Perinatal risk factors of adverse outcome in very preterm children: a role of initial treatment of respiratory insufficiency?

AIM: To investigate risk factors of adverse outcome in a cohort of very preterm children treated mainly with nasal continuous positive airway pressure (CPAP) during the neonatal course. METHODS: In Denmark, preterm children are treated with nasal CPAP as a first approach to respiratory support. A national prospective study of all infants with a birthweight below 1000 g or a gestational age below 28 wk born in 1994-1995 was initiated to evaluate this approach. Of the 269 surviving children 164 (61%) were not treated with mechanical ventilation in the neonatal period. A follow-up of the children at 5 y of age was conducted. Data from the neonatal period and the 5-y follow-up were analysed. RESULTS: In multivariate analyses including 250 children, a severely abnormal neonatal brain ultrasound scan was predictive of cerebral palsy (OR = 19.9, CI 95%: 6.1-64.8) and intellectual disability (OR = 6.2, CI 95%: 2.3-16.5). A high Clinical Risk Index for Babies (CRIB) score (OR = 2.4, CI 95%: 1.1-5.5) and chronic lung disease (OR = 2.8, CI 95%: 1.2-6.9) were predictive of intellectual disability. In univariate analyses mechanical ventilation was associated with cerebral palsy (OR=4.3, CI 95%: 1.7-10.8) and intellectual disability (OR = 2.2, CI 95%: 1.2-4.2), but the associations became insignificant in multivariate analyses including chronic lung disease and a severely abnormal ultrasound scan. CONCLUSION: The associations between neonatal risk factors and adverse outcome in our cohort were very similar to those found in other cohorts with another initial treatment of respiratory insufficiency. We found no significant adverse effects of mechanical ventilation beyond what could be explained by associations with chronic lung disease and IVH 3-4/PVL.

The Danish national study in infants with extremely low gestational age and birthweight (the ETFOL study): respiratory morbidity and outcome.

AIM: To describe and analyse neonatal care, short and long-term morbidity with special reference to ventilatory support and chronic lung disease (CLD) in a population-based study. METHODS: During 1994 and 1995 a prospective, nation-wide, multicentre study was conducted, comprising 477 liveborn infants with gestational age (GA) < 28 wk and/or birthweight < 1000 g. Of these, 407 infants received active treatment. The ventilatory treatment was based on the principle of permissive hypercapnia and early nasal continuous positive airway pressure (NCPAP) supplemented with surfactant and ventilator therapy in case of CPAP failure. RESULTS: Among actively treated infants 85% received CPAP and 23% mechanical ventilation from the first day of life. A total of 269 infants (56%) survived to discharge. Of these, 195 had a GA < 28 wk. One-hundred and five survivors with GA < 28 wk survived with NCPAP as sole respiratory support. In surviving infants, periventricular leucomalacia/intraventricular haemorrhage grade 3-4 was found in 10%, retinopathy of prematurity grade > 2 in 4%, and oxygen requirement at 36 and 40 wk of postmenstrual age (CLD) in 16 and 5%, respectively. Three infants either died of CLD (n = 1) or required oxygen therapy beyond 43 wk of postmenstrual age. Logistic regression analysis showed significant associations between oxygen requirement at 40 wk and GA, septicaemia, mechanical ventilation, symptomatic patent ductus arteriosus and Clinical Risk Index for Babies score. Only the two last-mentioned factors proved significant in infants with GA < 28 wk. No infant died after discharge and 253 (94%) were followed up at 2 y of corrected age; one or more moderate to severe impairments were found in 66 (26%) of the examined children. CONCLUSION: Ventilatory treatment in extremely premature and extremely low-birthweight infants based on early NCPAP and permissive hypercapnia may result in comparable survival rates and sensorineural outcome; however, the incidence of CLD seems lower than that reported on conventional treatment.

A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis.

Ustilago maydis, the causative agent of corn smut disease, is one of the most versatile model systems for the study of plant pathogenic fungi. With the availability of the complete genomic sequence there is an increasing need to improve techniques for the generation of deletion mutants in order to elucidate the functions of unknown genes. Here a method is presented which allows one to generate constructs for gene replacement without the need for cloning. The 5' and 3'-regions of the target gene are first amplified by PCR, and subsequently ligated directionally to a marker cassette via two distinct SfiI sites, providing the flanking homologies needed for homologous recombination in U. maydis. Then the ligation product is used as a template for the amplification of the deletion construct, which can be used directly for transformation of U. maydis. The use of the fragments generated by PCR drastically increases the frequency of homologous recombination when compared to the linearized plasmids routinely used for gene replacement in U. maydis.

Heterologous transposition in Ustilago maydis.

The phytopathogenic basidiomycete Ustilago maydis has become a model system for the analysis of plant-pathogen interactions. The genome sequence of this organism will soon be available, increasing the need for techniques to analyse gene function on a broad basis. We describe a heterologous transposition system for U. maydis that is based on the Caenorhabditis transposon Tc1, which is known to function independently of host factors and to be active in evolutionarily distant species. We have established a nitrate reductase based two-component counterselection system to screen for Tc1 transposition. The element was shown to be functional and transposed to several different locations in the genome of U. maydis. The insertion pattern observed was consistent with the proposed general mechanism of Tc1/mariner integration and constitutes a proof of principle for the first heterologous transposition system in a basidiomycete species. By mapping the insertion site context to known genomic sequences, Tc1 insertion events were shown to occur on different chromosomes, but exhibit a preference for non-coding regions. Only 20% of the insertions were found in putative open reading frames. The establishment of this system will permit efficient gene tagging in U. maydis and possibly also in other fungi.

Rehospitalization for respiratory syncytial virus infection in infants with extremely low gestational age or birthweight in Denmark.

AIM: To determine the risk of rehospitalization for respiratory syncytial virus (RSV) infection during the first 2 y of life in extremely preterm infants. METHODS: Records on all rehospitalizations during the first 2 living years of all infants born with gestational age <28 wk or birthweight <1,000g during 1994 and 1995 in Denmark were retrospectively reviewed. RESULTS: Among 240 eligible infants, 43 (18%) had been rehospitalized 48 times owing to RSV. In infants (n = 210) without CLD the risk of rehospitalization for RSV was 16%, whereas in infants with CLD (n = 30) it was 30% (p = 0.065). Eighteen infants (38%) required respiratory support (supplemental oxygen only 3, continuous positive airway pressure 14, mechanical ventilation 1). Apart from CLD the only factor that could be associated with increased risk of hospitalization for RSV was discharge during autumn (p = 0.05). No infant died from RSV infection. CONCLUSION: The high rate of rehospitalization for RSV in extremely preterm infants in Denmark, especially in infants with CLD, should lead to considerations concerning more widespread use of prophylaxis against RSV in these infants.

A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis.

The phytopathogenic basidiomycete Ustilago maydis produces sexual teliospores only after infection of its host plant, maize. To investigate the process of spore formation, we have isolated Ssp1, a protein that is abundantly expressed in mature teliospores. The corresponding gene, ssp1, is expressed at low levels in haploid sporidia; however, transcriptional levels are drastically induced in mature teliospores. Transcriptional regulation of ssp1 involves positive and negative promoter elements, and is subject to control by the cAMP signaling cascade and histone deacetylase-mediated repression. Ssp1 shows similarity to linoleate diol synthase, a fatty acid dioxygenase from the fungus Gaeumannomyces graminis. In agreement with this presumed function, Ssp1 is localized on lipid bodies in germinating teliospores, suggesting a role in the mobilization of storage lipids.

Identification of genes in the bW/bE regulatory cascade in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, the switch to filamentous growth and pathogenic development is controlled by a heterodimeric transcription factor consisting of the bW and bE homeodomain proteins. To identify genes in the regulatory cascade triggered by the bW/bE heterodimer, we have constructed strains in which transcription of the b genes is inducible by either arabinose or nitrate. At different time-points after induction, genes that are switched on or off were identified through a modified, non-radioactive RNA fingerprint procedure. From 348 gene fragments isolated initially, 48 fragments representing 34 different genes were characterized in more detail. After eliminating known genes, false positives and genes influenced in their expression profile by media conditions, 10 new b-regulated genes were identified. Of these, five are upregulated and five are downregulated in presence of the b heterodimer. Two do not share significant similarity to database entries, whereas the other eight show similarity to disulphide isomerases, exochitinases, cation antiporters, plasma membrane (H+)-ATPases, acyl transferases, a capsular associated protein of Cryptococcus neoformans, DNA polymerases X, as well as to a potential protein of Neurospora crassa. We demonstrate that in one of the early upregulated genes, the promoter can be bound by a bW/bE fusion protein in vitro. Interestingly, three out of the four genes that are downregulated by the b heterodimer appear upregulated after pheromone stimulation, suggesting a connection to the mating process.

[The idiopathic, neoplasmatic external biliary fistula]. / Samoistna, nowotworowa zewnetrzna przetoka pecherzyka zólciowego.

Authors present a case of 91 year old patient with spontaneous, neoplasmatic, external biliary fistula localized in right hypochondriac area, treated in the beginning as an abscess. The patient was operated because of the jaundice--cancer was discovered in the course of operation. Authors discussed diagnostic procedures and treatment.

A protein with similarity to the human retinoblastoma binding protein 2 acts specifically as a repressor for genes regulated by the b mating type locus in Ustilago maydis.

Pathogenic development in the corn smut fungus Ustilago maydis is controlled by a heterodimer of the two homeodomain proteins bE and bW which are encoded by the b mating type locus. The bE/bW heterodimer is thought to achieve its function as a transcriptional regulator of pathogenicity genes, either directly by binding to cis regulatory sequences or indirectly via a b-dependent regulatory cascade. In a screen for components of the b-dependent regulatory cascade we have isolated Rum1 (regulator U. maydis 1), a protein with similarities to the human retinoblastoma binding protein 2. Deletion of rum1 results in expression of several b regulated genes independently from their activation via the bE/bW heterodimer. rum1 mutant strains remain pathogenic, proliferate in planta, but fail to produce spores. The defect leads to an arrest in spore development at a defined stage before the spore wall is generated. Deduced from the highly conserved domain structure of Rum1 that includes a DNA-binding motif and a region known to facilitate the interaction with histone deacetylases, we propose that Rum1 functions as a transcriptional repressor through the modulation of chromatin structure.

Identification of a target gene for the bE-bW homeodomain protein complex in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW that form a heterodimeric complex when both proteins originate from different alleles. The heterodimer is presumed to be the central regulator for pathogenicity genes. Here, we show that a translational fusion protein comprising specific domains of bE1 and bW2 remains biologically active and binds to a sequence motif in the promoter of lga2, a gene located in the a mating type locus. This b binding sequence 1 (bbs1) is also recognized by the native bE1-bW2 heterodimer in vivo and mediates the b-dependent regulation of the lga2 gene. Our data demonstrate that the bE-bW heterodimer can act as a positive transcriptional regulator.

Regulation of pathogenic development in the corn smut fungus Ustilago maydis.

Abstract In Ustilago maydis, the b mating type locus constitutes the central regulatory domain for pathogenic development. The b locus encodes two homeodomain proteins that are thought to function as transcriptional regulators. Here we describe our current view of the different regulatory pathways in which the b locus is involved. We emphasize on the development of tools for the isolation of genes that are directly regulated by b homeodomain proteins.

Early nasal continuous positive airway pressure and minimal handling in the treatment of very-low-birth-weight infants.

Continuous positive airway pressure (CPAP) was introduced in 1971 and at that time welcomed as 'the missing link' between oxygen and ventilator treatment of premature infants. Originally CPAP was administered by tracheal tube (or head box) which because of the inherent risk of complications necessitated a cautious approach. New, more simple and less risky methods of application, such as nasal CPAP (N-CPAP), permitted earlier treatment which in randomized trials showed a reduction in inspired oxygen concentration, reduced need for mechanical ventilation and a reduction in the rate of death. Early N-CPAP/minimal handling today is an established first-line treatment in a number of centers in Denmark and Sweden, while N-CPAP outside Scandinavia apparently is used less often. However, recently the method has gained new interest as more publications have demonstrated that N-CPAP/minimal handling is both feasible and effective in most very-low-birth-weight infants. Early rescue treatment with fast-acting surfactant, given during a brief intubation, has increased the effectiveness of N-CPAP further. Descriptive studies on N-CPAP suggest that the risk of bronchopulmonary dysplasia may be lower than in conventional intensive treatment because of the relatively low need for mechanical ventilation. This question is at the present time being addressed in a randomized controlled trial in England.

[Frequency of admissions to neonatal care units of 1994 birth cohorts in Funen and Frederiksborg Counties]. / Hyppighed af indlaeggelser på neonatalafsnit fra fødselskohorterne 1994 i Fyns og Frederiksborg Amter.

The frequency of admission to neonatal intensive care units is higher in Denmark than in the UK. The organization of the paediatric departments varies from county to county in Denmark. To investigate possible differences due to the organization, the records for all hospitalized newborns in a one year period in two counties (FR and FU) were studied. The rate of prematurity was higher in county FU than in FR, but, surprisingly, the admission rate was lowest in county FU. For full term infants the highest admission rate was found in county FR, where more infants were treated with nasal continuous positive airway pressure compared to FU. Short admissions were more common in FR. The on-call paediatrician is a senior doctor in FU, whereas it is a junior doctor in FR, and this could be a possible explanation for the differences between the two counties.

Weight, length and head circumference standards based on a population of Danish newborn boys and girls in gestational weeks 25 to 43.

Growth standards of birth weight, birth length and birth head circumference were constructed based on a population of Danish newborn boys and girls in gestational weeks twenty-five to forty-three. Study populations were residents of the municipality of Odense for later gestational ages and residents of the county of Funen for early gestational ages. Strict selection criteria were employed.

The homeodomains of the heterodimeric bE and bW proteins of Ustilago maydis are both critical for function.

In Ustilago maydis, pathogenic development is controlled by the multiallelic b-mating type locus. b encodes two different homeodomain proteins, bE and bW, that dimerize only when they originate from different alleles. The heterodimer is thought to act as a transcriptional regulator of pathogenicity genes. To address the contribution of individual homeodomains to the function of the hetereodimer, amino acids presumed to be critical for the function of homeodomains were altered by introducing deletions and single amino acid substitutions. This analysis demonstrated that both homeodomains are essential for function of the bE/bW complex. All point mutations affecting conserved amino acids in bW resulted in non-functional proteins. For bE the mutational analysis revealed that certain features of the homeodomain, like base-specific contacts and contacts to the sugar phosphate backbone, contribute to function. However, the structural requirements for function of the bE homeodomain appear to be more flexible than those of the bW homeodomain. We compare our data with the related systems in Schizophyllum commune, Coprinus cinereus and Saccharomyces cerevisiae.

Single-chain fusions of two unrelated homeodomain proteins trigger pathogenicity in Ustilago maydis.

Pathogenic and sexual development of the fungus Ustilago maydis, the causal agent of corn smut disease, is regulated by heterodimerization of two unrelated homeodomain proteins bE and bW, both encoded by the multi-allelic b mating-type locus. This complex can only be formed if the two proteins are derived from different alleles. The heterodimer is believed to function as a transcriptional regulator that binds to target sites upstream of developmentally regulated genes. We have synthesized a translational fusion in which bE is tethered to bW by a designed flexible kink region. U. maydis strains expressing this synthetic b-fusion become pathogenic for corn illustrating that the single-chain fusion substitutes for the active bE/bW heterodimer. Synthetic b-fusions in which bE and bW originate from the same allele as well as fusions deleted for the dimerization domains were shown to be active while both homeodomains were required for function. Such active fusion proteins are expected to be instrumental in the identification of pathogenicity genes.

Isolation of a carbon source-regulated gene from Ustilago maydis.

We have isolated a carbon source-regulated gene from the phytopathogenic fungus Ustilago maydis by use of a promoter-probe vector. This gene, called crg1, is strongly induced by L-arabinose and efficiently repressed by D-glucose and D-xylose. The predicted 36.5-kDa mature crg1 gene product lacks similarity to known proteins but is likely to be secreted. Sequences required for regulated expression of a reporter gene are contained within a 3.6-kb fragment upstream of the crg1 gene. The promoter of crg1 fulfils requirements for an efficient controllable gene expression system in U. maydis

Control of mating and development in Ustilago maydis.

In the fungus Ustilago maydis, the ability to distinguish between partners that are of the same or of different mating type is controlled by two mating-type loci. One locus allows extracellular recognition though a pheromone-based system. After cell fusion, the other mating-type locus, which exists in multiple alleles, determines intracellular recognition. Each allele encodes a pair of homeodomain proteins that are active only in pairwise combinations in which the two partners originate from different alleles of the locus. Recent discoveries suggest that the underlying molecular recognition mechanism is the ability to form heterodimers. Whereas the proteins in all different allelic combinations interact, it is a specific feature of proteins from the same allele not to interact. This suggests the existence of a code for protein-protein recognition.

Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis.

In the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides. Only combinations of bE and bW of different allelic origin are active. We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon. By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles. Dimerization involves the N-terminal variable domains. Different point mutants of bE2 were isolated that function in combination with bW2. The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system. Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins. Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization.

Prolonged intrathecal chemotherapy replacing cranial irradiation in high-risk acute lymphatic leukaemia: long-term follow up with cerebral computed tomography scans and endocrinological studies.

UNLABELLED: Cranial irradiation in children with acute lymphatic leukaemia (ALL) decreases the risk of CNS relapse but is associated with serious long-term side-effects. We present the long-term outcome of 21 children with high-risk ALL who received prolonged intrathecal chemotherapy instead of the recommended cranial irradiation. Intrathecal triple therapy (methotrexate, hydrocortisone, and cytarabine) was administered every 2nd month throughout the maintenance phase. The average number of courses of intrathecal methotrexate was 8.7 and of triple 9.0. The 5-year event-free survival was 79%. No CNS relapses occurred. CT scan was performed at diagnosis, at cessation of therapy, and 3 years thereafter. No density abnormalities, pathological contrast enhancement, ventricular dilatation, or calcifications were found. One child showed cortical atrophy both at diagnosis and at cessation of therapy. There was a slight decrease in height SDS with time but no change in weight SDS. Delayed bone age was found in 5 children. No abnormalities of growth hormone, thyroid, adrenal, or gonadal function were observed. CONCLUSION: The study indicates that extended intrathecal chemotherapy in children with high-risk ALL may provide an effective protection from CNS relapses and is associated with a low risk of long-term side-effects.