[Gaussian distribution of intelligence in VLBW preterm infants at age 5: very low correlation with very low birth weight]. / Normalverteilung der Intelligenz von VLBW-Frühgeborenen mit 5 Jahren: sehr geringe Korrelation mit sehr geringem Geburtsgewicht.

BACKGROUND: Preterm infants with very low birth weight < 1500 g (VLBW) have a higher risk of developmental disorders. In addition to the common estimation of the mean intelligence values, we studied the distribution of intelligence at preschool age in VLBW infants and the risk factors influencing this distribution. PATIENTS AND METHODS: A prospective cohort study of 277 VLBW infants < 32 weeks born in 1991-1995 and treated according to a standardized regimen in one Perinatal Center was carried out, including measurement of intelligence (Kaufman-Assessment Battery for Children) at age 5. Statistical methods employed were: explorative data analysis, correlation, chi (2)- and t-tests; the tested variables were: small for gestational age (< third percentile), perinatal acidemia (umbilical arterial pH < 7.10), perinatal hypoxia (BE < - 10), hypothermia (< 36 degrees C), hypoglycemia after the first day of life (< 30 mg / dL), bronchopulmonary dysplasia (FiO (2) > 0.21 > or = 36 weeks), intraventricular hemorrhage, ventricular dilation, periventricular leukomalacia, seizures, abnormal acoustic evoked potentials, and hyperexcitability at discharge. RESULTS: The distribution of intelligence in 137 VLBW infants < 32 weeks (60 % follow-up rate) was similar to a symmetrical Gaussian bell curve. The intelligence increased very slightly with birth weight (Pearson correlation: 0.172; p = 0.045) and was significantly lower in children with hypoglycemia after the first day of life (- 13.35; 95 % confidence interval: - 20.08 to - 6.63; p = 0.002), hyperexcitability at discharge (- 16.28; 95 % confidence interval: - 25.26 to - 7.31; p = 0.005), and bronchopulmonary dysplasia (- 7.00; 95 % confidence interval - 11.71 to - 2.29; p = 0.039). CONCLUSIONS: At preschool age, the intelligence of VLBW infants is normally distributed and correlates only slightly with the very low birth weight. Hypoglycemia after the first day of life and bronchopulmonary dysplasia are risk factors for lower intelligence. Hyperexcitability at discharge seemed to represent a promising prognostic factor for a later intelligence reduction.

Focal congenital alveolar proteinosis associated with abnormal surfactant protein B messenger RNA.

Two siblings presented with typical clinical features of congenital pulmonary alveolar proteinosis (PAP). Necropsy of one sibling revealed scattered foci of the diagnostic histologic changes in the lung tissue. In contrast to infantile and adult PAP, focal distribution is uncommon in congenital PAP. Defective expression of the granulocyte-macrophage colony-stimulating factor receptor was ruled out. The surfactant protein B (SP-B) content in the lung tissue of the autopsied patient was low, and a deletion in the SP-B messenger RNA was detected. We speculate that the PAP in our patients was related to the reduced quantity and/or to the altered quality of SP-B.

Do we need new indications for ECMO in neonates pretreated with high-frequency ventilation and/or inhaled nitric oxide?

OBJECTIVE: High-frequency ventilation (HFV) and/or inhaled nitric oxide (iNO) has reduced ECMO in neonates. But, frequently, improvement with HFV/iNO is temporary and only prolongs lung injury without preventing ECMO. We tried to identify a threshold oxygenation index (OI) that predicts temporary or persistent improvement with HFV/iNO in neonatal ECMO candidates as early as possible. DESIGN: Cohort study of all neonates with OI > 40 during intermittent positive pressure ventilation between 1992 and 1997. The first treatment was HFV; at an OI > 40 during HFV, iNO was added; at an OI > 40 during HFV+iNO, ECMO was initiated. Temporary improvement was defined as secondary need for ECMO or fatal chronic lung disease without ECMO. SETTING: University hospital level III neonatal intensive care unit. MAIN RESULTS: Ten of the 34 neonates studied rapidly required ECMO despite HFV/iNO. Eleven neonates temporarily improved for 1-10 days before the OI was again > 40. Nine received ECMO, two were denied ECMO after mechanical ventilation > 14 days and died of chronic lung disease. Thirteen neonates persistently improved with HFV/iNO without ECMO. The OI before, at 24 h or 48 h of HFV/iNO did not predict temporary or persistent improvement. However, after 72 h of HFV/iNO, neonates with persistent improvement had lower OIs than those with temporary improvement [median OI 16 (4-24) vs 31 (20-40); P = 0.0004]. In all neonates with an OI > or = 25 after 72 h, HFV/iNO eventually failed (positive predictive value 100%, sensitivity 91 %, specificity 100%, positive likelihood ratio 91). CONCLUSION: For neonates pretreated with HFV/iNO, an OI > 40 is an inadequate ECMO indication. Based on our data we hypothesize that an OI > or = 25 after 72 h of HFV/ iNO is a better ECMO indication that avoids prolonged barotrauma.

Leukocyte and endothelial activation in a laboratory model of extracorporeal membrane oxygenation (ECMO).

An inflammatory response and a capillary leak syndrome frequently develop during the treatment of neonatal respiratory failure by extracorporeal membrane oxygenation (ECMO). The present study was performed to investigate leukocyte activation and endothelial cell dysfunction that are associated with prolonged contact of blood components with synthetic surfaces. Laboratory ECMO was performed with fresh human blood at 37 degrees C for 8 h (n = 6). Leukocyte activation was measured by L-selectin (CD62L) and CD18 integrin surface expression and by neutrophil-derived elastase release. To monitor endothelial activation, endothelial cell ICAM-1 (CD54) expression was measured in cultured endothelial cells from human umbilical veins (HUVEC) after incubation with plasma from the ECMO experiments. CD18 integrin expression was found significantly up-regulated on polymorphonuclear neutrophils and monocytes after 2-4 h of laboratory ECMO. L-selectin was reduced on both cell types during the total duration of the experiments. Soluble L-selectin (sCD62L) and total and differential leukocyte counts remained unchanged during the experiment. Neutrophil-derived elastase content was maximal after 8 h of ECMO. Plasma from the ECMO experiments did not induce ICAM-1 expression of cultured HUVEC. We conclude that prolonged contact with synthetic surfaces during ECMO activates phagocytes, which may contribute to the inflammatory response seen in ECMO-treated patients. Activated phagocytes do not accumulate in the extracorporeal system nor release humoral factors inducing ICAM-1 expression on endothelial cells.

Postnatal changes of extracellular volume, atrial natriuretic factor, and diuresis in a randomized controlled trial of high-frequency oscillatory ventilation versus intermittent positive-pressure ventilation in premature infants <30 weeks gestation.

OBJECTIVES: High-frequency oscillatory ventilation (HFOV) with a high lung volume strategy is an experimental mode of ventilating preterm infants aimed at achieving maximal alveolar recruitment Higher mean airway pressures are used during HFOV than during intermittent positive-pressure ventilation (IPPV), and the intrathoracic volume increase is relatively constant. Both factors increase the risk to depress organ blood flow and diuresis. Our objective was to test the hypothesis that high lung volume HFOV attenuates the postnatal reduction of extracellular volume in preterm infants by reducing plasma atrial natriuretic factor and diuresis. DESIGN: Prospective, randomized, controlled clinical trial. SETTING: University hospital, Level III neonatal intensive care unit. PATIENTS: Premature infants <30 wks gestation requiring intubation for respiratory distress syndrome within the first 6 hrs of life; 15 infants (gestational age, 26 [24-29] wks, birth weight 814 [452-1340] g) were randomized to HFOV, 19 infants (gestational age 27 [24-39] wks, birth weight 930 [644-1490] g) to IPPV. INTERVENTIONS: The randomized mode of ventilation was assigned within 1 hr after intubation. During HFOV mean airway pressure was increased as long as oxygenation improved and no lung overinflation was seen on chest radiograph. IPPV rates were > or =60/min. MEASUREMENTS AND MAIN RESULTS: We measured extracellular volume (sucrose dilution) and atrial natriuretic factor on Day 1 and Day 3. Mean airway pressure, body weight, diuresis, and fluid intake were measured daily. During HFOV mean airway pressure was higher at 12 hrs (median 7 cm H2O vs. 4 cm H2O; p = .001) and 24 hrs (median 6 cm H2O vs. 3 cm H2O; p = .01). In both groups, extracellular volume decreased between Day 1 and Day 3 (HFOV from 428 +/- 126 mL to 344 +/- 145 mL [p = .003], IPPV from 466 +/- 108 mL to 414 +/- 124 mL [p = .01]) and diuresis increased (HFOV, from 2.5 +/- 1.7 to 4.6 +/- 0.9 mL/kg/hr [p = .001]; IPPV, from 2.8 +/- 1.6 to 4.2 +/- 1.0 mL/kg/hr [p = .01]). Plasma atrial natriuretic factor was not decreased in the HFOV group. CONCLUSIONS: High lung volume HFOV as primary mode of ventilation in preterm infants <30 wks gestation did not result in unwanted fluid retention and a decrease in diuresis in the first days of life.

Randomized comparison of high-frequency ventilation with high-rate intermittent positive pressure ventilation in preterm infants with respiratory failure.

OBJECTIVE: In a randomized, controlled, multicenter trial, we tested the hypothesis that high-frequency ventilation (HFV) with a high lung volume strategy results in fewer treatment failures than intermittent positive pressure ventilation (IPPV) with high rates and low peak inspiratory pressures. STUDY DESIGN: Infants with a gestational age between >/=24 weeks and <30 weeks, requiring mechanical ventilation within 6 hours of birth, were randomly assigned to receive either IPPV or HFV until 240 hours after randomization, extubation, or meeting treatment failure criteria. Treatment failure, the primary end point, was determined when air leaks, an oxygenation index >35 to 45 (depending on gestational age), death, or chronic lung disease occurred. Chronic lung disease was defined as persistent requirement of mechanical ventilation, continuous positive airway pressure, or supplemental oxygen at a postmenstrual age of 36 weeks. Secondary end points included the incidence of intracranial hemorrhage. RESULTS: The third scheduled interim analysis led to termination of the trial after recruitment of 284 infants. Treatment failure criteria were met by 46% of infants receiving IPPV and 54% of infants receiving HFV (1-tailed primary hypothesis, P =.92; 2-tailed chi2 test, P =.15). Air leaks occurred in 31% and 42% (P =.042), CLD in 23% and 25%, and grade 3-4 intracranial hemorrhage in 13% and 14% of IPPV-treated and HFV-treated patients, respectively. The mortality rate before discharge was 10% in both groups. CONCLUSION: HFV with a high lung volume strategy did not cause less lung injury in preterm infants than IPPV with a high rate and low peak inspiratory pressures.

Pulmonary embolism and myocardial hypoxia during extracorporeal membrane oxygenation.

The treatment of a newborn with severe meconium aspiration by venoarterial extracorporeal membrane oxygenation (ECMO) was complicated by myocardial hypoxia with a marked decrease of myocardial contractility. The onset of the cardiac hypoxia was related to a pulmonary artery embolus. The origin of the embolus was a deep femoral vein thrombosis, caused by a central vein catheter, which was inserted 1 day before ECMO by venous cutdown. The possible pathophysiology of myocardial hypoxia in this patient is discussed, especially with regard to myocardial perfusion, supporting the hypothesis of coronary perfusion occuring with blood from the left ventricle and not from the arterial cannula in the aorta.

Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene.

The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Deltaycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

Tissue-specific and differential editing of the two ycf3 editing sites in maize plastids.

The hypothetical chloroplast open reading frame 3 (ycf3) of maize, consisting of three exons and two group II introns, contains two editing sites. Both of these sites were investigated with respect to the extent of editing in various tissues and different developmental stages. Northern blot analyses show nearly identical transcript patterns of ycf3 in all tissues investigated. In leaf plastids, both editing sites are completely edited, independent of light conditions and developmental stage. In non-leaf plastids, however, one editing site of ycf3 is only partially edited in unspliced transcripts and in one type of partially spliced transcripts. In different developmental stages of the same tissue, on the other hand, no differences in editing efficiency were found. These results indicate that, in partially spliced transcripts, different editing sites of one and the same gene can be edited with different efficiencies in a tissue-specific manner.

Occurrence of plastid RNA editing in all major lineages of land plants.

RNA editing changes posttranscriptionally single nucleotides in chloroplast-encoded transcripts. Although much work has been done on mechanistic and functional aspects of plastid editing, little is known about evolutionary aspects of this RNA processing step. To gain a better understanding of the evolution of RNA editing in plastids, we have investigated the editing patterns in ndhB and rbcL transcripts from various species comprising all major groups of land plants. Our results indicate that RNA editing occurs in plastids of bryophytes, fern allies, true ferns, gymnosperms, and angiosperms. Both editing frequencies and editing patterns show a remarkable degree of interspecies variation. Furthermore, we have found that neither plastid editing frequencies nor the editing pattern of a specific transcript correlate with the phylogenetic tree of the plant kingdom. The poor evolutionary conservation of editing sites among closely related species as well as the occurrence of single species-specific editing sites suggest that the differences in the editing patterns and editing frequencies are probably due both to independent loss and to gain of editing sites. In addition, our results indicate that RNA editing is a relatively ancient process that probably predates the evolution of land plants. This supposition is in good agreement with the phylogenetic data obtained for plant mitochondrial RNA editing, thus providing additional evidence for common evolutionary roots of the two plant organellar editing systems.

Mutations of cytochrome b6 in Chlamydomonas reinhardtii disclose the functional significance for a proline to leucine conversion by petB editing in maize and tobacco.

We have introduced a proline codon in place of a leucine codon at position 204 of the petB gene of Chlamydomonas reinhardtii. This gene modification mimics the presence of proline codons at the same position in the petB genes of maize and tobacco, which are subsequently edited to leucine codons at the RNA level. Following transformation, we observed no editing at this position in C. reinhardtii, independent of the type of proline codon we have used: the CCA codon, edited in maize, or a CCT codon. Strains carrying the introduced mutation were non phototrophic and displayed a block in photosynthetic electron transfer, consistent with a lack of cytochrome b6f activity. Thus the presence of a proline residue at position 204 in cytochrome b6 is detrimental to photosynthesis. We show that the mutant phenotype arose from a defective assembly of cytochrome b6f complexes and not from altered electron transfer properties in the assembled protein complex. Biochemical comparison of the proline-containing transformants with a cytochrome b6 mutant deficient in heme-attachment indicates that their primary defect is at the level of assembly of apocytochrome b6 with the bh heme, thereby preventing assembly of the whole cytochrome b6f complex.

25 years of respiratory support of newborn infants.

Respiratory support of newborn infants has changed in the last 25 years, because of new knowledge of patho-physiology, controlled studies of respiratory therapy and the realisation of perinatal centers. Respiratory support has changed from the "blow in--suck out" approach, inevitably leading to severe atelectasis, high morbidity and mortality to a now very sophisticated therapy with reduced mortality and morbidity also in very-low-birth-weight infants, who were hopeless patients 25 years ago. Major milestones of this development were the introduction of continuous distending pressure to surfactant deficient lungs, the high-frequency positive pressure ventilation with fine tuning of inspiratory and expiratory times, adjusted to individual time constants and the substitution of artificial surfactant. Techniques for the future, like HFO, NO-inhalation, proportional assist ventilation and liquid ventilation are presently investigated.

Transcriptionally active chromosomes (TACs) of barley chloroplasts contain the alpha-subunit of plastome-encoded RNA polymerase.

Transcriptionally active chromosomes (TACs) were isolated from mature chloroplasts of barley, from proplastids enriched in basal segments of barley primary foliage leaves, and from ribosome-deficient plastids of heat-bleached barley leaves. Immunological analysis with a specific antibody raised against the plastid rpoA gene product revealed that chloroplasts contain an immunoreactive protein of 38 kDa in the TAC fraction which appears to be identical to the alpha-subunit contained in the soluble RNA polymerase (sRNAP) fraction of the same chloroplasts. However, only traces of immunoreactive protein were detected in a TAC preparation derived from "proplastids". A positive correlation could be demonstrated between transcriptional activity and the amount of immunoreactive 38-kDa protein by analyzing different TAC fractions eluting at different times during gel filtration of a standard TAC preparation as well as in TAC preparations obtained under various detergent conditions.

RNA editing in plant mitochondria and chloroplasts.

In the mitochondria and chloroplasts of higher plants there is an RNA editing activity responsible for specific C-to-U conversions and for a few U-to-C conversions leading to RNA sequences different from the corresponding DNA sequences. RNA editing is a post-transcriptional process which essentially affects the transcripts of protein coding genes, but has also been found to modify non-coding transcribed regions, structural RNAs and intron sequences. RNA editing is essential for correct gene expression: proteins translated from edited transcripts are different from the ones deduced from the genes sequences and usually present higher similarity to the corresponding non-plant homologues. Initiation and stop codons can also be created by RNA editing. RNA editing has also been shown to be required for the stabilization of the secondary structure of introns and tRNAs. The biochemistry of RNA editing in plant organelles is still largely unknown. In mitochondria, recent experiments indicate that RNA editing may be a deamination process. A plastid transformation technique showed to be a powerful tool for the study of RNA editing. The biochemistry as well as the evolutionary features of RNA editing in both organelles are compared in order to identify common as well as organelle-specific components.

In vivo dissection of cis-acting determinants for plastid RNA editing.

Substitutional RNA editing changes single C nucleotides in higher plant chloroplast transcripts into U residues. To determine the cis-acting sequence elements involved in plastid RNA editing, we constructed a series of chloroplast transformation vectors harboring selected editing sites of the tobacco ndhB transcript in a chimeric context. The constructs were inserted into the tobacco plastid genome by biolistic transformation leading to the production of stable chimeric RNAs. Analysis of RNA editing revealed unexpected differences in the size of the essential cis elements or in their distance from the editing site. Flanking sequences of identical size direct virtually complete editing for one pair of editing sites, partial editing for a second and no editing at all for a third pair of sites. Serial 5' and 3' deletions allowed us to define the cis-acting elements more precisely and to identify a sequence element essential for editing site recognition. In addition, a single nucleotide substitution immediately upstream of an editing position was introduced. This mutation was found drastically and selectively to reduce the editing efficiency of the downstream editing site, demonstrating that position -1 is important for either site recognition or catalysis. Our results indicate that the editing of adjacent sites is likely to be mechanistically coupled. In no case did the presence in the plastome of the additional editing sites have any effect on the editing efficiency of the endogenous ndhB sites, indicating that the availability of site-specific trans-acting factors is not rate limiting.

Retinopathy of prematurity in infants of birth weight > 2000 g after haemorrhagic shock at birth.

BACKGROUND: The risk of retinopathy of prematurity (ROP) is associated with low birth weight and low gestational age. For ROP screening examination is recommended in infants weighing < or = 1500 g or of less than 32 weeks' gestational age. METHODS: From 1991 ROP screening was performed in 452 premature infants with either a birth weight < or = 1500 g (n = 303) or a birth weight > 1500 g (n = 149) and who required additional oxygen supplementation or underwent surgery with general anaesthesia before estimated term. RESULTS: Unexpectedly, three infants with birth weights between 2080 and 2325 g and a gestational age of 32 or 33 weeks developed stage 2 or 3 ROP. One of these underwent cryocoagulation. In three infants, preterm birth was induced by sudden placental abruption with severe prenatal blood loss followed by haemorrhagic shock. The umbilical cord packed cell volume was reduced to 0.14-0.19 (normal 0.43-0.63). All three infants underwent surgery with general anaesthesia within the first weeks of life. Of the remaining 449 infants none with a birth weight > 1650 g developed any stage of ROP. CONCLUSION: Severe prenatal blood loss requiring blood transfusions and surgery with general anaesthesia may induce higher stages of ROP even in infants with birth weights exceeding the usual screening criteria.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

A promiscuous chloroplast DNA fragment is transcribed in plant mitochondria but the encoded RNA is not edited.

The RNA editing processes in chloroplasts and mitochondira of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. An rpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrial rpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potential rpoB editing sites previously detected in maize were found to be edited in the rice chloroplast rpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events.

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC_ to CUU_), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing as another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

Editing of the chloroplast ndhB encoded transcript shows divergence between closely related members of the grass family (Poaceae).

The ndhB-encoded transcript from barley chloroplasts deviates from the genomic ndhB sequence by nine C-to-U transitions, which is the maximum number of editing events for a chloroplast mRNA reported so far. Comparison with ndhB transcripts from other chloroplast species shows that six of the nine editing sites observed in barley are structurally and functionally conserved in maize, rice and tobacco. The remaining three sites, however, show divergent patterns of conservation even within the three members of the grass family. The conservation of two of these sites in tobacco but not in the closely related graminean species suggests that divergence of the ndhB editing sites is caused by the loss of preexisting editing sites rather than by gain of new sites.