Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant.

BACKGROUND: Bleomycin (BLM)-induced lung injury is characterized by mixed histopathologic changes with inflammation and fibrosis, such as observed in human patients with bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Although no curative therapies for these lung diseases exist, stem cell therapy has emerged as a potential therapeutic option. Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent- and macrophage-like stem cells distributed in various adult and fetal tissues as stage-specific embryonic antigen-3-positive cells. They selectively home to damaged tissue by sensing sphingosine-1-phosphate and replace the damaged/apoptotic cells by in vivo differentiation. Clinical trials for some human diseases suggest the safety and therapeutic efficacy of intravenously injected human leukocyte antigen-mismatched allogenic Muse cells from adult bone marrow (BM) without immunosuppressant. Here, we evaluated the therapeutic effects of human Muse cells from preterm and term umbilical cord (UC), and adult BM in a rat BLM-induced lung injury model. METHODS: Rats were endotracheally administered BLM to induce lung injury on day 0. On day 3, human preterm UC-Muse, term UC-Muse, or adult BM-Muse cells were administered intravenously without immunosuppressants, and rats were subjected to histopathologic analysis on day 21. Body weight, serum surfactant protein D (SP-D) levels, and oxygen saturation (SpO2) were monitored. Histopathologic lung injury scoring by the Ashcroft and modified American Thoracic Society document scales, quantitative characterization of engrafted Muse cells, RNA sequencing analysis, and in vitro migration assay of infused Muse cells were performed. RESULTS: Rats administered preterm- and term-UC-Muse cells exhibited a significantly better recovery based on weight loss, serum SP-D levels, SpO2, and histopathologic lung injury scores, and a significantly higher rate of both Muse cell homing to the lung and alveolar marker expression (podoplanin and prosurfactant protein-C) than rats administered BM-Muse cells. Rats receiving preterm-UC-Muse cells showed statistically superior results to those receiving term-UC-Muse cells in many of the measures. These findings are thought to be due to higher expression of genes related to cell migration, lung differentiation, and cell adhesion. CONCLUSION: Preterm UC-Muse cells deliver more efficient therapeutic effects than term UC- and BM-Muse cells for treating BLM-induced lung injury in a rat model.

Comparison of Two Hepatitis B Vaccination Strategies Targeting Vertical Transmission: A 10-Year Japanese Multicenter Prospective Cohort Study.

In 1985, a hepatitis B (HB) vaccination strategy against vertical HB virus transmission was introduced in Japan that recommended vaccination of infants at two, three, and five months of age (delayed strategy). This schedule was revised in 2013, recommending to vaccinate at birth and at 1 and 6 months of age (non-delayed strategy). We aimed to compare the vertical HB virus transmission rates and immunogenic responses between these two vaccination strategies. This Japanese multicenter prospective cohort study included 222 infants born between 2008 and 2017 to serum hepatitis B surface (HBs) antigen (HBsAg)-positive mothers. During the study period, 136 and 86 infants received delayed and non-delayed strategies, respectively. A positive vertical HB virus transmission was defined as a positive serum HBsAg status. Seropositive immunogenic response was defined as a serum anti-HBs titer of ≥10 mIU/mL. Post-vaccination serum HBsAg positivity rates did not differ significantly between the delayed (0/136 [0.0%, 95% confidence interval, 0.0-2.7%]) and non-delayed (2/86 [2.3%, 95% confidence interval, 0.3-8.1%]) strategy groups. Seropositive immunogenic response rates were 100.0% (136/136) and 97.7% (84/86), respectively. Although this study was under-powered to detect a statistically significant result, no vertical HB virus transmission was observed in the delayed strategy.

A Novel Method for Measuring Serum Unbound Bilirubin Levels Using Glucose Oxidase-Peroxidase and Bilirubin-Inducible Fluorescent Protein (UnaG): No Influence of Direct Bilirubin.

The glucose oxidase-peroxidase (GOD-POD) method used to measure serum unbound bilirubin (UB) suffers from direct bilirubin (DB) interference. Using a bilirubin-inducible fluorescent protein from eel muscle (UnaG), a novel GOD-POD-UnaG method for measuring UB was developed. Newborn sera with an indirect bilirubin/albumin (iDB/A) molar ratio of <0.5 were classified into four groups of DB/total serum bilirubin (TB) ratios (<5%, 5-10%, 10-20%, and ≥20%), and the correlation between the UB levels and iDB/A ratio was examined. Linear regression analysis was performed to compare UB values from both methods with the iDB/A ratio from 38 sera samples with DB/TB ratio <5% and 11 samples with DB/TB ratio ≥5%. The correlation coefficient (r) between UB values and the iDB/A ratio for the GOD-POD method was 0.8096 (DB/TB ratio <5%, n = 239), 0.7265 (5-10%, n = 29), 0.7165 (10-20%, n = 17), and 0.4816 (≥20%, n = 16). UB values using the GOD-POD-UnaG method highly correlated with the iDB/A ratio in both <5% and ≥5% DB/TB ratio sera (r = 0.887 and 0.806, respectively), whereas a low correlation (r = 0.428) occurred for ≥5% DB/TB ratio sera using the GOD-POD method. Our GOD-POD-UnaG method can measure UB levels regardless of the presence of DB.

Efficacy of Valganciclovir Treatment Depends on the Severity of Hearing Dysfunction in Symptomatic Infants with Congenital Cytomegalovirus Infection.

Although earlier studies have shown that antiviral treatment regimens using valganciclovir (VGCV) improved hearing function in some infants with congenital cytomegalovirus (CMV) infection; its efficacy on the severity of hearing dysfunction is unclear. We conducted a prospective study among 26 infants with congenital CMV infections from 2009 to 2018. Oral VGCV (32 mg/kg/day) was administered for 6 weeks (November 2009 to June 2015; n = 20) or 6 months (July 2015 to March 2018, n = 6). Hearing function was evaluated by measuring the auditory brainstem response before VGCV treatment and at 6 months. Hearing dysfunction, defined as a V-wave threshold >40 dB, was categorized into: most severe, ≥91 dB; severe, 61⁻90 dB; and moderate, 41⁻60 dB. Hearing improvement was defined as a decrease of ≥20 dB from the pretreatment V-wave threshold. Of 52 ears in 26 infants with congenital CMV infection, 29 (56%) had hearing dysfunction, and of 29 ears, 16 (55%) improved after VGCV treatment. Although, 16 (84%) of 19 ears with moderate or severe hearing dysfunction improved after treatment (p < 0.001), 10 ears with the most severe form did not. In conclusion, VGCV treatment is effective in improving moderate and severe hearing dysfunction in infants with congenital CMV infection.

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Hyperbilirubinemia in Term Newborns Needing Phototherapy within 48 Hours after Birth in a Japanese Birth Center.

BACKGROUND: Hyperbilirubinemia in term newborns needing phototherapy within 48 hours after birth, early-onset hyperbilirubinemia, has not been evaluated in recent Japanese healthy birth centers. In this study, we sought to determine the cause of early-onset hyperbilirubinemia in a Japanese healthy birth center and to evaluate the 1992 Kobe University phototherapy treatment criterion requiring total serum bilirubin (TSB) and unbound bilirubin (UB). METHODS: In this retrospective observational study, we collected data on newborns diagnosed with early-onset hyperbilirubinemia between 2009 and 2016 at the Chibune General Hospital. Causes of the disease were investigated, as well as which index (TSB or UB) was used for treatment decisions. RESULTS: Overall, 76 term newborns were included in the analysis. Twenty-seven newborns (36%) found the cause (ABO blood type incompatibility [n=17, 22%], polycythemia [n=8, 11%], and cephalohematoma [n=2, 3%]). However, 49 newborns (64%) did not find any causes (i.e., idiopathic hyperbilirubinemia). Of these, 27 observed more than 5% weight loss from birth weight. Seventy (92%) newborns had abnormal TSB only, and 5 (7%) had abnormal TSB and UB values. Only 1 (1%) newborn with only abnormal UB values received phototherapy. CONCLUSIONS: Altogether, data from this Japanese healthy birth center suggest that many apparently healthy newborns with or without excessive weight loss develop early-onset hyperbilirubinemia. In the 1992 Kobe University phototherapy treatment criterion, TSB, not UB, was the main index used to make treatment decisions in these patients.

Nontypeable Haemophilus Influenzae Sepsis in a Term Neonate.

We reported a term newborn case of early onset sepsis caused by nontypeable Haemophilus Influenzae (NTHi) with massive bacterial invasion in the placenta. Based on the consistent results of maternal placental pathology and neonatal bacterial culture, we diagnosed this as vertical transmission of NTHi via vaginal delivery. In general, NTHi infections occur in preterm infants, and our term infant case is very unusual. In conclusion, clinicians should consider NTHi as a cause of neonatal sepsis, even in term infants.

A Case of Congenital Complete Atrioventricular Block Treated with Transdermal Tulobuterol.

Congenital complete atrioventricular block (CCAVB) is a condition in which the atria and ventricles beat independently of each other. CCAVB cases require permanent pacemaker implantation until adulthood. Nevertheless, consensus regarding postnatal medical therapy for bradycardia has not been reached. Here we report the case of a newborn with CCAVB, whose intractable bradycardia was successfully treated with transdermal tulobuterol. Tulobuterol is a selective ß2-adrenoceptor agonist, widely used safely as bronchodilator in children. It also has positive inotropic and chronotropic effect via ß1-adrenoceptors. We believe the tulobuterol patch can be used as an optional therapy for CCAVB where pacemaker implantation is not available.

Involvement of WNT Signaling in the Regulation of Gestational Age-Dependent Umbilical Cord-Derived Mesenchymal Stem Cell Proliferation.

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that is isolated initially from the bone marrow (BM) and subsequently almost all tissues including umbilical cord (UC). UC-derived MSCs (UC-MSCs) have attracted an increasing attention as a source for cell therapy against various degenerative diseases due to their vigorous proliferation and differentiation. Although the cell proliferation and differentiation of BM-derived MSCs is known to decline with age, the functional difference between preterm and term UC-MSCs is poorly characterized. In the present study, we isolated UC-MSCs from 23 infants delivered at 22-40 weeks of gestation and analyzed their gene expression and cell proliferation. Microarray analysis revealed that global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. WNT signaling impacts on a variety of tissue stem cell proliferation and differentiation, and its pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. Cell proliferation of preterm UC-MSCs was significantly enhanced compared to term UC-MSCs and counteracted by WNT signaling inhibitor XAV939. Furthermore, WNT2B expression in UC-MSCs showed a significant negative correlation with gestational age (GA). These results suggest that WNT signaling is involved in the regulation of GA-dependent UC-MSC proliferation.

Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN) 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs) are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt symptoms. Although recent translational studies of SMN-targeted therapies have revealed the very limited time window for effective SMA therapies during perinatal period, the exact time point when SMN shortage became evident is unknown in human samples. In this study, we analyzed SMN mRNA and protein expression during perinatal period by using umbilical cord-derived mesenchymal stem cells (UC-MSCs) obtained from preterm and term infants. METHODS: UC-MSCs were isolated from 16 control infants delivered at 22-40 weeks of gestation and SMA fetus aborted at 19 weeks of gestation (UC-MSC-Control and UC-MSC-SMA). FBs were isolated from control volunteer and SMA patient (FB-Control and FB-SMA). SMN mRNA and protein expression in UC-MSCs and FBs was determined by RT-qPCR and Western blot. RESULTS: UC-MSC-Control and UC-MSC-SMA expressed the comparable level of MSC markers on their cell surface and were able to differentiate into adipocytes, osteocytes, and chondrocytes. At steady state, SMN mRNA and protein expression was decreased in UC-MSC-SMA compared to UC-MSC-Control, as observed in FB-SMA and FB-Control. In response to histone deacetylase inhibitor valproic acid, SMN mRNA and protein expression in UC-MSC-SMA and FB-SMA was increased. During perinatal development from 22 to 40 weeks of gestation, SMN mRNA and protein expression in UC-MSC-Control was positively correlated with gestational age. CONCLUSION: UC-MSCs isolated from 17 fetus/infant of 19-40 weeks of gestation are expressed functional SMN mRNA and protein. SMN mRNA and protein expression in UC-MSCs is increased with gestational age during perinatal development.

Corrigendum: Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

This corrects the article DOI: 10.1038/jhg.2017.48.

Loss of Surfacten® during bolus administration with a feeding catheter.

BACKGROUND: Surfactant replacement therapy is widely used for treating neonatal respiratory distress syndrome, but insufficient evidence is available on the use of Surfacten® (S-TA). This study investigated the inadvertent loss of S-TA during instillation via feeding catheters with different bore sizes. METHODS: In this bench-based study, we measured the weight of syringes and tubes before and after surfactant treatment using a high-accuracy balance, and determined the amount of S-TA lost in tubes. We injected 120 mg of S-TA suspended in 4 or 3 mL into tubes followed with or without air boluses. Experiments were performed in triplicate. Percent weight loss of S-TA in each tube was calculated with or without air boluses. RESULTS: Percent weight loss of S-TA was significantly higher in larger-bore tubes (P < 0.01, overall ANOVA), and was significantly lower after air bolus flushing in 3 Fr, 4 Fr, and 5 Fr tubes (P < 0.005, respectively). The 3 mL S-TA suspensions had a significantly higher percent loss than the 4 mL S-TA suspensions when using 4 Fr and 5 Fr tubes, and the 5 Fr closed system (P < 0.05, respectively). CONCLUSIONS: Routine air bolus flushing effectively reduces S-TA loss in tubes. The 3 mL S-TA suspensions appear to be more susceptible to inadvertent S-TA loss during instillation. Therefore, caution is warranted for this procedure.

Evaluation of BiliCare™ transcutaneous bilirubin device in Japanese newborns.

BACKGROUND: Non-invasive transcutaneous bilirubin (TcB) monitoring has been widely used to screen for hyperbilirubinemia. TcB measured using the recently developed BiliCare™ system, however, has not been fully evaluated. METHODS: One hundred and seven TcB measurements were obtained from 82 Japanese newborns ≥35 weeks' gestational age within 2 weeks after birth. Measurements were taken at the scaphoid fossa, conchal cavity, and lobe of the ear using BiliCare. BiliCare TcB were compared with total serum bilirubin (TB) and TcB obtained using another bilirubinometer (JM-105™). RESULTS: Transcutaneous bilirubin measured at all three sites significantly correlated with TB (r = 0.91, 0.93, and 0.93 at the scaphoid fossa, conchal cavity, and lobe, respectively). The mean differences were 0.1, -0.3, and 3.6 at the scaphoid fossa, conchal cavity, and lobe, respectively. BiliCare TcB at the scaphoid fossa significantly correlated with that using the JM-105 (r = 0.91). The mean difference was 0.0. BiliCare, however, produced a significantly higher and lower TcB than the JM-105 for TB <7 and ≥15 mg/dL, respectively. CONCLUSIONS: Transcutaneous bilirubin measurements taken at the scaphoid fossa or conchal cavity using BiliCare were more reliable than those at the earlobe. BiliCare TcB differed from those of the JM-105, for TB <7 or ≥15 mg/dL.

Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

Adams-Oliver syndrome (AOS, OMIM; 100300) is a rare genetic disease characterized by aplasia cutis congenita, terminal transverse limb defects and cutis marmorata with vascular anomalies such as congenital heart defects. The etiology of this syndrome has remained largely unknown but defective Notch signaling during vascular formation has been suggested. Here we describe a sporadic Japanese newborn case with clinically diagnosed AOS. Trio whole-exome sequencing identified a de novo, novel, heterozygous missense mutation in the Delta-like 4 ligand gene (DLL4 c.572G>A, p.Arg191His) in the patient. DLL4 functions as a requisite ligand for NOTCH1 receptor, which is essential for vascular formation. Amino acid substitution of Arg191 to His was predicted by molecular models to interfere with direct binding between DLL4 and NOTCH1. DLL4 has recently been identified as a causative gene of an autosomal dominant type of AOS with milder symptoms. The case described here showed gradual recovery from skull defects after birth and no psychomotor developmental delay has been observed. This is the second report of an AOS case with DLL4 mutation, and the phenotypic characteristics between the two cases are compared and discussed.

Case of harlequin ichthyosis with a favorable outcome: Early treatment and novel, differentially expressed, alternatively spliced transcripts of the ATP-binding cassette subfamily A member 12 gene.

Harlequin ichthyosis (HI) is the most severe form of autosomal recessive congenital ichthyosis, with a high mortality rate. Recent advances in neonatal care and the early administration of retinoids have improved the survival rate of HI. Here, we present a case of HI who was successfully treated with early administration of etretinate and showed good prognosis. Next-generation sequencing identified novel mutations of the ATP-binding cassette subfamily A member 12 gene (ABCA12), c.5884+4_+5delAA and c.7239G>A, which caused skipping of exons 39 and 48, respectively. Transcripts with exon 48 skipping, which cause a deletion in the second ATP-binding cassette of ABCA12, were dominantly expressed in the skin. Besides the early administration of etretinate, the differential expression of the mutant protein with limited segmental deletion of ABCA12 may be related to the favorable outcome of our patient.

Gestational age-dependency of height and body mass index trajectories during the first 3 years in Japanese small-for-gestational age children.

Gestational age (GA) is thought to affect height growth in small-for-gestational age (SGA) children. However, the GA-specific trajectories in body mass index (BMI) and early appearances of adiposity rebound (AR) have not been fully investigated in a cohort of Japanese SGA children. A longitudinal cohort study was conducted with 1063 SGA children born in Kobe, Japan, with sufficient records from birth to 3 years of age. Subjects were divided into subgroups based on GA: 39-41 weeks GA (n = 723), 37-38 weeks GA (n = 256), 34-36 weeks GA (n = 62), and <34 weeks GA (n = 22). Height and BMI were assessed at 4 months, 9 months, 1.5 years, and 3 years of age. The catch-up rate for height was GA-dependent. Most children with 39-41 weeks GA (91%) caught up by 4 months of age; however, lower GA was associated with a slower elevation in the catch-up rate. The BMI trajectory during the first 3 years was also GA-dependent, with a change in GA dependency at a boundary of 37 weeks GA. Approximately 7% of SGA children had already developed AR before 3 years of age. In conclusion, growth patterns during infancy and early childhood in SGA children differ depending on GA.

Fluorescent protein-based detection of unconjugated bilirubin in newborn serum.

Increased serum levels of unconjugated bilirubin are associated with the development of brain damage in newborns. In current clinical settings, there are no methods for directly determining serum levels of unconjugated bilirubin. UnaG, a fluorescent protein from Japanese eel muscle that specifically binds to unconjugated bilirubin was used in this study. Linear regression analysis was carried out to compare unconjugated bilirubin levels measured by UnaG and conventional bilirubin oxidase methods. Unconjugated bilirubin levels in the serum of newborns who were untreated or treated with phototherapy were compared. Effects of interfering factors in the serum (conjugated bilirubin, hemoglobin, and lipid) on unconjugated bilirubin concentration measured by the UnaG method were also evaluated. Unconjugated bilirubin levels measured by the UnaG method were highly correlated with those determined by the bilirubin oxidase assay. Unconjugated bilirubin levels determined by bilirubin oxidase and UnaG assays were similar in serum samples containing conjugated bilirubin. The performance of the UnaG assay was unaffected by phototherapy and the presence of serum hemoglobin and lipid emulsion. These results demonstrate the clinical applicability of the UnaG method for direct measurement of unconjugated bilirubin levels in newborn serum.

Transcutaneous bilirubin monitoring predicts unexplained late-onset hemolysis in a very low birthweight infant.

BACKGROUND: In term infants, transcutaneous bilirubin (TcB) monitoring can be used to predict hemolytic hyperbilirubinemia. However, it is not clear whether the technique can also be used to predict unexplained late-onset hemolysis in very low birthweight (VLBW) infants. CASE PRESENTATION: The case was an infant with a birthweight of 1154 g who developed unexplained late-onset hemolysis at 8 days of age. The hyperbilirubinemia rapidly worsened, and therefore both phototherapy and exchange transfusion were performed. TcB levels were measured using the JM-105 jaundice meter and found to have increased by >3 mg/dL since before the onset, demonstrating for the first time that the device clearly detects changes in hemolytic rate. CONCLUSIONS: Although TcB levels did not correspond directly with total serum bilirubin levels in VLBW infants, the two values exhibited parallel changes in this case. Therefore, serial TcB monitoring may be useful in the early prediction of unexplained late-onset hemolysis in VLBW infants.

Extremely preterm infants small for gestational age are at risk for motor impairment at 3 years corrected age.

BACKGROUND: Few studies have targeted psychomotor development and associated perinatal risk factors in Japanese very low birth weight (VLBW) infants who are severely small for gestational age (SGA). DESIGN/SUBJECTS: A single-center study was conducted in 104 Japanese VLBW infants who were born preterm, due to maternal, umbilical cord, or placental abnormalities, between 2000 and 2007. Psychomotor development as a developmental quotient (DQ) was assessed using the Kyoto Scale of Psychological Development at 3 years corrected age. Severely SGA was defined as birth weight or length below -2 standard deviation values of the mean values at the same gestation. VLBW infants were divided into 2 subgroups based on gestational age at birth: ⩾28 weeks (n=64) and <28 weeks (n=40). DQs of infants with severe SGA were compared with those of infants who were appropriate for gestational age (AGA). Factors associated with developmental disabilities in VLBW infants with severe SGA (n=23) were determined. RESULTS: In the group born at ⩾28 weeks gestation, infants with severe SGA had normal DQ values and did not significantly differ from those with AGA. However, in the group born at <28 weeks gestation, severe SGA infants had significantly lower postural-motor DQ values than AGA infants. Gestational age <28 weeks was an independent factor for low postural-motor DQ, regardless of the cause of severe SGA or pregnancy termination. CONCLUSIONS: Extremely preterm newborns with severe SGA are at risk of motor developmental disability at age 3 years.