DNA-binding properties of the Erf protein of bacteriophage P22.

The phage P22 erf (essential recombination function) gene was placed in a small plasmid under the control of a strong, inducible promoter by manipulations in vitro. Erf protein was purified from induced cells, and characterized. Erf protein (monomer molecular weight 23,000) forms oligomers in solution. The carboxyl terminus is protease-sensitive: its removal generates a discrete amino-terminal fragment (molecular weight approximately 18,000) that also oligomerizes. At temperatures below 45 degrees C, Erf forms stable, discrete complexes with single-stranded DNA and supercoiled DNA, but not with relaxed double-stranded DNA. Binding to single-stranded DNA is stoichiometric; one Erf monomer binds approximately 15 bases of DNA, over the range of protein concentrations tested (2 to 100 micrograms/ml). At high temperatures (50 to 60 degrees C). Erf binds single- and double-stranded DNA, forming aggregates instead of discrete complexes. Heating and cooling in the absence of DNA produces a form of Erf that has single-stranded binding specificity, but forms aggregates on binding.

Efficient double-strand break-stimulated recombination promoted by the general recombination systems of phages lambda and P22.

To examine bacteriophage recombination in vivo, independent of such other processes as replication and packaging, substituted lambda phages bearing restriction site polymorphisms were employed in a direct physical assay. Bacteria were infected with two phage variants; DNA was extracted from the infected cells and cut with a restriction endonuclease. The production of a unique recombinant fragment was measured by Southern blotting and hybridization with a substitution sequence-specific probe. High frequency recombination was observed under the following conditions: the substituted lambda phages infected a wild-type host cell bearing a lambda repressor-expressing plasmid designed to shut down phage transcription and inhibit phage DNA replication as well. The same plasmid expressed the lambda red and gam genes. In addition, the host cell bore a second plasmid which expressed the EcoRI restriction-modification system. Both phage chromosomes possessed a single EcoRI site in the middle of the marked substitution sequence; however, as the site was modified in one of the parent phages, only the other partner was cut. Recombination was found to be dependent upon (1) red, (2) recA, (3) inactivation of the host recBCD function, either by Gam protein or by mutation and (4) double-strand breaks. The homologous recombination system of phage P22 could substitute for that of lambda.

Chronic lung disease following neonatal ventilation. II. Changing incidence in a geographically defined population.

The objective of this study was to examine the change in incidence of chronic lung disease following neonatal ventilation in a geographically defined population. Prospective data were collected over two 1-year periods (1987-1988 and 1990-1991) in the Trent Health Region, England. All infants were < or = 32 weeks gestation and/or < or = 1500g birthweight, born to mothers normally resident in the Trent Health Region. The principal outcome measures were mortality rate, presence of chronic lung disease, days of ventilation, and oxygen used by each infant. The proportion of low gestation, low birthweight babies was 1.5% in each period, made up of 897 and 925 babies from 61,050 and 63,350 births, respectively. There was a significant fall in mortality in infants of 750-1500g birthweight. However, the incidence of chronic lung disease (using either of two definitions) rose significantly between the two periods, with a corresponding large rise in the amount of respiratory care required. The contribution of various antenatal factors previously thought to be related to the development of chronic lung disease was examined. Birthweight and gestation were shown to be of overwhelming significance. We concluded that improvements in neonatal care, including the introduction of surfactant therapy, improved survival for some infants at the expense of an increased incidence of chronic lung disease. Clearly the hoped-for cost saving following the introduction of surfactant therapy has not occurred.

Bronchopulmonary infection with Moraxella catarrhalis in infants requiring extracorporeal membrane oxygenation.

Moraxella (formerly Branhamella) catarrhalis is now a well-recognized pathogen of the upper and lower respiratory tract. Four pediatric cases of life-threatening pneumonia requiring extracorporeal membrane oxygenation are presented. M. Catarrhalis was isolated within 48 hours of admission in three of the cases and within 24 hours of an acute deterioration in the fourth. We conclude that M. catarrhalis is either a significant pathogen in its own right, a marker of severe disease, or a secondary invader.

Chronic lung disease following neonatal ventilation. I. incidence in two geographically defined populations.

The objective of this study was to compare the incidence of chronic lung disease following neonatal ventilation in two geographically defined populations. Prospective data collection was carried out over a 1 year period from March 11, 1990 to February 28, 1991 in the Trent Health Region (England) and in British Columbia, Canada. All infants < or = 32 weeks gestation and/or < or = 1500 g birthweight born to mothers normally resident in either the Trent Health Region or British Columbia were included. The main outcome measures were mortality rate, presence of chronic lung disease, days of ventilation, and oxygen used by each infant. The proportion of shortened gestation, low birthweight babies was 1.5% in Trent and 1.2% in British Columbia (957 of 63,350 births in Trent and 526 of 45,333 births in British Columbia). There were no significant differences in mean birthweight or gestation between the two cohorts, but there was a trend towards lower mortality for infants 750-1500 g birthweight in British Columbia. The incidence of chronic lung disease (using either of two definitions) was significantly higher in British Columbia, with a corresponding greater amount of respiratory care required. This occurred despite higher use of antenatal steroids and surfactant therapy in the British Columbia group. We conclude that there are important clinical and resource implications resulting from the number of ventilator and oxygen days used by the preterm population in terms of planning of neonatal services. The role of individual treatment modalities in producing differences in the incidence of chronic lung disease warrants further study in the setting of a geographically defined population.

Optimising neonatal transfer.

Services for neonatal intensive care in the United Kingdom have evolved in a largely unplanned fashion. Units of different sizes provide various amounts of intensive care, and, with a few exceptions, there is little or no formal regional or subregional organisation. Chronic underresourcing and the salvaging of ever more complex infants have resulted in tertiary neonatal intensive care units operating at full capacity most of the time, a situation compounded by a chronic national shortage of nursing staff. These factors have in turn resulted in an increase in requirements for emergency perinatal transfers.

Postnatal growth retardation: a universal problem in preterm infants.

BACKGROUND: Previous data from this unit suggest that postnatal growth retardation (PGR) is inevitable in preterm infants. However, the study was performed in a single level III neonatal intensive care unit and applicability of the findings to other level III or level I-II special care baby units was uncertain. OBJECTIVES: To examine postnatal hospital growth and to compare growth outcome in preterm infants discharged from four level III tertiary care units and 10 level I-II special care baby units in the former Northern Region of the United Kingdom. SUBJECTS/METHODS: Preterm infants (< or = 32 weeks gestation; < or = 1500 g) surviving to discharge were studied. Infants were weighed at birth and discharge. Body weight was converted into a z score using the British Foundation Growth Standards. To ascertain the degree of PGR, the z score at birth was subtracted from the z score at discharge. Data were evaluated using a combination of split plot (level III v I-II=main factor; individual centre=subfactor) and stepwise regression analyses. Results were considered significant at p < 0.05. RESULTS: A total of 659 (level III, n = 335; level I-II, n = 324) infants were admitted over a 24 month period (January 1998-December 1999). No differences were detected in birth characteristics, CRIB score (a measure of illness in the first 24 hours of life), length of hospital stay, weight gain, weight at discharge, or degree of PGR between infants discharged from level III and level I-II units. Significant variation was noted in length of hospital stay (approximately 35%; p < 0.001), weight gain (approximately 33%; p < 0.001), weight at discharge (approximately 20%; p < 0.001), and degree of PGR (approximately 200%; p < 0.001) between the level III units. Even greater variability was noted in the duration of hospital stay (approximately 40%; p < 0.001), weight gain (approximately 60%; p < 0.001), weight at discharge (approximately 40%, p < 0.001), and degree of PGR (approximately 300%, p < 0.001) between the level I-II units. CONCLUSIONS: These data stress the variable but universal nature of PGR in preterm infants discharged from level III and I-II neonatal intensive care units and raise important questions about nutritional support of these infants before and after hospital discharge.

Normal range for blood flow velocity in cerebral arteries of newly born term infants.

A cross-sectional study of 128 healthy full term infants was made using duplex Doppler ultrasonography in order to establish a normal range for cerebral blood flow velocity (CBFV) in the first week of life. Recordings were made from both the anterior (ACA) and middle cerebral arteries (MCA). There was a statistically significant increase in CBFV in both the ACA and MCA over the first four days of life, which was particularly obvious in the first 24 h. There is a close relationship between measurement of CBFV from the ACA and the MCA, although the velocity tends to be higher in the MCA.

Shortfall of equipment for neonatal intensive care and the introduction of budget holding contracts.

As adequate allowance must be made for the costs of purchasing, maintaining, and updating equipment during the development of contracts the current standing of neonatal units with regard to available equipment was assessed. Data were collected as part of a one year prospective survey of the 17 perinatal units in the Trent region. Adequacy of provision of equipment for recognised intensive care cost was assessed using the recommendations of the British Paediatric Association and British Association of Perinatal Paediatrics. It was assumed that units without recognised intensive care cost had to be able to equip one cot to a standard of intensive care level 1 in the short term. Equipment more than 5 years old was considered likely to warrant replacement or major maintenance within the next two years. With these guidelines over 600,000 pounds would be required to provide sufficient equipment for all recognised level 1 intensive care cost and to allow units without funded cost to provide this level of care in the short term and to replace existing equipment more than 5 years old for these cost alone. This amount could be reduced by 25% by subdividing intensive care cost into levels 1 and 2, thereby reducing equipment requirements, but this would impair the units' ability to perform level 1 care at funded provision, which has already been shown to need expansion. Neither figure takes account of equipment requirements for infants requiring special care. In addition, no allowance has been made for purchase or update of ultrasound scanners or blood gas analysers. If the government's proposed reforms are to be implemented clinicians need to revise guidelines regarding essential equipment, and plans must be made to correct any existing shortfalls so that they do not become inherited financial liabilities for future budget holders.

Attitudes to viability of preterm infants and their effect on figures for perinatal mortality.

OBJECTIVE: To examine how local attitudes to management of extreme preterm labour can influence data on perinatal mortality. DESIGN: One year prospective study in a geographically defined population. SETTING: The 17 perinatal units of Trent region. PATIENTS: All preterm infants of less than or equal to 32 weeks' gestation in the Trent region. INTERVENTIONS: Infants who had been considered viable at birth were referred for intensive care; those who had been considered non-viable received terminal care. MAIN OUTCOME MEASURES: Whether each infant was born alive, dead, or alive but considered non-viable. RESULTS: Large differences were observed among units in the rates of delivery of infants of less than or equal to 27 weeks' gestation (rates varied from 7.2 to 0 per 1000 births). These differences were not present in the data relating to infants of between 28 and 32 weeks' gestation. The variation seemed to result from different approaches to the management of extreme preterm labour--that is, whether management took place in a labour ward or a gynaecology ward. CONCLUSIONS: Place of delivery of premature babies (less than or equal to 27 weeks' gestation) may influence classification and hence figures for perinatal mortality. In addition, the fact that the onus of judgment regarding viability and classification is often placed on relatively junior staff might also affect the figures for perinatal mortality. The introduction of a standard recording system for all infants greater than 500 g would be advantageous.

Effectiveness and safety of paediatric interventional cardiac catheterisation in a subregional cardiothoracic unit.

OBJECTIVE: To assess the effectiveness and safety of paediatric interventional cardiac catheterization during the development of the service. SETTING: Sub-regional Paediatric Cardiothoracic Centre. PATIENTS AND METHODS: All paediatric admissions for cardiac catheterisation between January, 1985 and December, 1992. Data were collected on all patients in whom interventional cardiac catheterisation was performed excluding babies undergoing balloon atrial septostomy. Results were compared with those reported previously by the larger centres. RESULTS: One hundred and seventy eight interventional procedures were performed in 158 patients, progressing from pulmonary valvuloplasty (1985) and aortic valvuloplasty (1986) to arterial duct occlusion and coil embolisation of shunts (1991). During the study period there was a rise in the number and variety of conditions for which interventional cardiac catheterisation was performed. In terms of morbidity, mortality and technical success, results compared favourably with those published from larger centres. CONCLUSIONS: Interventional cardiac catheterisation in children can be established effectively and safely in a relatively smaller set up.

Immunogenicity of a heptavalent conjugate pneumococcal vaccine administered concurrently with a combination diphtheria, tetanus, five-component acellular pertussis, inactivated polio, and Haemophilus influenzae type B vaccine and a meningococcal group C conjugate vaccine at 2, 3, and 4 months of age.

The immunogenicities of conjugate pneumococcal vaccines have been demonstrated when they are administered at 2, 3, and 4 months of age. There is a paucity of data on the immunogenicity of this vaccine when it is administered concurrently with other vaccines in the primary immunization schedule of the United Kingdom. We immunized 55 term infants at 2, 3, and 4 months of age with the seven-valent pneumococcal conjugate vaccine (PCV7), the meningococcal group C conjugate (MCC) vaccine, and the diphtheria, tetanus, five-component acellular pertussis, inactivated polio, and Haemophilus influenzae type b (DTaP(5)/IPV/Hib-TT) vaccine. The immune responses to the H. influenzae type b (Hib), MCC, and tetanus vaccines were measured at 2, 5, and 12 months of age; and the immune responses to PCV7 were measured at 2 and 5 months and then either at 12 months or following a 4th dose of PCV7. There were increases in the geometric mean concentrations (GMCs) of all antigens postimmunization. Greater than or equal to 90% of the infants achieved putatively protective levels postimmunization for all vaccine antigens except pneumococcal serotype 6B and Hib. The GMCs of the PCV7 serotypes increased following a 4th dose, although one infant had not reached putative levels of protection against serotype 6B. In conclusion, when infants were vaccinated according to the schedule described above, they had lower postprimary immunization responses to Hib, meningococcus group C capsular polysaccharide, and pneumococcal serotype 6B than the responses demonstrated by use of the other schedules. Despite this finding, there was a good response following a 4th dose of PCV7.

Responses to a conjugate pneumococcal vaccine in preterm infants immunized at 2, 3, and 4 months of age.

Preterm infants are at an increased risk of invasive pneumococcal disease infection and, additionally, have a diminished response to Haemophilus influenzae type b (Hib) conjugate vaccines. There are little data examining the response of preterm infants to a seven-valent pneumococcal conjugate vaccine (PCV7). We examined the responses of preterm infants immunized at 2, 3, and 4 months of age to PCV7. A total of 133 preterm and 54 term infants were immunized with PCV7 and the Neisseria meningitidis group C (MCC), diphtheria, tetanus, pertussis, polio, and Hib vaccines. Pneumococcal serotype-specific IgG was measured by enzyme-linked immunosorbent assay (ELISA) pre- and postimmunization and at 12 months or following a booster of PCV7. Term and preterm responses were compared using linear and logistic regression analyses. Term infants had higher preimmunization geometric mean concentrations (GMCs) for all serotypes. Preterm infants had lower postimmunization GMCs for serotype 23F. Gestational age affected postimmunization GMCs for serotypes 4, 6B, and 23F. Preterm infants were as likely to have levels of ≥0.35 µg/ml as term infants for all serotypes except 23F. The proportions of infants with titers of ≥0.35 µg/ml for all 7 serotypes were comparable between groups. A total of 28 of 29 term infants who received a booster had levels of ≥0.35 µg/ml for all serotypes. One infant had undetectable levels for serotype 6B. Of the 32 preterm infants boosted, 9 had levels of <0.35 µg/ml for 1 serotype, and 1 had levels of <0.35 µg/ml for 2 serotypes. In nonboosted infants, GMCs for all serotypes except 6B had fallen by 12 months of age. These results support the need for a booster dose in the second year of life.

Investigating the variations in survival rates for very preterm infants in 10 European regions: the MOSAIC birth cohort.

OBJECTIVE: To investigate the variation in the survival rate and the mortality rates for very preterm infants across Europe. DESIGN: A prospective birth cohort of very preterm infants for 10 geographically defined European regions during 2003, followed to discharge home from hospital. PARTICIPANTS: All deliveries from 22 + 0 to 31 + 6 weeks' gestation. MAIN OUTCOME MEASURE: All outcomes of pregnancy by gestational age group, including termination of pregnancy for congenital anomalies and other reasons, antepartum stillbirth, intrapartum stillbirth, labour ward death, death after admission to a neonatal intensive care unit (NICU) and survival to discharge. RESULTS: Overall the proportion of this very preterm cohort who survived to discharge from neonatal care was 89.5%, varying from 93.2% to 74.8% across the regions. Less than 2% of infants <24 weeks' gestation and approximately half of the infants from 24 to 27 weeks' gestation survived to discharge home from the NICU. However large variations were seen in the timing of the deaths by region. Among all fetuses alive at onset of labour of 24-27 weeks' gestation, between 84.0% and 98.9% were born alive and between 64.6% and 97.8% were admitted to the NICU. For babies <24 weeks' gestation, between 0% and 79.6% of babies alive at onset of labour were admitted to neonatal intensive care. CONCLUSIONS: There are wide variations in the survival rates to discharge from neonatal intensive care for very preterm deliveries and in the timing of death across the MOSAIC regions. In order to directly compare international statistics for mortality in very preterm infants, data collection needs to be standardised. We believe that the standard point of comparison should be using all those infants alive at the onset of labour as the denominator for comparisons of mortality rates for very preterm infants analysing the cohort by gestational age band.

Neonatal ventilation with inhaled nitric oxide vs. ventilatory support without inhaled nitric oxide for infants with severe respiratory failure born at or near term: the INNOVO multicentre randomised controlled trial.

BACKGROUND: Evidence from European centres to support the use of nitric oxide (NO) in mature newborns with evidence of severe respiratory failure is sparse. METHODS: Infants of >33 weeks' gestation, <28 days old, and with severe respiratory failure requiring ventilatory support were randomised to receive or not to receive inhaled NO (iNO). The study was not blinded. RESULTS: Sixty infants were recruited (29 allocated iNO, 31 no iNO) from 15 neonatal units in the UK, Finland, Belgium and the Republic of Ireland. 15/60 recruited babies died, and 8.1% of the survivors (4/45) were classified as severely disabled at 1 year. There was no statistically significant difference between the randomised groups in terms of the primary outcome of death or severe disability by the corrected age of 1 year (relative risk = 0.96 (95% confidence interval = 0.46-2.03); p = 0.86) (Fisher's exact p = 1.00). The costs of NO were outweighed by reduced extra corporeal membrane oxygenation costs in the iNO group. The mean total hospitalisation costs were lower in the iNO group, although the mean difference (1,697 pounds) was not statistically significant (95% confidence interval = -14,472 to 11,478). CONCLUSIONS: The results complement those of previous studies that suggest NO is cost-effective and reduces the need for extra corporeal membrane oxygenation in this group of babies. Overall survival rates compare unfavourably with results of US trials.

Towards safer neonatal transfer: the importance of critical incident review.

BACKGROUND: Critical incidents are common during the inter-hospital transfer of sick patients, and infants are an especially vulnerable group. AIMS: To examine the effect of critical incident review on the number of adverse events during inter-hospital transfer of sick infants. METHODS: Critical incidents over an eight year period are reported from a single neonatal transfer service before and after major service changes were made. The changes were instigated as part of ongoing critical incident reviews. RESULTS: Changes made as a result of critical incident review significantly reduced the number of incidents contributed to by poor preparation, transport equipment or clinical problems, ambulance delays, and ambulance equipment failure. CONCLUSIONS: The continuous process of critical incident reporting and review can reduce the number of adverse events during the transfer of critically ill infants.

Lymphocyte subsets in term and significantly preterm UK infants in the first year of life analysed by single platform flow cytometry.

This observational study describes the ranges observed for lymphocyte subsets for significantly preterm infants (<32 weeks) in the first year of life, measured by single platform flow cytometry and compared to identically determined subsets in term infants. After ethical approval 39 term and 28 preterm infants had lymphocyte subset analysis before and after their primary immunization series. Median values with 5th and 95th percentiles of absolute counts and percentages are presented for total lymphocytes, T cells, NK cells, B cells, cytotoxic T cells, helper T cells, dual positive T cells, activated T cells, activated T helper cells (including T regulatory cells), pan memory T cells, pan naive T cells, memory helper T cells, naive helper T cells and the T helper/suppressor ratio. The lymphocyte profile of the preterm infants differed from that of the term infants.

Neonatal Ventilation With Inhaled Nitric Oxide Versus Ventilatory Support Without Inhaled Nitric Oxide for Preterm Infants With Severe Respiratory Failure: the INNOVO multicentre randomised controlled trial (ISRCTN 17821339).

BACKGROUND: Although inhaled nitric oxide (iNO) may be a promising treatment for newborn infants with severe respiratory failure, the results from 3 previous small trials were inconclusive. METHODS: Infants of <34 weeks' gestation, <28 days old, and with severe respiratory failure requiring ventilatory support were randomized to receive or not receive iNO. The study was not blinded. FINDINGS: Recruited were 108 infants (55 allocated to receive iNO and 53 not allocated to receive iNO) from 15 neonatal units in the United Kingdom and Republic of Ireland. Fifty-nine percent (64 of 108) died, and 84% of the survivors (37 of 44) had signs of some impairment or disability, 9 (20%) of them classified as severely disabled. There was no evidence of an effect of iNO on the primary outcomes: death or severe disability at 1 year corrected age (relative risk [RR]: 0.99; 95% confidence interval [CI]: 0.76 to 1.29); death or supplemental oxygen on expected date of delivery (RR: 0.84; 95% CI: 0.68 to 1.02); or death or supplemental oxygen at 36 weeks' postmenstrual age (RR: 0.98; 95% CI: 0.87 to 1.12). There was a trend for infants allocated to the iNO group to spend more time on the ventilator (log rank: 3.6), on supplemental oxygen (log rank: 1.4), and in hospital (log rank: 3.5) than those allocated to receive no iNO. This pattern predominantly reflected the infants who died. Mean total costs at 1 year corrected age were significantly higher in the iNO group, partly because of the costs of the gas but mainly because of the difference in initial hospitalization costs. INTERPRETATION: Evidence of prolongation of intensive care and increased costs of such care, without clear beneficial effects, implies that iNO cannot be recommended for preterm infants with severe hypoxic respiratory failure.

Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12.

Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.