Chest compression and/or epinephrine at birth for preterm infants <32 weeks gestational age: matched cohort study of neonatal outcomes.

BACKGROUND: Studies of outcomes of preterm infants after the receipt of extensive cardiopulmonary resuscitation (CPR) at birth have yielded varied results. OBJECTIVE: To compare adverse outcome (death or severe morbidities) of preterm infants <32 weeks gestational age (GA) who received chest compressions with or without administration of epinephrine at birth with those who did not receive either. DESIGN/METHOD: Data were retrospectively analyzed from a database for preterm infants <32 weeks GA discharged from hospital between July 2004 and October 2007. CASES: Infants who received chest compression with or without administration of epinephrine during the initial resuscitation. Matched cohort: Infants who did not receive extensive CPR at birth (matched for GA, sex and admission date). PRIMARY OUTCOME: Death or any of three severe morbidities (grade 3 or 4 intraventricular hemorrhage or periventricular leukomalacia; retinopathy of prematurity > stage 2 or chronic lung disease). RESULT: Sixty-six cases and 156 matched infants were identified. There were no baseline differences between groups except Apgar and severity of illness scores. Median (interquartile range) duration for chest compression (n=66) was 60 (30 to 180 s) and number of epinephrine doses (n=29) was 1 (1 to 3). Logistic regression confirmed significantly higher risk of adverse outcome among cases compared with matched controls (58 vs 37%; P=0.04, adjusted odds ratio 2.23, 95% confidence interval 1.04, 4.77). CONCLUSION: Infants born prematurely who met criteria for extensive CPR at birth experienced higher risk of combined adverse outcome, including death or severe neurological injury, severe retinopathy of prematurity or bronchopulmonary dysplasia.

Definition of the human N-myc promoter region during development in a transgenic mouse model.

The N-myc oncogene directs organogenesis, and gene amplification is associated with aggressive forms of neuroblastoma, a common malignant tumor in children. N-myc is expressed in fetal epithelium, and expression decreases markedly postnatally. To localize sequences responsible for directing expression, we have analyzed the human N-myc promoter. We noted previously that N-myc promoter regions 5' to exon 1 directed reporter gene expression in all cell lines, including those without detectable N-myc transcripts. However, when promoter constructs included 3' exon 1 and the 5' portion of intron 1, reporter activity was detected only when there was expression of the endogenous gene. To determine the role of this "tissue-specific region" in directing expression during development, we generated transgenic mice carrying N-myc promoter lacZ minigenes that contained 5' N-myc promoter elements alone or the promoter linked to the 3' exon 1/5' intron 1 tissue-specific region. Animals lacking the tissue-specific exon 1/intron 1 region showed beta-galactosidase expression in the CNS, but expression was not observed in other organs in which endogenously derived N-myc transcripts were seen. Within the CNS, transgene expression was seen mainly in the olfactory system and was not observed in other areas in which expression of the murine gene has been noted. In contrast, no transgene expression was observed in any of the animals carrying the tissue-specific exon 1/intron 1 region. Thus, sequences that direct expression within the olfactory system were contained within our 5' promoter transgene, whereas sequences that guide the ubiquitous expression of N-myc during organogenesis lie outside the regions studied here. Finally, the exon 1/intron 1 region seems to act in a dominant fashion to repress expression in the CNS from the immediate 5' N-myc promoter.

A novel intron element operates posttranscriptionally To regulate human N-myc expression.

Precisely regulated expression of oncogenes and tumor suppressor genes is essential for normal development, and deregulated expression can lead to cancer. The human N-myc gene normally is expressed in only a subset of fetal epithelial tissues, and its expression is extinguished in all adult tissues except transiently in pre-B lymphocytes. The N-myc gene is overexpressed due to genomic amplification in the childhood tumor neuroblastoma. In previous work to investigate mechanisms of regulation of human N-myc gene expression, we observed that N-myc promoter-chloramphemicol acelyltransferase reporter constructs containing sequences 5' to exon 1 were active in all cell types examined, regardless of whether endogenous N-myc RNA was detected. In contrast, inclusion of the first exon and a portion of the first intron allowed expression only in those cell types with detectable endogenous N-myc transcripts. We investigated further the mechanisms by which this tissue-specific control of N-myc expression is achieved. Using nuclear run-on analyses, we determined that the N-myc gene is actively transcribed in all cell types examined, indicating a posttranscriptional mode of regulation. Using a series of N-myc intron 1 deletion constructs, we localized a 116-bp element (tissue-specific element [TSE]) within the first intron that directs tissue-specific N-myc expression. The TSE can function independently to regulate expression of a heterologous promoter-reporter minigene in a cell-specific pattern that mirrors the expression pattern of the endogenous N-myc gene. Surprisingly, the TSE can function in both sense and antisense orientations to regulate gene expression. Our data indicate that the human N-myc TSE functions through a posttranscriptional mechanism to regulate N-myc expression.

Expression of the c-Myc protein in childhood medulloblastoma.

PURPOSE: The purpose of this study was to determine the incidence of c-Myc protein expression in medulloblastoma/primitive neuroectodermal tumor (MB/PNET) and to identify mechanisms in addition to c-myc gene amplification that lead to increased protein expression. METHODS: We analyzed c-myc gene copy number, mRNA level and protein expression in a panel of MB/PNET cell lines. C-Myc protein levels were assessed in tumor specimens and cell lines using immunohistochemical staining with a c-Myc-specific monoclonal antibody. RESULTS: Southern analysis confirmed c-myc gene amplification in the D425 MED cell line and re-arrangement of one allele in D283 MED, which was analyzed further and appeared to represent a small deletion 3' of exon 3. C-myc transcript levels were dramatically elevated in both lines. Using a c-myc probe, fluorescence in situ hybridization (FISH) showed c-myc present in 3 tandem copies at 8q24 in D283 MED and multiple copies as double minutes in D425 MED. Immunohistochemistry showed c-Myc protein expression in 9 of 10 tumors and all cell lines, regardless of gene amplification status or level of mRNA expression. CONCLUSIONS: c-Myc protein expression is common in MB/PNET tumor specimens and cell lines. Elevated protein levels are observed in the absence of amplification, suggesting that multiple mechanisms of c-myc dysregulation may be involved in MB/PNET. These studies support a role for c-Myc in the development of this common childhood tumor.

Autoregulation of the human N-myc oncogene is disrupted in amplified but not single-copy neuroblastoma cell lines.

Amplification of the N-myc gene is a significant adverse prognostic factor in neuroblastoma, a common childhood tumor. In non-transformed cells, myc expression is controlled through an autoregulatory circuit, through which elevated Myc protein levels lead to down-regulation of myc transcription. The precise mechanism of myc gene autoregulation is unknown. Loss of c-myc autoregulation has been documented in transformed cells from a number of different lineages, but N-myc autoregulation has not yet been investigated. In neuroblastoma, the increased N-Myc protein produced by amplified tumors would be expected to silence N-myc transcription if the autoregulatory loop were intact. To determine whether N-myc autoregulation is operative in human neuroblastoma, and to localize cis-acting elements which mediate N-myc autosuppression, we transfected a series of N-myc 5' promoter constructs into a panel of human neuroblastoma cell lines carrying one or multiple copies of N-myc. The transfected promoter was equally active in single-copy and amplified lines. Significant promoter activity in the presence of abundant Myc protein in amplified neuroblastoma lines indicates that autoregulation is disabled in this subset of tumors. To investigate whether single-copy lines produce insufficient N-Myc protein to trigger autosuppression yet retain an intact autoregulatory circuit, we transfected neuroblastoma lines with 5' promoter constructs in the presence of a c- or N-myc expression vector. Overexpression of c- or N-Myc resulted in diminution of activity of both the transfected promoter and the endogenous N-myc gene in single-copy, but not amplified lines. Using a series of 5' promoter-deletion minigenes, we localized a cis-acting element required for autoregulation close to the transcription start sites. While the precise mechanism of autosuppression remains unknown, we demonstrated that Myc is incapable of silencing the adenovirus major late promoter (AdMLP) in neuroblastoma cells, indicating that Myc suppression of its own promoter and the AdMLP involve distinct components. These studies provide the first systematic investigation of autoregulation in neuroblastoma, and indicate that single-copy neuroblastoma lines produce insufficient N-Myc protein to activate downstream effector(s) of autosuppression; the autoregulatory circuit is otherwise intact. Amplified lines, in contrast, have lost autoregulation.

Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes.

Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis-acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti-NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5' IgH sequences, a rearranged VDJ region, the 5' matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3' IgH sequences (an alpha constant region and the 3' enhancer) and either the original VDJ segment or a substituted T cell receptor beta segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3' enhancer.

Outer surface lipoproteins of Borrelia burgdorferi stimulate nitric oxide production by the cytokine-inducible pathway.

The outer surface lipoproteins of Borrelia burgdorferi, OspA and OspB, stimulate the production of nitric oxide (NO) by murine bone marrow-derived macrophages from BALB/c, C3H/HeN, and C3H/HeJ mice. Gamma interferon (IFN-gamma) caused a three- to fivefold enhancement of this production of NO, and the L-arginine analog N-guanidino-monomethyl L-arginine inhibited it. Activation of transcription of the inducible NO synthase gene in stimulated macrophages was demonstrated by reverse transcriptase rapid PCR. Although IFN-gamma increased the amount of NO produced in macrophage cultures, it did not cause transcription of the inducible NO synthase gene greater than that seen with the Borrelia proteins. OspA and OspB also induced the production of high levels (40 to 150 ng/ml) of IFN-gamma in cultures of macrophages incubated with interleukin-2 (IL-2)-elicited cells from normal (T and NK cells) and scid (NK cells) mice but not in macrophages or IL-2-elicited cells cultured individually. This suggests that OspA stimulated macrophage production of cytokines, which, in turn, stimulated the production of IFN-gamma by NK and T cells. Reverse transcriptase rapid PCR demonstrated that OspA and sonicated B. burgdorferi stimulated production of several inflammatory cytokines in macrophage cultures, including IL-1, IL-6, IL-12, IFN-beta, and tumor necrosis factor alpha. As tumor necrosis factor alpha, IFN-beta, and IL-12 are potent activators of IFN-gamma production by T and NK cells, their presence in these cocultures could be responsible for the IFN-gamma production. Lymphocytes from infected C3H mice also produced IFN-gamma when stimulated with B. burgdorferi; thus, immune cells may also modulate NO responses. The generation of NO during infection with B. burgdorferi may be important, as NO has potent antimicrobial properties. NO can also be involved in pathological inflammatory processes in which its generation is detrimental to the host. Thus, the colocalization of B. burgdorferi lipoproteins, NO-producing cells, and regulatory cytokines may determine the outcome of infection.

Normal human B lymphocytes and mononuclear cells respond to the mitogenic and cytokine-stimulatory activities of Borrelia burgdorferi and its lipoprotein OspA.

Borrelia burgdorferi produces potent cell-activating molecules capable of stimulating polyclonal proliferation and immunoglobulin production by murine B lymphocytes and cytokine production by a variety of cell types. These stimulatory molecules function in infected mice, resulting in elevated levels of circulating immunoglobulins and serum interleukin-6. We have recently demonstrated that the purified outer surface lipoproteins OspA and OspB possess these properties. To assess their possible involvement in human disease, we determined whether cells from normal human donors could respond to these activities. Normal human B lymphocytes but not T lymphocytes proliferated when incubated with either sonicated B. burgdorferi or purified OspA. Sonicated B. burgdorferi was efficient at stimulating immunoglobulin M production by human mononuclear cell cultures; however, purified OspA was relatively inactive. Both sonicated B. burgdorferi and purified OspA stimulated production of high levels of interleukin-6 by mononuclear cells. These findings extend our observations with the mouse model and suggest that the stimulatory lipoproteins could indeed be involved in the symptoms and pathologies of human infection with B. burgdorferi.

Sequential changes in pulmonary epithelial permeability after birth in term and preterm rabbits.

We assessed sequential changes in the permeability properties of the pulmonary epithelium in spontaneously breathing, newborn term (30 days of gestation) and preterm (28 days) rabbit pups, using the rate of pulmonary clearance of 99mTc-DTPA (MW = 492) as an index of permeability. In term rabbits, clearance was faster at 1 h of age than at hourly timepoints thereafter (p < 0.05). In preterm rabbits, clearance rates measured from 1 to 5 h after birth were not quite significantly different (p = 0.0519) although the trend to slower clearance with increasing time after birth was significant. When term and preterm rabbits were compared, clearance was similar at 1 h after birth but was faster at both 2 and 3 h in the preterm rabbits (p < 0.05). Pulmonary epithelial permeability appears to be increased in the immediate postnatal period and the duration of increased permeability is longer in preterm rabbits. Because lung water content at birth is greater in the preterm rabbits, we speculate that the permeability changes may be associated with clearance of fetal lung liquid.

99mTc-DTPA clearance in preterm lambs. Effect of surfactant therapy and ventilation.

Surfactant therapy and high-frequency oscillatory ventilation (HFO) may minimize damage to the pulmonary epithelium of surfactant-deficient newborns. Using pulmonary clearance of insufflated, aerosolized 99mTc-DTPA (molecular weight 492) as an index of lung epithelial permeability, we examined the effects of 300 mg bovine lipid extract surfactant (S) administered at birth to preterm lambs ventilated by either HFO or conventional mechanical ventilation (CMV). Four groups of lambs, delivered by cesarean section at 129 to 133 days of gestation, were studied: (1) HFO + S, (2) CMV + S, (3) HFO, and (4) CMV. 99mTc-DTPA clearance was assessed at 2, 4, and 5.5 h after birth. Surfactant treatment improved oxygenation and lung pressure-volume relationships, with oxygenation best maintained by the combination of HFO + S. All groups had similar biexponential clearance curves at the three time points, however, and there was no significant difference in the mean rates of clearance (k) between the four groups at 2 h (k = 6.03 +/- 0.60 [SEM], 7.04 +/- 1.46, 5.67 +/- 0.91, and 7.23 +/- 0.97 %/min for Groups 1, 2, 3, and 4, respectively), 4 h (k = 6.95 +/- 0.77, 5.60 +/- 0.51, 6.39 +/- 0.64, and 6.78 +/- 1.71 %/min), and 5.5 h (k = 7.43 +/- 0.78, 6.08 +/- 0.80, 7.86 +/- 0.90, and 7.95 +/- 0.66 %/min). These data suggest that neither surfactant nor HFO significantly alters pulmonary epithelial permeability to a small radiolabeled molecule in preterm lambs.

The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA.

The complete nucleotide sequence of hepatitis C virus (HCV) cloned from the liver tissue of a Taiwanese patient with post-transfusion type C hepatitis was determined. The 5' end of HCV genomic RNA was located 341 nucleotides upstream from the initiation codon for the viral polyprotein open reading frame. The 5' end of the viral antigenomic RNA was shown to have 13 consecutive As. Thus the 3' terminus of the viral genome is a stretch of U which ends about 50 nucleotides downstream from the stop codon of the large open reading frame. The nucleotide sequence homology between this HCV strain and two Japanese isolates was 90.5 and 90.7%, respectively. Homology with the United States strain, however, was only 77.8%. Accordingly, the indigenous Taiwanese HCV strain is of the same subtype as the Japanese isolates. Novel features of the viral genome termini are possibly relevant to HCV genome replication.