Hsin-Chun Huang

miRNA-125b regulates TNF-α production in CD14+ neonatal monocytes via post-transcriptional regulation

Neonates, although deficient in cell immunity, frequently reveal sepsis with augmented proinflammatory reactions. Here, we found that neonatal monocytes produced significantly higher TNF‐α mRNA and protein than adult monocytes. Assessment of the transcriptional factor found no significant difference of NF‐κB p65 level between neonatal and adult monocytes. Addition of Act D to access the half‐life of TNF‐α mRNA revealed no significant difference of the LPS‐induced TNF‐α mRNA half‐life between them, whereas CHX increased neonatal TNF‐α mRNA significantly. This suggests that a post‐transcriptional mechanism involves the augmentation of TNF‐α production by neonatal monocytes. To examine whether miRNA was involved in the post‐transcriptional regulation, differential displays of miRNA array between neonatal and adult MNCs were performed, along with the discovery of hsa‐miR‐103, hsa‐miR‐125b, hsa‐miR‐130a, hsa‐miR‐454‐3p, and hsa‐miR‐542‐3p, which were greater than a twofold decrease or increase after LPS treatment for 4 h. The functional validation identified that miR‐125b decreased significantly in association with higher TNF‐α expression by neonatal monocytes after LPS stimulation. Transfection of the miR‐125b precursor into neonatal monocytes significantly repressed the TNF‐α mRNA and protein expression, suggesting that miR‐125b negatively regulates TNF‐α expression in neonatal monocytes. Modulation of miRNA expression may be used to regulate TNF‐α production in newborns with altered proinflammatory reactions.

IFN-α production by human mononuclear cells infected with varicella-zoster virus through TLR9-dependent and -independent pathways

Understanding the defense mechanisms of the host of an organism is important for infection control. In previous studies, we demonstrated that interferon-α (IFN-α), but not IL-12, was produced by human peripheral blood mononuclear cells infected with varicella-zoster virus (VZV). Here, we investigated what kind of cell(s) and which signal molecule(s) are involved in IFN-α production. Using cell isolation and ELISA, we found that plasmacytoid dendritic cells (pDCs) were responsible for IFN-α production during VZV infection. We also found that Toll-like receptor 9 (TLR9) was involved in VZV-induced IFN-α production because inhibitory CpG oligodeoxynucleotide inhibited IFN-α production. UV-inactivated VZV-induced IFN-α production was lower than that of active VZV, indicating another TLR9-independent pathway. Further studies demonstrated that double-stranded RNA-dependent protein kinase, but not DNA-dependent protein kinase was involved in VZV-induced IFN-α production. Together, these results suggest that pDCs play an important role in IFN-α production during VZV infection through TLR9-dependent and -independent pathways.

Partial Protein-Hydrolyzed Infant Formula Decreased Food Sensitization but Not Allergic Diseases in a Prospective Birth Cohort Study

Background: Exposure to cow’s milk protein in early infancy could lead to increased rates of allergic diseases later in life. We investigated whether feeding a protein-hydrolyzed formula (HF) in the first 6 months of life decreased allergic diseases up to 36 months later. Methods: Newborns who had at least 1 first-degree family member with a history of atopy and could not breast-feed were enrolled. They were fed with HF or cow’s milk infant formula (CM) for at least 6 months via an open-label protocol and were monitored prospectively at 6, 18 and 36 months of age to assess allergy sensitization and allergic diseases. Results: A total of 1,002 infants were enrolled and 679 infants were consistently fed the same formula for the first 6 months of life (345 HF and 334 CM). The percentage of food sensitization (especially to milk protein) was significantly lower in the HF group than in the CM group at 36 months (12.7 vs. 23.4%, p = 0.048). There was no significant difference in the prevalence of aeroallergen sensitization between the groups. Occurrence of allergic diseases during the first 3 years of life was significantly correlated with aeroallergen sensitization, but not to food allergen sensitization, parental atopy or feeding types. Conclusions: Infants fed with HF during the first 6 months of life had a significantly lower percentage of sensitization to milk protein allergens, but not allergic diseases during the first 3 years of life. Avoidance of cow’s milk protein alone in infancy is not enough to decrease rates of allergic diseases.

Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children.

To study differences in the development of immunity, leukocytes from cord blood are often compared with those from adult peripheral blood. Western blot analysis is a common method for detecting proteins. In this study, we investigated the reliability of using different housekeeping proteins (β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) as internal controls for different leukocyte subpopulations from infants, children, and adults. Our results showed that the expression levels of β-actin and β-tubulin were much lower in cord blood leukocytes than in adult leukocytes, and this expression pattern persisted in children up to 3 years old. Further study revealed that the β-actin expression level in newborns was especially lower in CD14-positive monocytes. However, cord blood and adult peripheral blood monocytes had similar expression levels of β-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low β-actin expression level in neonatal monocytes. Thus, researchers should carefully assess the appropriate use of housekeeping gene-encoded proteins as internal standards to normalize samples for comparisons of different leukocyte populations from subjects of different ages. In this study, we determined that GAPDH was a more reliable internal control than others in Western blot analysis for comparing the development of immunity among infants, children, and adults.

Late-presenting congenital diaphragmatic hernia in childhood

Aim:  To characterize the clinical manifestations of late‐presenting diaphragmatic hernia and its associated anomalies, diagnostic methods and outcomes.

l-Arginine modulates neonatal lymphocyte proliferation through an interleukin-2 independent pathway

In cases of arginine depletion, lymphocyte proliferation, cytokine production and CD3ζ chain expression are all diminished. In addition to myeloid suppressor cells, polymorphonuclear cells (PMN) also exert T‐cell immune suppressive effects through arginase‐induced l‐arginine depletion, especially during pregnancy. In this study, we investigated how arginase/l‐arginine modulates neonatal lymphocyte proliferation. Results showed that the neonatal plasma l‐arginine level was lower than in adults (48·1 ± 11·3 versus 86·5 ± 14·6 μm; P = 0·003). Neonatal PMN had a greater abundance of arginase I protein than adult PMN. Both transcriptional regulation and post‐transcriptional regulation were responsible for the higher arginase I expression of neonatal PMN. Exogenous l‐arginine enhanced neonate lymphocyte proliferation but not that of adult cells. The RNA‐binding protein HuR was important but was not the only modulation factor in l‐arginine‐regulated neonatal T‐cell proliferation. l‐Arginine‐mediated neonatal lymphocyte proliferation could not be blocked by interleukin‐2 receptor blocking antibodies. These results suggest that the altered arginase/l‐arginine cascade may be one of the mechanisms that contribute to altered neonatal immune responses. Exogenous l‐arginine could enhance neonate lymphocyte proliferation through an interleukin‐2‐independent pathway.

Identification of immunodeficient molecules in neonatal mononuclear cells by proteomic differential displays.

Human newborns are known to be susceptible to microbial infection. This susceptibility is generally attributed to immaturity of the newborn immune system. However, the mechanisms for impaired immunity in newborns are still incompletely defined. In this study, we sought to elucidate the protein differential display between adult and neonatal mononuclear cells (MNC) using a proteomic approach. MNC samples from cord blood and adult peripheral blood were subjected to 2‐D PAGE analysis. Differential protein displays between cord blood and adult MNC were determined and validated. There were 34 differentially expressed proteins between cord blood and adult MNC identified by 2‐D PAGE. The differentially displayed proteins were clustered into two major signal pathways, cellular processing and purine metabolism. After validation by Western blot, we found more abundant arginase‐1 (ARG1) and Rho GDP‐dissociation inhibitor 2 (RhoGDI2), while less adenosine deaminase (ADA) and β‐actin in cord blood MNC. In functional validation, we found that lower ADA was proven to enhance the TNF‐α production by cord blood monocytes. The results from this study discovered the proteomic displays for altered immunity between adult and neonatal MNC that support a understanding of the correction of impaired immune response in newborns.

Congenital diaphragmatic hernia in the neonatal period: review of 21 years' experience.

BACKGROUND Despite advances in therapeutic modalities, congenital diaphragmatic hernia (CDH) still accounts for significant neonatal mortality. This study aimed to describe the demographic features, clinical experiences of postnatal care, and differences between non-survivors and survivors with CDH. METHODS We retrospectively reviewed medical records of neonates with CDH admitted to Kaohsiung Chang Gung Memorial Hospital over a 21-year period. Neonates with diaphragmatic eventration and those transferred after surgery were excluded. RESULTS A total of 24 live-born neonates fulfilled the study criteria; 13 (54%) were boys and 11 (46%) were girls. Eight (33%) patients were prenatally diagnosed. The mean gestational age was 38.8 +/- 1.8 weeks (range, 35-41 weeks). Twenty-three (96%) had Bochdalek hernia [19 (83%) left-sided, 4 (17%) right-sided], and one (4%) had right-sided Morgagni hernia. Additional major congenital anomalies were identified in five patients (21%). The overall mortality was 21% (5/24); all deaths occurred before surgery. Statistically significant differences between survivors and non-survivors were found for right-sided CDH, low 1-minute and 5-minute Apgar scores, and low pH of the first arterial blood gas. Deaths were attributed to severe persistent pulmonary hypertension, unresponsiveness to aggressive resuscitation at birth, and major associated malformations. CONCLUSION Seventy-nine percent of our CDH patients survived to hospital discharge. Resuscitation by a skilled neonatology team to prevent low Apgar scores and low pH, careful evaluation of other anomalies, and overcoming pulmonary hypertension might improve the survival rate. Recognizing unfavorable factors in CDH may help clinicians manage the critical care of these babies.

Correlation of augmented IL-8 production to premature chronic lung disease: implication of posttranscriptional regulation.

Despite that advances in neonatal medicine have significantly reduced the early mortality of premature infants, a considerable number of them are still prone to develop chronic lung disease (CLD) later. To find a method of early prevention, we investigated the efficacy of using certain early proinflammatory responses to predict the development of CLD. In the present study, 34 premature infants who required endotracheal intubation within 4 h of birth were recruited for analysis of IL-8, IL-10, and TNF-α levels in their bronchoalveolar lavage (BAL) fluid and blood. It was found that level of IL-8 but not TNF-α or IL-10 in initial BAL fluid was significantly correlated to neutrophils in the BAL and inversely correlated to the gestational age of prematurity. Elevation of IL-8 level in BAL on the first day of life was correlated to the development of CLD. Further studies showed that neonatal cord blood released significantly higher IL-8 but lower TNF-α levels after stimulation by endotoxin. The augmented IL-8 mRNA expression in cord blood was inhibited by actinomycin D but enhanced by cycloheximide, suggesting that IL-8 production is controlled by de novo transcriptional induction as well as posttranscriptional up-regulation of IL-8 by neonatal leukocytes, relating to the development of CLD. Thus, an appropriate modulation of initial IL-8 production in premature infants might be beneficial for the prevention of the development of CLD.

Expressed breast milk for procedural pain in preterm neonates: a randomized, double‐blind, placebo‐controlled trial

To determine whether expressed breast milk (milk) reduces procedural pain associated with heel lancing in preterm neonates.