Genome complexity in acute lymphoblastic leukemia is revealed by array-based comparative genomic hybridization.

Chromosomal abnormalities are important for the classification and risk stratification of patients with acute lymphoblastic leukemia (ALL). However, approximately 30% of childhood and 50% of adult patients lack abnormalities with clinical relevance. Here, we describe the use of array-based comparative genomic hybridization (aCGH) to identify copy number alterations (CNA) in 58 ALL patients. CNA were identified in 83% of cases, and most frequently involved chromosomes 21 (n=42), 9 (n=21), 6 (n=16), 12 (n=11), 15 (n=11), 8 (n=10) and 17 (n=10). Deletions of 6q (del(6q)) were heterogeneous in size, in agreement with previous data, demonstrating the sensitivity of aCGH to measure CNA. Although 9p deletions showed considerable variability in both the extent and location, all encompassed the CDKN2A locus. Six patients showed del(12p), with a common region encompassing the ETV6 gene. Complex CNA were observed involving chromosomes 6 (n=2), 15 (n=2) and 21 (n=11) with multiple regions of loss and gain along each chromosome. Chromosome 21 CNA shared a common region of gain, with associated subtelomeric deletions. Other recurrent findings included dim(13q), dim(16q) and enh(17q). This is the first report of genome-wide detection of CNA in ALL patients using aCGH, and it has demonstrated a higher level of karyotype complexity than anticipated from conventional cytogenetic analysis.

Detection of authentic SV40 DNA sequences in human brain and bone tumours.

This report summarizes our follow-up studies of SV40 DNA sequences in human brain tumors of early childhood and our confirmation of the presence of SV40 DNA in human osteosarcomas. We examined brain tumors and osteosarcoma samples by the polymerase chain reaction (PCR) using primers from four separated regions of the SV40 genome. Sequence analysis confirmed that authentic SV40 DNA was present. The regulatory region of each tumor-associated viral DNA was of archetypal length (non-duplicated enhancer); sequence variation was noted at the extreme C-terminus of the large T-antigen (T-ag) genes. Infectious SV40 was recovered from one brain tumor. We sequenced the entire early genomic region from three human isolates of SV40 and two laboratory strains originally recovered from monkeys. The predicted amino acid sequence of the large T-ags showed remarkable sequence conservation among isolates, except for a small variable region identified at the C-terminus of the protein. There were no human-isolate-specific changes detected that could serve to distinguish a human variant of SV40 nor were any tumor-type-specific viral markers observed. Based on these data, we conclude that authentic SV40 is associated with some human brain and bone tumors and that multiple SV40 strains can infect humans.

Sequence analyses of human tumor-associated SV40 DNAs and SV40 viral isolates from monkeys and humans.

SV40 DNA has been found associated with several types of human tumors. We now report a sequence comparison of SV40 DNAs from pediatric brain tumors and from osteosarcomas with viral isolates from monkeys and from humans. We analyzed the entire genomic sequences of five isolates, Baylor and VA45-54 strains from monkeys and SVCPC, SVMEN, and SVPML-1 recovered from humans, and compared them to the reference virus SV40-776. The viral sequences were highly conserved, but isolates could be distinguished by variations in the structure of the viral regulatory region and in the nucleotide sequence of the variable domain at the C-terminus of the large T-antigen gene. We conclude that multiple strains of SV40 exist that can be identified on the basis of sequences in these regions of the viral genome. The isolates were more similar to each other and to the Baylor strain than to the reference strain SV40-776. Human isolates SVCPC and SVMEN were found to be identical. The DNAs present in some human brain and bone tumors were authentic SV40 sequences. Many of the C-terminal T-ag sequences associated with human tumors were unique, but some sequences were shared by independent sources. There was no compelling evidence for human-specific strains of SV40 or for tumor type-specific associations, suggesting that SV40 has a relatively broad host range. The source of the viral DNA found in human tumors remains unknown.

Natural isolates of simian virus 40 from immunocompromised monkeys display extensive genetic heterogeneity: new implications for polyomavirus disease.

Simian virus 40 (SV40) DNAs in brain tissue and peripheral blood mononuclear cells (PBMCs) of eight simian immunodeficiency virus-infected rhesus monkeys with SV40 brain disease were analyzed. We report the detection, cloning, and identification of five new SV40 strains following a quadruple testing-verification strategy. SV40 genomes with archetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment and a single 72-bp enhancer element) were recovered from seven animal brains, two tissues of which also contained viral genomes with nonarchetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment as well as a variable duplication within the enhancer region). In contrast, PBMC DNAs from five of six animals had viral genomes with both regulatory region types. It appeared, based on T-antigen variable-region sequences, that nonarchetypal virus variants arose de novo within each animal. The eighth animal exclusively yielded a new type of SV40 strain (SV40-K661), containing a protoarchetypal regulatory region (lacking a duplication within the G/C-rich segment of the regulatory region and containing one 72-bp element in the enhancer region), from both brain tissue and PBMCs. The presence of SV40 in PBMCs suggests that hematogenous spread of viral infection may occur. An archetypal version of a virus similar to SV40 reference strain 776 (a kidney isolate) was recovered from one brain, substantiating the idea that SV40 is neurotropic as well as kidney-tropic. Indirect evidence suggests that maternal-infant transmission of SV40 may have occurred in one animal. These findings provide new insights for human polyomavirus disease.

SV40 DNA in human osteosarcomas shows sequence variation among T-antigen genes.

Authentic simian virus 40 (SV40) has been detected in association with human choroid plexus and ependymoma tumors, and SV40-like DNA sequences have been found in some human osteosarcomas. We report here an analysis of human osteosarcoma samples for the presence of SV40 DNA using PCR and primers directed at 4 distinct sites of the SV40 genome, coupled with sequence analysis. Authentic SV40 DNA sequences were detected in 5 of 10 osteosarcoma tumor samples. The SV40 regulatory region in each case was identical and of archetypal length (non-duplicated enhancer), as is usually found in natural isolates of SV40 from monkeys and in human brain tumors. A section of the gene that encodes a viral late gene product (VP1) was detected in 5 of 10 tumors and had an exact match with the known sequence of SV40. Two separated segments of the large T-antigen (T-ag) gene were found in the same 5 tumors. Analysis of the DNA sequences encoding the T-ag carboxy terminus revealed sequence variation among the tumors, as observed previously in viral DNA associated with human brain tumors. There does not appear to be a preferential association of a T-ag variable domain sequence with a given tumor type. No sequences from the regulatory region of human polyomaviruses JCV and BKV were detected in the bone tumors. We also noted less efficient recovery of SV40 DNA from tumor samples fixed in paraffin as compared to frozen tumors. Our results confirm the presence of SV40 DNA in human bone tumors and, based on the sequence variation observed for the carboxy terminus of the T-ag gene, suggest that there is not a specific SV40 strain associated with human osteosarcomas.

Identification of a variable region at the carboxy terminus of SV40 large T-antigen.

The entire early regions of three human isolates of simian virus 40 (SV40), as well as two laboratory strains recovered from monkeys, were sequenced. The early coding region of each isolate contains a number of nucleotide differences when compared to the reference strain SV40-776. These differences result in some changes in the predicted amino acid sequence of the unique region of small t-antigen and in the carboxy (C) terminus of large T-antigen. The amino acid sequence of the remainder of large T-antigen was absolutely conserved among all isolates. Thus, SV40 large T-antigen contains a variable domain at the C-terminal end of the molecule.

The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane.

The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor.

The E3-10.4K protein of adenovirus is an integral membrane protein that is partially cleaved between Ala22 and Ala23 and has a Ccyt orientation.

The Ad2 E3-10.4K protein is required together with the E3-14.5K protein to down-regulate the epidermal growth factor receptor in adenovirus-infected cells. Both proteins are also required to prevent tumor necrosis factor cytolysis under certain conditions. 10.4K is a 91 amino acid membrane-associated protein that migrates as two bands, upper and lower, on SDS-PAGE. We show here that the upper band is the primary translation product which initiates at AUG2173 in the E3 transcription unit of Ad2. The upper band is processed slowly (greater than 4 hr to complete) into the lower band by proteolytic cleavage between residues Ala22 and Ala23 by a microsome-associated protease. The upper and lower bands become equal in abundance, after which they are very stable. The N-terminus of the in vivo-derived upper band is not blocked to sequencing and it retains its initiating Met. 10.4K has a hydrophobic domain (H1) near its N-terminus that is probably a signal sequence for membrane insertion; cleavage of this signal is atypical because it was not cotranslational in vivo and it was not complete. 10.4K has a second hydrophobic domain (H2) located within residues 35-60. H2 appears to be a transmembrane (stop transfer) domain because both the upper and the lower 10.4K bands remained associated with membranes after extraction at pH 11.5, because both bands were extracted into the detergent phase with Triton X-114, and because both bands were only partially reduced in size when 10.4K-containing microsomes were digested with proteinase K. These proteinase K-digested bands were immunoprecipitated with an antipeptide antiserum against residues 19-34 but not with an antiserum against residues 68-80 or 77-91, indicating that both 10.4K bands are orientated in the membrane with the C-terminus in the cytoplasm. We conclude that the lower band of 10.4K is a type I bitopic membrane protein and suggest that the upper band is a polytopic membrane protein with both the H1 and the H2 hydrophobic domains spanning the membrane.

The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor.

In adenovirus-infected cells, the epidermal growth factor receptor (EGF-R) is internalized from the cell surface via endosomes and is degraded, and the E3 10,400-dalton protein (10.4K protein) is required for this effect (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now report that both the E3 10.4K and E3 14.5K proteins are required for this down-regulation of EGF-R in adenovirus-infected cells. Down-regulation of cell surface EGF-R was demonstrated by results from several methods, namely the absence of EGF-R autophosphorylation in an immune complex kinase assay, the inability to iodinate EGF-R on the cell surface, the formation of endosomes containing EGF-R as detected by immunofluorescence, and the degradation of the metabolically [35S]Met-labeled fully processed 170K species of EGF-R. No effect on the initial synthesis of EGF-R was observed. This down-regulation was ascribed to the 10.4K and 14.5K proteins through the analysis of cells infected with rec700 (wild-type), dl748 (10.4K-, 14.5K+), or dl764 (10.4K+, 14.5K-) or coinfected with dl748 plus dl764. Further evidence that the 10.4K and 14.5K proteins function in concert was obtained by demonstrating that the 10.4K protein was coimmunoprecipitated with the 14.5K protein by using three different antisera to the 14.5K protein, strongly implying that the 10.4K and 14.5K proteins exist as a complex. Together, these results indicate that the 10.4K and 14.5K proteins function as a complex to stimulate endosome-mediated internalization and degradation of EGF-R in adenovirus-infected cells.

Effects of alterations of inspiratory and expiratory pressures and inspiratory/expiratory ratios on mean airway pressure, blood gases, and intracranial pressure.

Twenty neonates requiring mechanical ventilation for respiratory failure, including 13 with hyaline membrane disease, were studied to assess the effects of alterations in ventilator settings on mean airway pressure (MAP), blood gases, and intracranial pressure (ICP). The study involved random alterations in peak inspiratory pressure (PIP), positive end-expiratory pressure (PEEP), and inspiratory/expiratory ratio while MAP, PaO2, ICP, and end-tibal PCO2 were continuously monitored. The results showed a significant relationship between MAP and PaO2 that was expressed as the change in PaO2 per millimeter of mercury change in MAP (delta PaO2/delta MAP) with a mean delta PaO2/delta MAP of 4.92. The delta PaO2/delta MAP was highest for changes in PEEP (6.08), followed by PIP (5.07), and inspiratory/expiratory ratio (1.9). There was a significant relationship between alterations in PEEP and PIP vs PaCO2 and pH. Increases in PEEP and decreases in PIP resulted in an elevated PaCO2 and a lowered pH, and decreases in PEEP and increases in PIP resulted in a decreased PaCO2 and an elevated pH. There was no significant relationship between MAP and ICP, but there was a significant association between delta ICP and delta PaCO2 during alterations in PIP (r = .64, P less than .001). Increases in PEEP will lead to the greatest increase in PaO2 per change in MAP, followed by increase in PIP and inspiratory/expiratory ratio using a pressure-limited ventilator.

Continuous transcutaneous oxygen monitoring in the critically ill neonate. A controlled clinical trial.

Ten neonates with respiratory distress requiring mechanical ventilation and supplemental oxygen were studied during a continuous 24-h period to determine the value of continuous transcutaneous oxygen (PtcO2) monitoring. All 10 infants were continuously monitored during the study with a Clark-type skin electrode (Litton) and 5 of the 10 also had a catheter-tip oxygen electrode in place in the umbilical artery to measure umbilical artery O2 (PuaO2). The results of these two forms of monitoring were not available for the care of the infant during the study period. Hypoxia, as defined by a PO2 of less than 50 torr, occurred for an average of 237 +/- 51 min/24 h from continuous PtcO2 monitoring as compared with 146 +/- 33 min/24 h by estimation from arterial blood gas (PaO2) (p less than 0.05). Hyperoxia, a PO2 of greater than 75 torr, occurred 69 +/- 16 min/24 h in the continuous group and 113 +/- 26 min/24 h from PaO2 estimations. Severe hypoxia, a PO2 of less than 30 torr, was not observed from PaO2 estimations, but was seen for an average 32 +/- 15 min/24 h from the PtcO2 monitoring. These latter differences were not significant. Correlation between PaO2 and PtcO2 values (r = 0.93) was greater than the correlation between PaO2 and PuaO2 (r = 0.81). PtcO2 = 19.7 +/- 0.74 X PuaO2, and the correlation coefficient between PtcO2 and PuaO2 was 0.64. Continuous oxygen monitoring revealed significantly longer periods of hypoxia than that observed from blood gas estimations alone and its use in the low birth weight infant should result in more rational ventilatory therapy and in fewer episodes of hypoxia.

Neonatal nasotracheal intubation: an evaluation.

A retrospective review of 100 surviving infants, all requiring nasotracheal intubation in the neonatal period for greater than 24 hr. was performed to assess the morbidity of this form of airway management. Seventy infants needed only one intubation, 22 were intubated twice and 8 infants required 3 intubations. No infant had evidence of laryngeal or tracheal sequelae, either in the immediate newborn period or on follow-up. Nasotracheal intubation by an experienced practitioner with appropriate tube fixation and toilet coupled with the use of low pressure ventilation and a consistent extubation routine will result in very low long-term tracheal morbidity in the neonate.

Postextubation atelectasis: a retrospective review and a prospective controlled study.

To determine the role of chest physiotherapy in the prevention of postextubation atelectasis in neonates intubated for greater than 24 hours, a retrospective survey compared the incidence of this complication in a newborn intensive care unit prior to and following the institution of a routine of chest physiotherapy. Eight of 23 infants extubated developed atelectasis in the "pre-physio" period, whereas only one collapse occurred in 20 infants treated with a routine of physiotherapy at extubation (P less than 0.025). Subsequently a prospective controlled trial compared the use of a routine of physiotherapy at extubation with no physiotherapy. Eight of 21 infants not receiving physiotherapy developed postextubation atelectasis and none of 21 infants receiving physiotherapy developed atelectasis (P less than 0.01). Seventy-six percent of the collapses involved the right upper lobe. A vigorous program of chest physiotherapy, including postural drainage emphasizing the positions of the right upper lobe and chest vibrations, will significantly reduce the incidence of postextubation atelectasis.