, as well as by alleviating oxidative stress.

Severe Vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation.

Hyperoxia causes acute lung injury along with an increase of oxidative stress and inflammation. It was hypothesized that vitamin E deficiency might exacerbate acute hyperoxic lung injury. This study used alpha-tocopherol transfer protein knockout (alpha-TTP KO) mice fed a vitamin E-deficient diet (KO E(-) mice) as a model of severe vitamin E deficiency. Compared with wild-type (WT) mice, KO E(-) mice showed a significantly lower survival rate during hyperoxia. After 72 h of hyperoxia, KO E(-) mice had more severe histologic lung damage and higher values of the total cell count and the protein content of bronchoalveolar lavage fluid (BALF) than WT mice. IL-6 mRNA expression in lung tissue and the levels of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in both lungs and BALF were higher in KO E(-) mice than in WT mice. It was concluded that severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress or inflammation.