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We analyzed data from an ongoing registry to determine time to improvement in oxygenation in preterm and late preterm or term neonates with hypoxic respiratory failure and pulmonary hypertension receiving inhaled nitric oxide (iNO) in Japan.Registry neonates received iNO ≤7 days after birth (February 26, 2010, to October 9, 2012). Efficacy and safety profile data were collected up to 96 h after iNO initiation and, if necessary, every 24 h thereafter and before iNO discontinuation. Patients were stratified by gestational age (GA), oxygenation index (OI), and shunt direction at baseline.Data were evaluated for 1106 neonates (431 with a GA34 weeks and 675 with a GA of ≥34 weeks). Sixty percent of patients had improved OI; rates were similar for those with GAs of34 versus ≥34 weeks (61% vs 59%). Overall, mean time to improvement was 11.4 h and tended to be shorter in the groups with a GA34 weeks versus ≥34 weeks (9.2 vs 12.9 h). Thirty percent of responding neonates required1 h to achieve improvement in oxygenation. Neonates with higher baseline OI had the greatest decrease in OI during the first hour of treatment. The mortality rate was higher among iNO-treated patients with a baseline OI ≥25 versus those with OI ≥15 to25 (25% vs 12%; P = 0.0073).iNO treatment provided acute, sustained improvement in oxygenation in neonates with GAs34 and ≥34 weeks; 70% of patients had improvement within 1 h, but the remaining 30% took1 h to respond. Initiation of iNO at lower OIs was associated with reduced mortality compared with higher OI.

Background Transplanted organs from female donors are associated with less favorable prognoses and outcomes. This study aimed to determine whether donor gender affects levels of serum terminal complement component C5a and oxidative stress in pediatric living related liver transplantation (LRLTx) recipients. Material/Methods The subjects were 43 patients (20 males and 23 females) who underwent LRLTx during childhood (age range 1.2 years to 14.4 years; mean age 5 years). Serum samples were taken during the patients’ regular outpatient visits after LRLTx. Serum C5a was measured using the specific human C5a ELISA kit. Serum total hydroperoxide (TH) and biological antioxidative potential (BAP) were measured using the free radical analytic system, and the oxidative stress index (OSI) was calculated as the ratio of TH to BAP. Serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), gamma-glutamyl transpeptidase (γGTP), and lactate dehydrogenase (LDH) were also measured as part of a typical outpatient examination for such patients. Results C5a serum levels were higher in the 29 recipients who received their grafted livers from female donors than in the 14 recipients who received their grafted livers from male donors. Recipients who received their grafted livers from female donors had higher incidence of post-LTx (liver transplantation) complications. Female recipients from female donors showed the highest serum GPT and GOT levels, but this difference was only significant when compared to the female recipients from male donors (41.4±9.8 IU/L vs. 17.3±1.8 IU/L for GPT and 42.2±7.5 IU/L vs. 23.4±2.2 IU/L for GOT; P Conclusions Pediatric LRLTx patients who receive their grafts from female donors exhibit higher levels of serum C5a that probably plays a role in the immunological response against grafted livers from female donors in LTx.

Background: Phototherapy with radiation of 460–490 nm wavelengths provides the most potent therapeutic effect for neonatal jaundice. However, the efficacy of phototherapy has been estimated using single-wavelength detectors with sensitivity at approximately 460 nm. Cyclobilirubin formation capacity (CFC), which comprises the sum of the irradiance values from three wavelengths multiplied by their specific coefficients, has been proposed as an alternative marker to evaluate the efficacy of phototherapy. This study aimed to test whether two types of phototherapy devices with distinct spectral characteristics provide similar therapeutic effects on adjustment of device-to-patient distances to deliver similar CFCs. Methods: Using a three-wavelength spectroradiometer, CFCs and footprints of the light-emitting diode and fluorescent tube devices were assessed. Having determined the device-specific distances that ensured similar CFCs, 32 newborn infants, requiring phototherapy for hyperbilirubinemia, were randomized into the light-emitting diode and fluorescent tube groups. The total serum bilirubin levels before and after phototherapy were assessed. Results: The light-emitting diode and fluorescent tube devices had comparable CFCs at distances of 60 and 50 cm, respectively. Phototherapy reduced the total serum bilirubin levels from 18.1 to 14.6 mg/dL and from 19.1 to 15.1 mg/dL in the light-emitting diode and fluorescent tube groups, respectively. The two groups did not differ significantly with respect to the patients' clinical backgrounds, serum bilirubin levels, or changes before and after phototherapy. Conclusion: At similar CFCs, the two phototherapy devices reduced the total serum bilirubin levels by comparable amounts. Hence, determining CFCs may help predict phototherapy efficacy. This may ensure better safety and greater efficacy of the treatment for newborn infants. Key Words: bilirubin, cyclobilirubin, irradiance, light-emitting diode, spectroradiometer

Perinatal hypoxia-ischemia (HI) frequently causes white-matter injury, leading to severe neurological deficits and mortality, and only limited therapeutic options exist. The white matter of animal models and human patients with HI-induced brain injury contains increased numbers of oligodendrocyte progenitor cells (OPCs). However, the origin and fates of these OPCs and their potential to repair injured white matter remain unclear. Here, using cell-type- and region-specific genetic labeling methods in a mouse HI model, we characterized the Olig2-expressing OPCs. We found that after HI, Olig2+ cells increased in the posterior part of the subventricular zone (pSVZ) and migrated into the injured white matter. However, their oligodendrocytic differentiation efficiency was severely compromised compared with the OPCs in normal tissue, indicating the need for an intervention to promote their differentiation. Erythropoietin (EPO) treatment is a promising candidate, but it has detrimental effects that preclude its clinical use for brain injury. We found that long-term postinjury treatment with a nonerythropoietic derivative of EPO, asialo-erythropoietin, promoted the maturation of pSVZ-derived OPCs and the recovery of neurological function, without affecting hematopoiesis. These results demonstrate the limitation and potential of endogenous OPCs in the pSVZ as a therapeutic target for treating neonatal white-matter injury.

Periventricular leukomalacia is recognized as the leading cause of cerebral palsy in preterm infants. To clarify the prevalence of periventricular leukomalacia and cerebral palsy in Japan, a nationwide survey was performed. The prevalence of periventricular leukomalacia in the group of surviving preterm infants of gestational ages less than 33 weeks born in 2007 was 2.7% (78/2883) on ultrasound diagnosis, and 3.3% (92/2824) on magnetic resonance imaging. The prevalence of cerebral palsy was 4.3% (125/2883) on clinical diagnosis. In our previous study, the prevalences of periventricular leukomalacia in 1990-1991, 1993-1994, 1996, and 1999 were 4.8%, 4.9%, 4.9%, and 5.3% on ultrasound, and 7.9%, 7.7%, 6.9%, and 7.3% on magnetic resonance imaging, respectively. The prevalence of periventricular leukomalacia has decreased significantly in Japan.

The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury.

Background/Aims: To investigate the long-term efficacy and safety of two doses (33 and 67 µg/kg/day) of growth hormone (GH) in short Japanese children born small for gestational age (SGA). Methods: 96 children born SGA (age 3 to Results: After 208 weeks, change in height SDS from baseline (least square (LS) means (SE)) was 1.01 (0.47) and 1.99 (0.67) in the UC 33 and UC 67 µg/kg/day groups, respectively. After 260 weeks, change in height SDS from baseline was 1.22 (0.51) and 2.01 (0.64) in the 33 and 67 µg/kg/day groups, respectively. Insulin-like growth factor-1 levels were significantly higher in the groups receiving 67 µg/kg/day but largely remained within normal limits (–2 to +2 SDS). Conclusion: Long-term continuous GH treatment was well tolerated and effective in improving height SDS. Improvements were dose-dependent and significantly higher at 67 than 33 µg/kg/day.

Systemic infection in the newborn (neonatal sepsis) is the most common cause of neonatal mortality. Neonatal sepsis is complicated by pulmonary hypertension. In this study, we analyzed the effect of edaravone, a free radical scavenger that is known to reduce the production of inflammatory mediators, such as tumor necrosis factor α (TNFα), on pulmonary hypertension. Experimental and sham groups were drawn from 19 three-day-old piglets; 5 underwent a modified procedure of cecal ligation and perforation (CLP) (CLP group), 8 underwent CLP followed 30 min later by edaravone intravenous administration (edaravone group), and 6 did not undergo CLP and did not receive edaravone (sham group). To evaluate the pulmonary blood pressure despite the sepsis-induced low cardiac output, mean arterial blood pressure (mABP), mean pulmonary arterial pressure (mPAP), and comparative pulmonary hypertension ratio (mPAP/mABP) were determined. Serum TNFα levels were measured before the procedure and at 1, 3, and 6 h after. The mPAP levels were higher in the CLP group at 9 h compared to the edaravone group. The mPAP/mABP ratio was lower in the edaravone and sham groups compared to the CLP group at 6 and 9 h. TNFα in the edaravone and sham groups were lower at 1 and 3 h compared to that in the CLP group. In all animals, mPAP/mABP at 6 h correlated with serum levels of TNFα at 1, 3, and 6 h. These findings suggest that edaravone ameliorates the severity of pulmonary hypertension in a neonatal sepsis model by reducing serum TNFα levels.

ABO-incompatible liver transplantation (LTx) is becoming more common in response to the paucity of liver allografts. Several studies have expressed concern about the effect of ABO compatibility on graft survival.To evaluate the differences in serum cytokine levels between ABO-incompatible (ABO-i) and ABO-compatible (ABO-c; includes ABO-compatible and identical) pediatric LTx recipients during regular outpatient follow-up. Note that, in the field of organ transplantation, transplants are categorized as incompatible, compatible or identical; accordingly, these are the terms we use in the paper.A clinical outpatient study measuring serum transforming growth factor (TGF)-β1, interferon (IFN)-γ, interleukin (IL)-2 and IL-10 in 43 living related liver transplantation (LRLT) recipients, of whom 36 received ABO-c LRLT (34 were ABO-identical and 2 were non-identical) and 7 ABO-i LRLT. Serum glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase and bilirubin were measured as part of the patients' regular follow-up visits.There were no differences between the ABO-c and ABO-i groups in terms of recipient's age [mean 12.6 vs. 11.1 years (y)], post-LTx duration (mean 7.3 vs. 7.3 y), donor's age (mean 35.5 vs. 34.6 y), body weight (28.9 ± 2.9 vs. 27.9 ± 6.9 kg), or gender (19 female and 17 male vs. 4 female and 3 male). Serum TGF-β1, IFN-γ and IL-2 were significantly higher in the ABO-i group than in the ABO-c group. IL-10, however, did not differ between the two groups. There was a tendency toward higher γGTP levels in the ABO-i group, but this difference did not reach significance.ABO-incompatible LRLTx patients have higher serum TGF-β1, IFN-γ and IL-2 levels as measured at regular outpatient visits. As a result, they face a higher risk of T-helper 1 cell polarization, which could make graft rejection more likely.

To evaluate effects of endothelin receptor antagonist ETR-P1/fl in a neonatal sepsis model. Eighteen anesthetized and mechanically ventilated 3-day-old piglets were divided into three groups. Six piglets received cecal ligation and perforation (CLP group). Six piglets were administrated a continuous infusion of ETR-P1/fl (0.05 mg/kg/h), an antisense homology box-derived peptide with an endothelin A receptor antagonist effect, starting 30 min after CLP (ETR-P1/fl group). Six piglets acted as the sham group. Mean arterial pressure (MAP), heart rate, cardiac output, arterial blood gas, body temp (BT), serum nitrite and nitrate (NOx), tumor necrosis factor (TNF)-α, and high-mobility group box 1 (HMGB-1) were measured before CLP and at 1, 3, 6, and 9 h after CLP. Cecal ligation and perforation exposure evoked a state of shock and showed deteriorated cardiac output, pulmonary hypertension, decreased MAP, low oxygen saturation, and base excess (BE) with elevated TNF-α, NOx, and HMGB1. ETR-P1/fl administration resulted in higher MAP at 6 and 9 h after CLP, less negative BE, lower mean pulmonary arterial pressure (mPAP)/MAP ratio at 9 h after CLP, and lower TNF-α, NOx, and HMGB-1 compared to the CLP group. BT showed no differences between the groups. Survival time in the ETR-P1/fl group was longer than in the CLP group (18.9 ± 2.3 h vs. 9.0 ± 0.8 h, p

Rotavirus, one of the major causes of severe gastroenteritis in children, occasionally causes central nervous system complications. Recently several patients with acute encephalitis/encephalopathy due to rotavirus associated with cerebellar signs and symptoms have been reported. The condition is characterized by disturbances of consciousness at onset and cerebellar signs and symptoms such as hypotonia, ataxia, dysmetria, and speech disorders, including mutism, slow speech, and dysarthria at convalescence. We report two patients (3-year-old girl, 2-year-old boy) who developed acute encephalitis due to rotavirus and showed cerebellar signs and symptoms. Both patients had characteristic history of consciousness disturbances subsequent to several days of diarrhea, vomiting and fever, and cerebellar symptoms such as hypotonia, ataxia, dysmetria, and speech disorders during the recovery period. Electroencephalography showed diffuse high-voltage delta wave activity in each patient. Brain magnetic resonance imaging showed cerebellar edema in the acute phase followed by cerebellar atrophy on follow-up images in both patients. In the first patient, diffusion-weighted images (DWI) revealed high signals at the left cerebellar peduncle region and apparent diffusion coefficient (ADC) maps showed decreased ADC values of the lesion in the acute phase. The first patient had dysmetria at 1-year follow-up. However, she had normal motor and cognitive functions and could lead her daily life without impairment. In the second patient, no further symptoms were apparent at 1-year follow-up. Acute encephalitis/encephalopathy due to rotavirus with cerebellar signs and symptoms might be diagnosed on DWI, by demonstrating decreased ADC values in acute phase.

We report two heterosexual sibling cases of mild non-specific myopathy, with bilateral occipital cortical dysplasia and diffuse white matter hyperintensity on brain magnetic resonance imaging (MRI). The histological examination of the muscle in the elder sister revealed non-specific myopathic changes and no reductions of alpha-dystroglycan and laminin alpha2 expressions. The characteristic findings in the occipital lobe on brain MRI in both cases suggested cobblestone lissencephaly. Disrupted structure of the glia limitans and pial basement membrane complex, by a cause other than an alpha-dystroglycan and laminin alpha2 abnormality, may be the cause of the cortical dysplasia in the sibling cases.

Background and objective: The antiviral neuraminidase inhibitor oseltamivir (OSV) is used to treat influenza. The macrolide clarithromycin (CAM) is used to treat bacterial infections and has anti-inflammatory and immunomodulatory activities. This retrospective study investigated the immunomodulatory effects of CAM in children presenting with influenza A. Methods: The study recruited 40 children with acute influenza, and grouped them according to the treatment received: 5-day treatment with OSV (n = 14), CAM (n = 8), OSV + CAM (n = 12) and untreated (n = 6). The before and after treatment comparisons were made of the level of secretory IgA (sIgA) against influenza A virus (H3N2) and (H1N1), total sIgA, viral RNA copy numbers in nasopharyngeal aspirates and disease symptoms. Results: Infection induced anti-viral mucosal sIgA in the nasopharyngeal aspirates of most patients of all treatment groups. Particularly prominent increases in the levels were found in the CAM and OSV + CAM groups. Low induction of anti-viral sIgA was observed in the OSV group, but the addition of CAM to OSV augmented sIgA production and restored local mucosal sIgA levels. The frequency of residual cough in the OSV + CAM group was significantly lower than in the other groups including the group treated with OSV. Conclusions: CAM boosted the nasopharyngeal mucosal immune response in children presenting with influenza A, even in those treated with OSV who had low production of mucosal anti-viral sIgA, and alleviated the symptoms of influenza.

Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased. In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-kappaB inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-kappaB p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, L-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-kappaB signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF.

We report a two-year and one-month-old immunocompetent boy who developed aphasia and right hemiparesis eight months after mild varicella with only a few vesicles. Magnetic resonance images and angiography demonstrated mixed acute and old infarctions of the bilateral middle cerebral arteries. VZV-DNA was detected on polymerase chain reaction analysis of cerebral spinal fluid (CSF). He was treated with intravenous acyclovir and edaravone, and his speech and motor functions had almost recovered after two months. Cerebral lesions of the bilateral middle cerebral artery territories and virus DNA detection from CSF are rare in VZV-related vasculopathy and suggest incomplete immunoresponse to varicella in this patient.

Background: The underlying neurologic disorders of status epilepticus (SE) in childhood remain poorly characterized. Methods: We reviewed 249 consecutive patients with SE, aged 1 month to 18 years, who were referred to Tottori University Hospital from 1984 to 2002. After exclusion of SE patients with acute symptomatic etiology and progressive encephalopathy, such as acute encephalitis/encephalopathy, meningitis, head trauma, or metabolic disorders, we analyzed 112 patients, aged 3 months to 14 years, and focused on the epilepsy classification and perinatal brain damage in these patients. Results: Major underlying neurologic disorders were non-symptomatic epilepsy (41 patients, 36.6%), perinatal brain damage (15 patients, 13.4%), non-syndromic mental retardation (17 patients, 15.2%), and congenital disorders including chromosomal abnormalities (13 patients, 11.6%). In non-symptomatic epilepsy, childhood epilepsy with occipital paroxysms (Panayiotopoulos syndrome, 11 patients) and severe myoclonic epilepsy in infancy (SMEI, 6 patients) were common and had high recurrence rates (81.8% and 66.7%, respectively). In patients with a history of perinatal brain damage, preterm birth, neonatal seizure, asphyxia, and neonatal hypoglycemia were frequent. Neonatal hypoglycemia and neonatal seizure were related to the recurrence of SE (100% and 87.5%, respectively). They were mostly diagnosed as symptomatic occipital lobe epilepsy. Parieto-occipital paroxysms were associated with a high recurrence rate of SE (80.6%). Conclusions: Although the underlying neurologic disorders of SE are heterogeneous, three specific epileptic syndromes (Panayiotopoulos syndrome, SMEI, and symptomatic occipital lobe epilepsy secondary to neonatal hypoglycemia and neonatal seizure) were the most common causes of SE and were associated with higher recurrence rates.

Periventricular leukomalacia is a major neuropathology in preterm infants associated with adverse motor and cognitive outcome. The cerebral blood flow volume of the internal carotid artery and the vertebral artery was measured by ultrasonography at the neck in 36 low-birth-weight infants with gestational age of 25–34 weeks in order to investigate the pathophysiology of cerebral white-matter injury: 30 infants, normal and 6 infants, diagnosed as PVL. The mean blood flow velocity and diameter of each vessel were measured at postnatal days from day 0 to day 70. The intravascular flow volume was determined by calculating the mean blood flow velocity and the cross-sectional area. The mean blood pressures were recorded and PaCO 2 was determined. The total blood flow volume was significantly lower in infants with PVL than in normal infants on days 0, 1, 21, 28, 35, 42, and 63. The mean blood pressure was significantly lower in infants with PVL than in normal infants on days 7, 14, 21, 28, and 42. We suggest that the total cerebral blood supply is decreased in cases of PVL in the few days after birth and from day 21 to day 42. The results of the present study suggest that a dip in the blood flow volume in the few days after birth might result in subsequent PVL.

Adrenocorticotrophic hormone (ACTH) therapy is an established treatment for West syndrome. However, some patients may relapse after this therapy, for whom there is no established treatment. We describe 3 patients with symptomatic West syndrome and multiple, poor prognostic factors who relapsed after initial ACTH therapy. They were treated with a second round of ACTH therapy, i.e., daily intramuscular injection for 2-3 weeks and subsequent withdrawal, alternative days for 1 or 2 weeks, every 3 days for 1 or 2 weeks, followed by weekly or biweekly for/=1 year. Clinical seizures and hypsarrhythmia resolved in all 3 patients within 4 weeks, and these clinical improvements continued through a second round of ACTH therapy. Two patients developed other types of seizures and aggravation of paroxysms on electroencephalography, but no hypsarrhythmia, soon after termination of ACTH therapy. In the other patient, although electroencephalographic findings remained normal during weekly ACTH therapy, focal sharp waves with irregular slow waves appeared after the injection interval became biweekly. After a second round of ACTH therapy, all patients exhibited developmental progress, particularly in gross motor development and visual functions. No serious adverse events occurred during treatment. Long-term weekly ACTH therapy is a potentially effective treatment option for relapsed West syndrome.

Females with salt-wasting (SW) 21-hydroxylase deficiency (21OHD) may present with mild external genitalia virilization, despite complete or almost complete enzyme inactivation. We therefore analyzed genotype/phenotype correlation in 13 Japanese female patients with SW 21OHD. Criteria for classification into the SW phenotype included history of a salt-losing crisis with documented hyponatremia, hyperkalemia, and markedly elevated plasma renin activity. Urologists and pediatricians determined the Prader genital stage and classified the location of the vaginal entrance into the common urogenital sinus as low, moderate, or high. CYP21A2 gene, coding for 21-hydroxylase, was analyzed with Southern blotting and direct sequencing. Genotypes were categorized into four mutation groups, based on the degree of enzymatic activity (N, complete enzyme inactivation; groups A,2%, B, 3-7%, and C30%). Basal androgen levels were available from only six out of thirteen patients, so we could not relate androgen levels with the severity of external genitalia virilization. We compared the degree of external genitalia virilization with genotype. The severity of external genitalia virilization varied from Prader stage 1 to 4. One patient who presented with Prader 1 had a genotype consistent with Group B. In addition, discordance between Prader classification and the location of the vaginal entrance was noted; one patient classified as Prader 4 showed low vaginal entrance, while another patient classified as Prader 3 showed high vaginal entrance. The degree of the impairment of 21-hydroxylase activity does not correlate with the severity of virilization of the external genitalia in female patients with the SW type of 21OHD.

The single-strand conformation polymorphism (SSCP) procedure has been applied in routine testing for hereditary diseases. Temperature, running buffer, gel composition, and fragment length can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes, such as the dihydropyrimidine dehydrogenase (DPYD) gene, which codes the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). The authors have optimized the condition of SSCP with an automated system (GenePhor system, GE Healthcare UK Ltd.) to screen genetic polymorphisms in the DPYD gene. The efficiency of the method was evaluated using 21 positive controls (DNA samples with polymorphisms in the DPYD gene, previously characterized) and DNA samples from 35 Japanese. Results showed that the use of three different running buffers (pH 7.4, 8.3, and 9.0) in combination with other optimized conditions (10% polyacrylamide gel, 60-90 min at constant 900 V at 5 degrees C) resulted in a high polymorphism detection rate (95.3%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for pharmacogenetic studies on 5FU.

Newborn males are more sensitive to brain injury than newborn females are. The aim of the present study was to find an explanation for this. We used the neuron-specific enolase (NSE) levels in the cerebrospinal fluid (CSF) for the classification of 32 newborns (19 males and 13 females) on their fifth postnatal day. The NSE levels were higher than normal (8.4 +/- 1.6 ng/mL) in 10 newborn males and 6 females and were, respectively, considered asphyxiated male and female groups. The remaining newborns, 9 males and 7 females, had normal CSF levels of NSE and were considered normal newborn male and female groups. The CSF samples were measured for 12 cytokines, using a cytokine array kit, and for total hydroperoxide and biological antioxidant potentials (BAPs), using the free radical analytic system. Among the 12 cytokines measured, only interleukin 8 (IL-8) was properly detected. The CSF levels of IL-8 were higher in the asphyxiated newborn females than in the other three groups. The mean CSF levels of BAPs in the asphyxiated newborn females were higher compared with the other three groups, but significance was detected only in comparison with the BAP levels in the CSF samples of the normal newborn males. There were no differences in total hydroperoxide levels among the groups. There are sex-related differences in the CSF levels of IL-8 and antioxidants in asphyxiated newborns, with higher levels in newborn females; this might contribute in the sexual dimorphism regarding the fact that females have better protection from brain injury than the males.

Tandem mass spectrometry (MS/MS) has become a prominent method for screening newborns for diseases such as organic acidemia and fatty acid oxidation defects, although current methods cannot separate acylcarnitine isomers. Accurate determination of dicarboxylic acylcarnitines such as methylmalonylcarnitine and glutarylcarnitine has not been carried out, because obtaining standards of these acylcarnitines is difficult. We attempted the individual determinations of acylcarnitines with isomers and dicarboxylic acylcarnitines by applying high-performance liquid chromatography (HPLC). Chromatographic separation was performed by gradient elution using a mixture of 0.08% aqueous ion-pairing agent and acetonitrile as the mobile phase. Mass transitions of m/z 161.8--84.8 for carnitine and m/z 164.8--84.8 for deuterated carnitine were monitored in positive ion electrospray ionization mode. One carnitine and 16 acylcarnitines were quantified. The limit of quantitation (LOQ) was 0.1 micromol/L for methylmalonylcarnitine and 0.05 micromol/L for the other acylcarnitines. Intra-day and inter-day coefficients of variance (CVs) were8.3% and8.8%, respectively, for all acylcarnitines in serum, and both were9.2% in urine. Mean recoveries were90% for all acylcarnitines. Human samples were quantified by this method. After addition of deuterated acylcarnitines as internal standards, acylcarnitines in serum or urine were extracted using a solid-phase extraction cartridge. In healthy adult individuals, isobutyryl-, 2-methylbutyryl- and isovalerylcarnitine were detected in serum and urine. Dicarboxylic acylcarnitines were detected in urine. High concentrations of methylmalonylcarnitine and propionylcarnitine were found in both the serum and the urine of a patient with methylmalonic acidemia. The described HPLC/MS/MS method could separate most acylcarnitine isomers and quantify them, potentially allowing detailed diagnoses and follow-up treatment for those diseases.

West syndrome (WS) is a generalized epileptic syndrome of infancy and early childhood with various etiologies, and consists of a triad of infantile spasm, arrest or regress of psychomotor development and specific electroencephalogram (EEG) pattern of hypsarrhythmia. WS had been believed to be refractory, but recent evidence supports effectiveness of adrenocorticotropic hormone (ACTH) treatment. The ACTH treatment, however, has a problem that it is often accompanied by adverse autonomic symptoms. We therefore examined heart rate variability (HRV) for assessing cardiac autonomic functions in WS and prospectively observed the changes during ACTH treatment. We studied 15 patients with WS and 9 age-matched controls during sleep (EEG stage 2). Compared with controls, the patients with WS were greater in the low-frequency component (LF) of HRV, an index reflecting sympatho-vagal interaction (p = 0.02), but were comparable for high-frequency component (HF) and LF-to-HF ratio (LF/HF), indices reflecting cardiac vagal activity and sympathetic predominance, respectively. During ACTH treatment, heart rate decreased (p < 0.01), LF and HF increased (p < 0.01), and LF/HF did not differ significantly. These results indicate that WS might be accompanied by autonomic changes and that ACTH treatment enhances parasympathetic function and causes bradycardia.

Despite administration of vitamin K (VK), some infants show lower activity of VK-dependent coagulation factors and they could develop intracranial hemorrhage. For preventing VK deficiency bleeding (VKDB) in infants, oral administration of VK and a screening test for VK deficiency are carried out in Japan. For the screening, the total activity of VK-dependent coagulation factors is measured using a commercial product, Normotest. This study was undertaken to clarify the importance of the following genetic and environmental factors on the coagulation status in one-month-old infants: two polymorphisms in the factor VII gene, -323P0/10 (a 10-bp insertion in the promoter region at position -323) and R353Q (the replacement of arginine [R] with glutamine [Q] at residue 353) and sex, age, gestational age, birth weight, and feeding regimen. Two hundred Japanese infants (34.6 +/- 4.0 days old) were screened for VK-dependent coagulation activity with Normotest and were genotyped for the two polymorphisms. Among the subjects screened, 18 infants (9%) carried the P10 allele and 26 (13%) carried the R353Q allele. Multiple regression analysis showed that the 10-bp inserted (P10) allele or the Q allele was associated with the lower coagulation activities. The coagulation activities for the R/Q genotype were significantly lower than those for the R/R genotype and those for the P0/P10 genotype were significantly lower than those for the P0/P0 genotype. Therefore, infants who carry the P10 allele or the Q allele show lower activity of VK-dependent coagulation factors. These infants may have a higher risk of VKDB manifestation.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is rare among Asian individuals, and the clinical course and biochemical findings remain unclear. We report herein a 3-year-old Japanese girl with MCADD. The diagnosis was suggested by acylcarnitine profiles and confirmed by enzyme activity and genetic analysis after clinical presentation. Our described method with high-performance liquid chromatography/tandem mass spectrometry allows quantification of levels of n-octanoylcarnitine (C8-N) and other isomers (e.g. valproylcarnitine). We examined the patient's acylcarnitine profiles in serum and urine samples during carnitine loading and 14-hr fasting tests with/without carnitine supplementation. Under hypocarnitinemia, serum level of C8-N was 0.16 micromol/l and C8-N/decanoylcarnitine (C10) ratio was 1.8, which did not correspond to the diagnostic criteria for MCADD. However, intravenous carnitine loading test (100 mg/kg/day for 3 days and 50 mg/kg/day for 1 day) led to increased serum C8-N levels and urinary excretion was obvious, strongly suggesting MCADD. In the fasting test with carnitine supplementation, marked production of acylcarnitines (C8-N > C2 >> C6 > C10) was found, compared to the fasting test without carnitine supplementation. These results indicate that carnitine supplementation may be useful for detoxification of accumulated acylcarnitines even in an asymptomatic state. Moreover, the one-point examination for serum C8-N level and/or C8-N/C10 ratio may make the diagnosis of MCADD difficult, particularly in the presence of significant hypocarnitinemia. To avoid this pitfall, attention should be given to serum levels of free carnitine, and carnitine loading may be demanded in hypocarnitinemia.

In this study, seven patients with cryptogenic West syndrome were subjected to short-duration adrenocorticotrophic hormone (ACTH) therapy. ACTH was administered daily for 7 to 12 days (mean 10 days), and then was withdrawn. Daily single dose was 0.022 to 0.027 mg/kg/day (0.024 mg/kg/day, 0.96 IU/kg), total dose was 0.17 to 0.28 mg/kg (0.23 mg/kg, 9.0 IU/kg), and treatment lag was 12 to 105 days (median 14 days). ACTH therapy controlled tonic spasms with no serious side effects in all patients, but two patients still had electroencephalographic abnormality. The intelligence quotients or developmental quotients of six patients with short treatment lag were 79 to 110 at the age of 2 to 6 years; only one patient with long treatment lag had a developmental quotient of 60. This new short-duration ACTH therapy could yield better cognitive outcomes for cryptogenic West syndrome.

Oxidant/antioxidant imbalance plays an important role in septic shock. The present study examined changes in circulating oxidative components in a neonatal sepsis model. Subjects were 14 newborn mixed-strain piglets randomly divided into two groups: a cecal ligation and perforation (CLP) model (n = 7) and sham (n = 7). Blood samples for total hydroperoxide (TH), biological antioxidant potential (BAP), tumor necrosis factor (TNF) alpha, interleukin (IL)-6, and IL-10 were collected pre-CLP and at 1, 3, and 6 h post-CLP. TH and BAP levels at 1 h post-CLP were significantly higher in the CLP group than in the sham group. In the CLP group, TH decreased gradually and reached baseline levels by 6 h post-CLP, while BAP remained elevated. Linear correlations were identified between serum TH and BAP at 1 h post-CLP, serum TH and TNF-alpha at 1 h post-CLP, and BAP and IL-6 at 6 h post-CLP. Changes in and correlations between circulating oxidative and inflammatory state components in a neonatal sepsis model were clarified. This is the first study to reveal that the presence of oxidant/antioxidant imbalance in sepsis and septic shock changes during the disease course.

Background: In Japan, palivizumab was approved in 2002 for prophylaxis of severe respiratory syncytial virus disease in high-risk infants. In order to evaluate the efficacy and safety of this drug, a questionnaire survey was conducted. Methods: A questionnaire was sent to member institutions of the Japan Neonatologist Association. The subjects were premature infants who were considered possible candidates for treatment with palivizumab. Results: A total of 6302 case reports, including those of 2806 infants receiving palivizumab (group P) and 3496 infants not receiving palivizumab (group NP), respectively, were retrieved. Background characteristics revealed significant lower gestational age (GA) and birthweight for group P (P < 0.0001). Sex ratio did not differ significantly, while use of oxygen and mechanical ventilation in the neonatal intensive care unit, and presence of chronic lung disease were significantly higher for infants in group P (P < 0.0001). When comparison of hospitalization rate for respiratory symptoms was performed with stratification by eligibility criteria, in the group of infants born at 29–35 weeks GA the hospitalization rate was 4.0% and 5.7% in groups P and NP, respectively (P < 0.05). Multivariate analysis also showed that prophylaxis with palivizumab was the only variable that significantly decreased rate of hospitalization (odds ratio 0.630, P= 0.0053). The incidence of adverse events associated with the administration of palivizumab was low. Conclusion: In this non-randomized questionnaire survey, multivariate analysis showed that palivizumab significantly decreased the rate of hospitalization due to respiratory symptoms for infants born prematurely at 29–35 weeks GA. These data confirmed the efficacy and safety of palivizumab.

Study objective Compared with control infants, those who will be future victims of sudden infant death syndrome (SIDS) show a decreased arousability during sleep, with fewer cortical arousals and more-frequent subcortical activations. These findings suggest an incomplete arousal process in victims of SIDS. Prone sleep position, a major risk factor for SIDS, has been reported to reduce arousal responses during sleep. The present study was undertaken to evaluate whether the prone sleep position impairs the arousal process in healthy infants. Methods Twenty-four healthy infants were studied polygraphically during 1 night; 12 infants regularly slept supine and 12 infants regularly slept prone. Infants were matched for sex, gestational age, and age at recording. Arousals were differentiated into subcortical activations or cortical arousals, according to the presence of autonomic and/or electroencephalographic changes. Frequencies of subcortical activations and cortical arousals were compared in the prone- and the supine-sleeping infants. Results Compared with supine sleepers, prone sleepers had significantly fewer cortical arousals during rapid eye movement (REM) sleep (p = .043). There were no significant differences in cortical arousals between the 2 groups during non-REM sleep. No significant differences were seen in the frequencies of subcortical activations during both REM and non-REM sleep between supine and prone sleepers. The ratio of cortical arousal to subcortical activation showed no significant differences between the prone and the supine sleepers. Conclusions Prone sleep position decreased the frequency of cortical arousals but did not change the frequency of subcortical activations, as has been previously found in SIDS victims. These results suggest specific pathways for impairment of the arousal process in SIDS victims.

Adenosine is a physiologically active molecule produced locally in many sites of the body to regulate various cell functions. Measurement of levels of the factor in organs and biological fluids provides clues to its role and we reported an accurate quantitative high-performance liquid chromatography method for urinary adenosine requiring no preliminary sample preparation, other than filtration. Analyses were performed isocratically with a reversed-phase and a molecular exclusion columns connected by a column switch. Each sample was analyzed automatically in 35 min. Linearity could be verified up to 1,000 micromol/L (r = 0.999) and recovery of adenosine was 94.6-98.0%. The coefficients of variation (CV) were established to be 0.56-1.32%, intra-assay, and 1.61-4.67%, inter-assay. Based on analyses of healthy individuals at different ages, we are here able to provide age-related values, infants (1.51 +/- 0.71 micromol/mmol creatinine) and children (1.06 +/- 0.36 and 0.83 +/- 0.27 micromol/mmol creatinine; aged 1-5 and 6-10 years), excreting significantly higher amounts of adenosine than adults (0.44 +/- 0.08 micromol/mmol creatinine). We also measured urinary adenosine from patients suffering from metabolic disease or severe respiratory failure and found that unfavorable pathophysiologic conditions are associated with appreciable elevation of adenosine.

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2-5% of Caucasian populations, but at higher frequencies (18-23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.

Inosine triphosphate pyrophosphohydrolase (ITPase) is an enzyme that catalyzes the conversion of inosine triphosphate (ITP) to inosine monophosphate and pyrophosphate. In Caucasian populations it is reported that the frequency of cases showing decreased ITPase activity is 5%. The structure of ITPA gene along with five single nucleotide polymorphisms has been reported in Caucasians. We examined ITPase activity and frequency of two polymorphisms (94C > A and IVS2 + 21A > C) in 100 Japanese individuals. Among these individuals, we observed that three cases with zero activity were homozygote for 94C > A, and were accompanied by abnormal accumulation of ITP in erythrocytes. The cases included in the low ITPase activity group were heterozygote for 94C > A polymorphism. The activity of the heterozygote cases was approximately 27% of the mean value of the wild type. The allele frequency of the 94C > A polymorphism was 0.155, which was 2.6 times higher than that of the Caucasians (0.06). The IVS2 + 21A > C was not detected in Japanese cases, although it occurred with a frequency of 0.130 in Caucasians. Furthermore, we identified a novel mutation IVS2 + 68T > G in intron 2 in the case with the lowest enzyme activity in the 94C > A wild type. Since the frequency of ITPA 94C > A polymorphism is higher in the Japanese population than that in Caucasians, it is more important to examine ITPA 94C > A polymorphism in the Japanese population to prevent thiopurine drug toxicity. Pretherapeutic screening of individuals for ITPA polymorphisms should be considered for safer and more tolerable treatment with thiopurine drugs.

Of 27,000 infants whose sleep-wake characteristics were studied under the age of 6 months, 38 died unexpectedly 2-12 weeks after the sleep recording in a pediatric sleep laboratory. Of these infants, 26 died of sudden infant death syndrome (SIDS), and 12 of definitely identified causes. The frequency and duration of sleep apneas were analysed. Sleep recordings and brainstem histopathology were studied to elucidate the possible relationship between sleep apnea and neuropathological changes within the arousal system. Immunohistochemical analyses were conducted using tryptophan hydroxylase (TrypH), a serotonin synthesizing enzyme, and growth-associated phosphoprotein 43 (GAP43), a marker of synaptic plasticity. The terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was used for apoptosis. The pathological and physiological data were correlated for each infant. In the SIDS victims, statistically significant positive correlations were seen between the number of TrypH-positive neurons in the dorsal raphe nucleus of the midbrain and the duration of central apneas (p = 0.03), between the number of TUNEL-positive glial cells in the pedunculopontine tegmental nucleus (PPTN) and the average number of spines in GAP43-positive neurons in the PPTN (p = 0.04). These findings in the dorsal raphe nucleus of the midbrain and PPTN, that play important roles in the arousal pathway suggest a possible link between changes in arousal and SIDS.

This study evaluated whether the ratio of the mean flow velocities in the middle cerebral artery (MCA) and the internal carotid artery (ICA) of neonates in the first days of life can be used to identify future neurodevelopmental disabilities.We observed 127 term neonates without congenital malformations, chromosomal aberrations, intracranial hemorrhage, or early onset sepsis. The mean cerebral blood flow velocities were measured in the right and left ICAs and in the right and left MCAs with a Doppler flowmeter once from day 1 to day 3. The Vm ratio was defined as the mean velocity in the right and left MCAs/mean velocity in the right and left ICAs. Neurologic examinations were performed at 12 months of age in the outpatient follow-up clinic to detect cerebral palsy (CP), and the subjects were divided into 4 groups according to the diagnosis of hypoxic-ischemic encephalopathy (HIE) and neurologic prognosis: HIE- and normal neurologic function, HIE- and CP, HIE+ and normal neurologic function, and HIE+ and CP.The Vm ratio in infants with the HIE- diagnosis and CP was significantly lower than that in infants with the HIE- diagnosis without CP (P.05). There was no significant difference between the Vm ratios in infants with the HIE+ diagnosis without CP and infants with the HIE+ diagnosis and CP.The Vm ratio might be a useful index in estimating neurologic outcome at birth, especially in neonates without the diagnosis of HIE.

Ornithine aminotransferase (OAT) deficiency (MIM: 258870) is a rare congenital metabolic disorder characterized by gyrate atrophy of the choroid and retina. Here, we report a 37-year-old male with gyrate atrophy of the choroid and retina who has been treated for 18 years. At the age of 7 years, the patient consulted an ophthalmologist due to progressive loss of vision. A large atrophied area was observed in his retina, and OAT deficiency was suspected. At the age of 19 years, amino acid analysis revealed high serum ornithine levels (1,140 nmol/ml), with the normal range being 40-100 nmol/ml. He was treated with vitamin B(6) 300 mg/day for 6 months, which successfully reduced his serum ornithine levels by 20-30%. For 18 years since, his serum ornithine levels have been maintained with vitamin B(6) medication. There was no further impairment of vision or increase in the atrophied area, as judged by ophthalmoscopic examination. OAT activity was undetected in white blood cells of the patient and was 105% and 45% of normal values in his wife and son, respectively. OAT gene analysis revealed a novel mutation of Gly237Asp in exon 7 (710GA) in both alleles of the patient, while his son was a heterozygote for the mutation. Notably, this novel mutation is associated with a vitamin B6-responsive phenotype. Therefore, early diagnosis and treatment with vitamin B(6) may prevent loss of vision in some patients with OAT deficiency.

Purpose The purpose of this study was to evaluate the cerebral blood flow of the posterior cerebral arteries (PCAs) in neonates in relation to the onset of periventricular leukomalacia (PVL). Methods Among 57 low-birth-weight neonates studied, 7 were diagnosed with PVL with cyst formation on sonography and MRI. The mean cerebral blood flow velocity (CBFV) was measured in all the neonates by Doppler sonography through the posterior fontanel separately in the right and left PCA at days 0, 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 42, 56, and 70 following birth. Results In the 7 neonates with PVL the mean CBFV in the right PCA was significantly lower than that in neonates without PVL at days 10, 14, 21, 28, 42, 56, and 70; the mean CBFV in the left PCA of neonates with PVL was significantly lower than that in those without PVL at days 7, 10, 14, 21, 28, 42, 56, and 70. CBFV measured in neonates without PVL exhibited a gradual increase postnatally. In contrast, CBFV values for neonates with PVL plateaued after day 5 or 7. Conclusions The serial measurement of PCA CBFV postnatally may prove useful as a predictor of the development of PVL. © 2004 Wiley Periodicals, Inc. J Clin Ultrasound 33:24–28, 2005

Recently, a novel deoxyribonucleic acid (DNA) virus, designated SEN virus (SENV), was discovered and strong associations between the two SENV variants (SENV-D and SENV-H) and non-A to E hepatitis were reported. To clarify the character of SENV infection in children, we investigated the detection rates of serum SENV DNA by polymerase chain reaction (PCR) among children with non-A to C hepatitis, with histories of transfusions, with neither histories of transfusions nor liver diseases (control), and among pregnant women. SENV-D was detected in 60% of fulminant hepatitis, 5% of acute hepatitis, 11% of chronic hepatitis, 13% of controls, and 15% of pregnant women. SENV-H was detected in none of fulminant hepatitis, 5% of acute hepatitis, none of chronic hepatitis, 2% of controls, and 12% of pregnant women. No significant difference was found for SENV-D between acute or chronic hepatitis and controls, however SENV-D detection rate in fulminant hepatitis was significantly higher than that in controls (P

To study the effect of exchange transfusion on cytokine profiles in a patient with necrotizing enterocolitis, the levels of 12 cytokines and serum calprotectin were measured among exchange transfusion. A male extremely low birth weight infant was in non-compensated shock and diagnosed stage 3 necrotizing enterocolitis. Exchange transfusion was performed for critical condition, refractory hypotension and disseminated intravascular coagulation. After exchange transfusion, the patient's blood pressure increased and stabilized. Then an enterostomy was performed and revealed necrosis of the ascending colon. Of the cytokines examined, interleukin-8 and serum calprotectin were high before exchange transfusion and decreased after exchange transfusion.

Persistent pulmonary hypertension of the newborn is a clinical syndrome associated with a variety of cardiopulmonary diseases. Serial evaluation of pulmonary circulation and cardiac function is important, but available imaging techniques have been limited. A new Doppler index combining systolic and diastolic time intervals (the Tei index, which is a simple and noninvasive measurement) has been reported to be useful for the assessment of global cardiac function in adults and children. The purpose of this study was to test the effectiveness of the Tei index in prospectively assessing ventricular function and pulmonary circulation in a newborn piglet model with hypoxic pulmonary hypertension. One-day-old piglets (1.1-1.6 kg) were intubated and prepared for the experiments under room air and hypoxia. A complete two-dimensional Doppler echocardiographic examination was performed. Common hemodynamic variables were measured continuously throughout the study. The right ventricle (RV) Tei index under hypoxia (fraction of inspired oxygen = 0.10) was significantly higher than the value under air ventilation (medians, 0.38 versus 0.56; p < 0.05). Moreover, there was a significant correlation between RV Tei index and mean pulmonary artery pressure and positive linear correlation between individual changes in RV Tei index and changes in mPAP (r2 = 0.799, p < 0.05). We conclude that the Tei index is useful for assessing the function of the RV and the left ventricle and pulmonary circulation in a newborn piglet model with hypoxic pulmonary hypertension. These results suggest that the Tei index will become an objective method of assessing patients with persistent pulmonary hypertension of the newborn.

Objective: To clarify whether combination treatment with desmopressin (DDAVP) and thiazide was clinically effective in a patient with congenital nephrogenic diabetes insipidus (CNDI), we evaluated the treatment in a 7-year-old boy with CNDI who had demonstrated a partial response to DDAVP. Method: Both volume of urine and the presence of nocturia were determined during treatment. Result: Neither the usual therapy of a low-salt diet and a thiazide nor intranasal therapy with a large dose of DDAVP was effective. However, combination treatment resulted in a decrease in urinary volume and the disappearance of nocturia. Conclusion: DDAVP coupled with thiazide may be useful for CNDI in patients who have shown a partial response to DDAVP.

We analyzed the PHKA2 gene in four Japanese families with hepatic phosphorylase kinase (PhK) deficiency. Mutational analysis of PHKA2 cDNA was performed by reverse-transcribed polymerase chain reaction (RT-PCR) and direct sequencing, and each mutation was confirmed on the genomic DNA. In boys with low erythrocyte PhK activity (i.e., x-linked liver glycogenosis [XLG] type I), deletion of exon 2 (splice site mutation of 79-1 G > T) or nonsense mutation of Q1169X or R497X was identified. However, missense mutation of R295C was identified in one boy with normal erythrocyte PhK activity (i.e., XLG type II). This mutation was not found in 100 control alleles, and was considered responsible for presentation of the XLG type II phenotype. Excluding Q1169X, all mutations detected in this study represented novel mutations. All mothers were found to be heterozygous carriers of the mutations. Gene analysis was confirmed to represent a useful procedure for diagnosing XLG type II, for which liver biopsy had previously been required to detect hepatic PhK deficiency.

Background: Persistent pulmonary hypertension of the newborn (PPHN) commonly appears as a complication of several pulmonary and non-pulmonary diseases. The hypoxia possibly inhibits Ca 2 + ′ dependent K + channels, thus resulting in membrane depolarization of pulmonary smooth muscle cells, which leads to the opening of Ca 2 + channels and Ca 2 + entry, resulting in contraction of the vascular smooth muscle. However, magnesium (Mg 2 + ) is an antagonist of Ca 2 + . We studied the effect of magnesium sulfate on the treatment of hypoxia-induced pulmonary hypertension and compared to the site of action of nitric oxide (NO). Methods: Zero-day-old piglets were used in each experiment. The effects of Mg 2 + were tested in each hypoxic, normoxic and hyperoxic states. Once the desired physical state was achieved, Mg 2 + was administered at a dose of 100 mg/kg approximately every 10 min. In order to determine the exact mechanism of the Mg 2 + , Nw-nitro-L-arginine (LNNA), a NO synthase-inhibitor, was administered simultaneously with Mg 2 + in some of the experiments. Results: There was a significant correlation between the percent reduction of the pulmonary arterial pressure (PAP) caused by magnesium and the level of oxygen (O 2 ) present in the pulmonary artery. The greatest amount of reduction was seen in the hypoxic condition where the least amount of O 2 is found. A further reduction in the PAP was seen when NO was given at the end of the Mg 2 + trials. There was no significant reduction seen in the systemic arterial pressure. Conclusion: Inhaled NO further reduced the PAP in piglets already treated with Mg 2 + .