Yohei Matsuzaki

Conditional deletion of Abca3 in alveolar type II cells alters surfactant homeostasis in newborn and adult mice

ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3(Δ/Δ)) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3(Δ/Δ) mice survived after birth. Surviving Abca3(Δ/Δ) mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ∼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3(Δ/Δ) mice. In adult Abca3(Δ/Δ) mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells.

Curcumin and genistein additively potentiate G551D-CFTR

BACKGROUND The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. METHODS We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. RESULTS Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. CONCLUSIONS The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy.

Usefulness of measurement of urinary N-terminal pro-brain natriuretic peptide in neonatal period

N-terminal pro-brain natriuretic peptide (NT-proBNP) is an N-terminal peptide dissociated from proBNP when BNP is generated from proBNP. Its molecular size is about 8500 kDa, and it has no physical activity. It is a sensitive marker for the early diagnosis and evaluation of heart failure, as its blood level increases from a mild stage of cardiac dysfunction. Serum NT-proBNP in childhood was reported by Schwachtgen. Some references have investigated the usefulness of urinary NT-proBNP, however, it is still very controversial. We measured the serum and urinary NT-proBNP levels in neonates, and evaluated their correlation and the usefulness of urinary NT-proBNP. The 36 serum and urine samples were collected at the age of 0–25 days from 9 neonates with a birthweight of 659–3330 g (median: 2864 g), who were born at 25–41 (median: 37) weeks’ gestation at Keio University Hospital between October 2007 and June 2008. Each NT-proBNP concentration was determined by ElectroChemi-Luminescence Immunoassay using modular analytics (Roche Diagnostics Co. Ltd, Tokyo, Japan). The serum and urinary sample needed is just 200 mL, and it takes about 20 min for analysis. Neonates who showed severe neonatal asphyxia, congenital heart disease, congenital malformation, or chromosomal anomaly were excluded. Urine samples were collected within 2 h before or after blood collection. Written informed consent was obtained from the neonates’ parents prior to the study. Serum level of NT-proBNP was in agreement with the normal range in the neonatal period reported by Schwachtgen. A significant correlation with a coefficient of determination of 0.548 was observed in the regression equation obtained by the least squares method between the serum and urinary NT-proBNP levels. The serum and urinary level of NT-proBNP was not normally distributed in each group. Furthermore, a low level of NT-proBNP was frequently observed, and we evaluated these data with a natural logarithm. As shown in Figure 1, similar significant correlation with a coefficient of determination of 0.703 was observed by the same method. NT-proBNP is considered to be excreted primarily via the kidney, and its metabolic routes via the muscle, liver, etc., have also been reported in adults. However, in neonates, the metabolic route via the muscle or liver may be negligible because of the small muscle mass and immature metabolic function of the liver. Blood samples cannot be collected in sufficient amounts from neonates, such as premature infants. The results of this study suggest that the cardiac load can be evaluated non-invasively according to the trend of changes in the urinary NT-proBNP level in neonates, especially extremely low-birthweight infants.

SOX9 dimerization domain mutation mimicking type 2 collagen disorder phenotype

The classification of bone dysplasia has relied on a clinical/radiographic interpretation and the identification of specific genetic alterations. The clinical presentation of the SOX9 mutation and type 2 collagen disorders overlap with the Pierre-Robin sequence and talipes equinovarus, but the former is often accompanied by the bent long bones. In its milder form, the SOX9 mutation is not necessarily associated with the bent long bones. Here, we report a patient with the Pierre-Robin sequence and talipes equinovarus who did not exhibit either bent long bones or scapular hypoplasia; thus, this patient was instead classified as having a type 2 collagen disorder. Despite this phenotypic presentation, the proposita was found to have a de novo SOX9 mutation. The peculiar location of the mutation within the dimerization domain might account for the relatively mild phenotypic effect of the SOX9 mutation to a degree that is compatible with a clinical diagnosis of type 2 collagen disorder, except for a developmental delay. We concluded that mutations in SOX9 can mimic a type 2 collagen disorder-like phenotype.

Discordant fetal phenotype of hypophosphatasia in two siblings

Hypophosphatasia (HPP) is an autosomal recessive metabolic disorder with impaired bone mineralization due to mutations in the ALPL gene. The genotype‐phenotype correlation of this disorder has been widely described. Here, we present two affected siblings, whose fetal phenotypes were discordant. A 31‐year‐old Japanese woman, G0P0, was referred to our institution because of fetal micromelia. After obstetric counseling, the pregnancy was terminated at 21 weeks’ gestation. Post‐mortem radiographs demonstrated severely defective mineralization of the skeleton. The calvarial, spinal, and tubular bones were mostly missing. Only the occipital bones, mandible, clavicles, ribs, one thoracic vertebra, ilia, and tibia were relatively well ossified. The radiological findings suggested lethal HPP. Genetic testing for genomic DNA extracted from the umbilical cord identified compound heterozygous mutations in the ALPL gene (c.532T>C, p.Y178H; c.1559delT, p.Leu520Argfs*86). c.532T>C was a novel variant showing no residual activity of the protein by the functional analysis. The parents were heterozygous carriers. In the next pregnancy, biometric values on fetal ultrasonography at 20 and 26 weeks’ gestation were normal. At 34 weeks, however, a small chest and shortening of distal long bones came to attention. The neonate delivered at 41 weeks showed serum ALP of <5U/L. Radiological examination showed only mild thoracic hypoplasia and metaphyseal mineralization defects of the long bones. ALP replacement therapy was introduced shortly after birth, and the neonate was discharged at day 22 without respiratory distress. Awareness of discordant fetal phenotypes in siblings with HPP precludes a diagnostic error, and enables early medical intervention to mildly affected neonates.

Metabolic acidosis due to continuous drainage of massive chylous pleural effusion in two neonates

There are two types of metabolic acidosis: one resulting from acid accumulation, and the other from HCO3 loss. The latter type is often associated with renal or intestinal HCO3 loss. We report two neonates who presented with metabolic acidosis due to continuous drainage of chylous pleural effusion and required sodium bicarbonate. Case 1: after normal pregnancy and threatened premature labor, baby 1 was born at 31 weeks 2 days of gestation with a birthweight of 1344 g. Because there was no spontaneous breathing, the child was put on a respirator. Bilateral pleural effusion gradually increased, and chest drainage was started at the age of 16 days. Because chylous pleural effusion fluctuated between 100 and 500 mL per day, physiological saline and albumin were given continuously to maintain the circulating plasma volume. The baby was diagnosed with congenital myotonic dystrophy, and the pleural effusion was considered associated with this condition. Octreotide and prednisolone were given to reduce the pleural effusion but no response was observed, and the baby died at the age of 126 days. Metabolic acidosis persisted throughout the clinical course. Arterial blood gas analysis before sodium bicarbonate was as follows: pH, 7.066; pCO2, 56.8 mmHg; base excess, -12.9 mEq/L; Na, 133 mEq/L; K, 5.2 mEq/L; Cl, 111 mEq/L; anion gap (AG), 9.9 mEq/L. Normal anion gap suggested that the metabolic acidosis was due to HCO3 loss. Stools were small in quantity, excluding the loss of HCO3 from the gastrointestinal tract. The urinary pH was 4.91 at a time when the serum pH was 7.03, 5.04 at a time when the serum pH was 7.38, excluding renal tubular acidosis. The dose of sodium bicarbonate required for pH adjustment changed with fluctuations in the amount of pleural effusion, suggesting HCO3 loss into the pleural effusion. It is not possible to measure HCO3 in the pleural effusion directly. Instead, we measured pH of the pleural effusion several times, and this was close to serum pH. For example, pH of pleural effusion was 6.992 at a time when the serum pH was 7.066, and 7.393 at a time when the serum pH was 7.336. We therefore estimated HCO3 loss assuming that HCO3 in the pleural effusion is the same as serum HCO3. The estimated HCO3 loss into the pleural effusion approximated the HCO3 dose given (Fig. 1a). Case 2: baby 2 developed pleural effusion from 30 weeks of gestation, and underwent thoracentesis with basket catheter placement. The infant was born at 33 weeks 4 days of gestation with a birthweight of 2055 g, and was placed on a respirator. Chest drainage for pleural effusion was started at the age of 2 days. Pleural effusion fluctuated between 100 and 500 mL per day, and the circulating plasma volume was adjusted with physiological saline, albumin, fresh frozen plasma (FFP), and gammaglobulin. At the age of 37 days or later, physiological saline was replaced with Lactated Ringer. The pleural effusion subsided after octreotide, thoracic duct ligation, and intrapleural infusions of autologous blood, and the baby was discharged at the age of 111 days. As in baby 1, metabolic acidosis with a normal anion gap was observed, requiring supplementation with sodium bicarbonate. Clinical and laboratory findings excluded HCO3 loss from the kidney or gastrointestinal tract. The estimated HCO3 loss into the pleural effusion approximated the HCO3 dose given (Fig. 1b). Metabolic acidosis with a normal anion gap is generally associated with HCO3 loss in the kidney or gastrointestinal tract. Siegler et al. reported that a 3-month-old infant with massive chylous pleural effusion resulting from postoperative thoracic duct injury presented with metabolic acidosis. The two infants reported here differed regarding the cause of chylous pleural effusion, the underlying disease, and the clinical course, but the clinical and laboratory findings suggest that the loss of HCO3 into the pleural effusion led to metabolic acidosis. We speculate that the continuous drainage of a large amount of chylous pleural effusion resulted in a reduction in plasma HCO3, and the circulating plasma volume was maintained with physiological saline and albumin, leading to dilutional metabolic acidosis. Baby 2 did not require sodium bicarbonate at the age of 23 days or later because of FFP use. At the age of 37 days or later, physiological saline was replaced with Lactated Ringer. This may have eliminated the need for sodium bicarbonate, but it was difficult to evaluate the effect of Lactated Ringer because of the decrease of pleural effusion. In the present study we estimated HCO3 loss to the pleural effusion by using serum HCO3, because serum pH always nearly equaled that of pleural effusion. But this does not mean that serum HCO3 concentration always equals that of the pleural effusion. This is a limitation of the study. It is hoped that a method to measure HCO3 concentration of the pleural effusion Correspondence: Emi Okishio, MD, Division of Neonatology, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: e_oxy@yahoo.co.jp Received 22 January 2012; revised 6 June 2012; accepted 20 June 2012. bs_bs_banner

Hepatocyte growth factor levels of cord blood in healthy term newborns

Hepatocyte growth factor (HGF) has been reported recently to be a prognostic factor of acute pulmonary disorders. Regarding the clinical relationships of HGF with neonates, Khan et al reported the cord blood serum HGF level in neonates in 1996.1 Since the serum HGF level in normal neonates was 0.66 to 1.33 ng/dl, which was about three times higher than the adult reference value, and increased with the gestational age, they …

Surfactant protein A in gastric fluid at birth as a useful marker of differentiation diagnosis between respiratory distress syndrome and transient tachypnea of the newborn.

In premature babies born after 30 weeks’ gestation, it is hard to differentiate respiratory distress syndrome (RDS) from other respiratory disorders, such as transient tachypnea of the newborn (TTN), based on chest X-ray and clinical findings. Surfactant protein A (SP-A) is synthesized in alveolar type II cells. It is known that the maturity of the fetal lung can be estimated by measuring SP-A concentration in amniotic fluid. However, amniotic fluid collection at birth is difficult. As an index of the maturity of the fetal lung at birth, the microbubble test is commonly employed in Japan. While some studies have investigated the SP-A concentration in gastric fluid instead of amniotic fluid at birth, there is no publication written in English, and cut-off values vary due to limited data. In this study, we performed a large-scale, retrospective analysis to examine the association between the gastric fluid concentration of SP-A and clinical symptoms in 179 neonates born in our hospital. We reviewed the cut-off concentration of SP-A in the gastric fluid of neonates with RDS. The subjects were 206 normal-/premature-birth neonates born in the neonatal intensive care unit (NICU) of Keio University Hospital from November 2005 to October 2010. Immediately after birth, gastric fluid was collected from these neonates using a gastric tube to measure the concentration of SP-A employing an enzyme immunoassay (Sysmex Corporation, Hyogo, Japan). The subjects were divided into two groups by their clinical symptoms: the RDS and non-RDS groups. Statistical analysis was performed using the Mann–Whitney U-test. We reviewed 179 neonates, excluding four with meconium aspiration syndrome and 23 in whom gastric fluid collection was impossible (RDS group, seven; non-RDS group, 16). Sixty-six of the 179 neonates were diagnosed with RDS clinically, and 113 with non-RDS disorders. There were significant differences in the birthweight and gestational age between the RDS and non-RDS groups. There were no differences in the sex, prenatal steroid therapy, or cesarean section rate (Table 1). The median concentrations of SP-A of gastric fluid in the RDS and non-RDS groups were 310 and 1870 ng/mL, respectively. There was a significant difference between the two groups, employing the Mann–Whitney U-test (Fig. 1). We conducted a large-scale study involving 179 neonates. In the RDS group, the SP-A concentration in gastric fluid was significantly lower. In this study, the subjects were divided into the RDS and non-RDS groups based on conventional clinical evaluation. Therefore, while clinical features suggested RDS in some cases, the SP-A concentrations were high. In non-RDS, a diagnosis of non-RDS disorders, such as TTN, was made, but the SP-A concentrations were low. In particular, among neonates with SP-A concentration in gastric fluid between 500 and 2000 ng/mL, RDS and non-RDS neonates may have been mixed in the two groups. The concentration of SP-A in gastric fluid may become a useful test parameter for clarifying the pathogenesis in such cases. Several studies reported that surfactant-like