Identifying the essential knowledge and skills for Neonatal-Perinatal Medicine: a systematic analysis of practice.

The knowledge and skills expected for board certification in Neonatal-Perinatal Medicine (NPM) should reflect the clinical practice of neonatology. First, a 14-member panel of practicing neonatologists, convened by the American Board of Pediatrics (ABP), drafted a practice analysis document which identified the practice domains, tasks, knowledge, and skills deemed essential for clinical practice. NPM fellowship program directors provided feedback via online survey resulting in revisions to the document. During the second phase of the project, the panel organized testable knowledge areas into content domains and subdomains to update the existing ABP NPM content outline. All ABP board-certified neonatologists were asked to review via online survey, and results were used to guide final revisions to the content outline. The NPM practice analysis document and the updated NPM content outline should serve as helpful resources for educators, trainees, and practicing neonatologists.

Bacterial killing is enhanced by exogenous administration of lysozyme in the lungs.

OBJECTIVE: Lysozyme, a 14-kDa protein, is one of the most abundant antimicrobials in the lungs. Its concentration in airway surface sufficient to kill several bacterial pathogens in vitro. The purpose of this study was to determine if administration of exogenous lysozyme would further enhance bacterial killing in vivo. METHODS: To assess the effect of acute lung infection on endogenous lysozyme protein levels, mice were infected by intratracheal instillation of Pseudomonas aeruginosa and bronchoalveolar (BAL) fluid assessed for lysozyme concentration and for muramidase activity. In order to inform in vivo testing, species-specific bacterial killing efficacy was determined by incubating mucoid P. aeruginosa with 2×105 units of chicken lysozyme, human lysozyme or with vehicle at 37°C for 2hours. Subsequently, mice challenged with intratracheally-administered mucoid P. aeruginosa, were reintubated and injected with 2×105 Units of native human lysozyme, recombinant human lysozyme or with vehicle. Lung bacterial burden was enumerated subsequently. RESULTS: The concentration of lysozyme protein in BAL fluid from mice challenged with mucoid clinical isolate of P. aeruginosa was increased 4-fold at 6hours post-infection. Quantitative culture showed that the number of recoverable bacteria was significantly decreased by both chicken and human lysozyme compared to vehicle but human lysozyme was significantly more effective than chicken egg lysozyme. In vivo, 24hours post-infection quantitative culture of lung homogenates showed that the number of viable bacteria recovered from mice treated with either native or recombinant lysozyme was decreased with 0.76±0.25×104 and 0.84±0.16×104, respectively, vs. 7.0±2.52×104 CFU/g protein in mice treated with HBSS, both P<0.05. CONCLUSIONS: These results indicate that endogenous lysozyme is increased during acute lung infection and that early administration of exogenous lysozyme further enhances bacterial killing in vivo.

The lost art of intubation: assessing opportunities for residents to perform neonatal intubation.

OBJECTIVE: The objective of this study is to assess the opportunities afforded to and competence of pediatric residents in performing neonatal endotracheal intubations. STUDY DESIGN: The records of all intubations performed on neonates over a 3-year period at a university-based birthing hospital were reviewed to assess the relationships between outcomes, types of providers and the setting of intubations. RESULT: A total of 785 attempts were made during 362 intubations. Pediatric residents were given the opportunity to intubate 38% of the cohort (n=137) and were successful on 21% of the attempts. Residents were more likely to perform intubation in the neonatal intensive care unit (vs delivery room; P<0.001), in non-emergency situations (P<0.001), and on older (P<0.001) and larger (P=0.07) infants. CONCLUSION: Opportunities for residents to intubate neonates were few and their success rate was low. In the current care paradigm, it is doubtful if trainees can be sufficiently skilled in endotracheal intubation during residency. Residents that plan to pursue procedure-intensive subspecialties may benefit from other models for training.

Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice.

To assess the role of lysozyme in pulmonary host defense in vivo, transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated. Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar (BAL) lavage fluid was increased 2- or 4-fold relative to that in WT mice. Lung structure and cellular composition of BAL were not altered by the expression of lysozyme. Lysozyme activity in BAL was significantly increased (6.6- and 17-fold) in 5-wk-old animals from each transgenic line. To determine whether killing of bacteria was enhanced by expression of rat lysozyme, 5-wk-old transgenic mice and WT littermates were infected with 10(6) CFU of group B streptococci or 10(7) CFU of a mucoid strain of Pseudomonas aeruginosa by intratracheal injection. Killing of group B streptococci was significantly enhanced (2- and 3-fold) in the mouse transgenic lines at 6 h postinfection and was accompanied by a decrease in systemic dissemination of pathogen. Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines (5- and 30-fold). Twenty-four hours after administration of Pseudomonas aeruginosa, all transgenic mice survived, whereas 20% of the WT mice died. Increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo, and was associated with decreased systemic dissemination of pathogen and increased survival following infection.

Altered surfactant protein B levels in transgenic mice do not affect clearance of bacteria from the lungs.

To determine the role of surfactant protein B (SP-B) in bacterial clearance from the airways, three groups of mice expressing different levels of SP-B were studied: wild-type mice, hemizygous SP-B mice, and SP-B overexpressing transgenic mice. SP-B levels in overexpressing mice were increased 5-fold relative to hemizygous mice and 2- to 3-fold over wild-type littermates. Mice from each group were infected intratracheally with the common airway pathogens, group B streptococci or Pseudomonas aeruginosa. There was no significant difference in the number of recoverable viable bacteria at 6 h (group B streptococci and P. aeruginosa) and at 24 h (P. aeruginosa) among the three groups. Similarly, systemic dissemination of bacteria was not different among the three groups for both pathogens and at both time points. We conclude that SP-B levels in vivo do not influence clearance of bacteria from the lungs.

Neonatal jaundice. Strategies to reduce bilirubin-induced complications.

Neonatal hyperbilirubinemia is the most common reason for hospital readmission in the first 2 weeks of life. Kernicterus is still relatively uncommon but has been on the rise with the institution in the 1990's of aggressive early postnatal discharge policies. Bilirubin-induced complications can be prevented by instituting a neonatal jaundice protocol to identify infants at risk for significant hyperbilirubinemia, by ensuring adequate parental education and preparedness, and by implementing a good neonatal tracking system for follow-up care. Hyperbilirubinemia is easily treated with phototherapy, which can be administered at home in selected infants.

Surfactant protein B (SP-B) -/- mice are rescued by restoration of SP-B expression in alveolar type II cells but not Clara cells.

Surfactant protein B (SP-B) mRNA and protein are restricted to alveolar Type II and Clara cells in the respiratory epithelium. In order to investigate the function of SP-B in these distinct cell types, transgenic mice were generated in which SP-B expression was selectively restored in Type II cells or Clara cells of SP-B -/- mice. The 4.8-kilobase murine SP-C promoter was used to generate 3 transgenic lines which expressed human SP-B in Type II cells (mSP-C/hSP-B). Likewise, the 2.3-kilobase murine CCSP promoter was used to generate two transgenic lines which expressed human SP-B in Clara cells (mCCSP/hSP-B). mSP-C/hSP-B and mCCSP/hSP-B transgenic mice were subsequently bred to SP-B +/- mice in order to selectively express SP-B in Type II cells or Clara cells of SP-B -/- mice. Selective restoration of SP-B expression in Type II cells completely rescued the neonatal lethal phenotype in SP-B -/- mice. Expression of SP-B in some, but not all Type II cells of SP-B -/- mice, allowed postnatal survival, but resulted in significantly altered lung architecture and function. Selective restoration of SP-B expression in Clara cells of SP-B -/- mice resulted in respiratory dysfunction and invariable neonatal death, related to the complete absence of mature SP-B peptide in these mice. These results indicate that expression and processing of the SP-B proprotein to the mature peptide in Type II cells is absolutely required for lung function in vivo and that SP-B expression in Clara cells cannot substitute for this function.

Rescue of SP-B knockout mice with a truncated SP-B proprotein. Function of the C-terminal propeptide.

The function of the 102-amino acid C-terminal propeptide of surfactant protein B (SP-B) was analyzed by characterizing the phenotype associated with loss of expression of this peptide domain in transgenic mice. A construct encoding the signal peptide, N-terminal propeptide, and mature peptide of human SP-B (hSP-BDeltac) was cloned under the control of the 3.7-kilobase human SP-C promoter and injected into fertilized eggs of the FVB/N mouse strain. Founder mice expressing the hSP-BDeltac transgene were bred with heterozygous SP-B knockout mice (SP-B +/-). Offspring containing the transgene and one allele of mouse SP-B were identified and subsequently crossed to generate a transgenic line that expressed SP-BDeltac in a null background (SP-B(-/-)/hSP-BDeltac(+/+)). Expression of hSP-BDeltac in SP-B(-/-) mice was restricted to type II cells and resulted in a 2-fold increase in mature SP-B relative to wild type littermates. These mice survived without any evidence of respiratory problems and had normal lung function, normal alveolar surfactant phospholipid pool sizes, and typical tubular myelin indicating that the 102-residue C-terminal propeptide of SP-B is not required for normal structure and function of extracellular surfactant. However, proteolytic processing of the SP-C proprotein was perturbed resulting in the accumulation of a processing intermediate, Mr = 11,000, similar to the phenotype detected in SP-B(-/-) mice; furthermore, lamellar bodies in type II cells of SP-B(-/-)/hSP-BDeltac(+/+) mice were much larger than in the wild type animal and saturated phosphatidylcholine content in lung tissue was significantly increased although the incorporation of choline into saturated phosphatidylcholine was normal. Collectively, these results demonstrate a role for the C-terminal propeptide of SP-B in SP-C proprotein processing and the maintenance of lamellar body size. The C-terminal propeptide may be an important determinant of intracellular surfactant pool size.

Structural requirements for targeting of surfactant protein B (SP-B) to secretory granules in vitro and in vivo.

Human surfactant protein B (SP-B) is synthesized by type II cells as a 381-residue preproprotein which is proteolytically processed to a 79-residue mature peptide and targeted to lamellar bodies for secretion. To identify secretory granule targeting determinants, constructs encoding the SP-B preproprotein (SP-B), COOH-terminally deleted SP-B (SP-BDeltaC), the NH2-terminal propeptide (SP-BN), and a chimeric molecule consisting of albumin and the mature peptide (ALB/SP-BM) were transfected into AtT-20 and PC12 cells. Pulse-chase studies demonstrated that 10-30% of SP-B and SP-BDeltaC remained in cells in an endoglycosidase H-resistant form. Secretion of stored SP-B was stimulated by forskolin/12-O-tetradecanoylphorbol-13-acetate and intracellular SP-B was localized to secretory granules by immunoelectron microscopy. In contrast, SP-BN and ALB/SP-BM were constitutively secreted and not detected in secretory granules. Specific processing of SP-B was not detected in either AtT-20 or PC12 cells. Expression of SP-BDeltaC in transgenic mice resulted in secretion of fully processed mature SP-B, indicating correct processing and targeting of this construct in vivo. We conclude that 1) SP-B processing occurs in a cell-specific manner, 2) the proprotein contains secretory granule targeting determinants that are not cell-specific, 3) the NH2-terminal propeptide and the mature peptide are required for targeting SP-B to lamellar body, and 4) the COOH-terminal propeptide is not required for processing or sorting of SP-B.

Macrosomic infants of nondiabetic mothers and elevated C-peptide levels in cord blood.

C-peptide concentrations in the cord blood of 29 macrosomic neonates born of nondiabetic mothers were higher than in 23 control infants whose birth weight was appropriate for gestational age, and there was a significant direct correlation between birth weight and C-peptide concentration. Six of the macrosomic infants studied (20%) had hypoglycemia in the first 24 hours of life, compared with none of the infants born with appropriate weight. We conclude that chronic fetal hyperinsulinemia may be one of the causes of macrosomia and neonatal hypoglycemia in infants of nondiabetic mothers.