Children's Hospital Foundation

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5'-CACGTG-3'). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.

A GREAT shift in medical interests has occurred in recent years paralleling a shift in causes of morbidity and mortality in the United States and other Western countries. In the first half of this century the medical sciences were chiefly occupied with studies of environmental pathogenic factors affecting man in postnatal life. After the solution, in those years, of many infectious and nutritional problems, resulting in effective preventive measures, a concern with pathogenic factors acting in prenatal life developed. It has become clear that many diseases and disorders that manifest themselves after birth are to a certain extent determined before . . .

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.

THE present study was designed to investigate the viral etiology of respiratory illness in infants and young children. One of the technics employed for isolation of the virus was the recently described hemadsorption method of Vogel and Shelokov.1 , 2 With this technic, several strains of Asian influenza A virus were isolated, but the majority of agents recovered were not influenza A, B or C. The noninfluenza agents appear to comprise two newly recognized groups of myxoviruses,3 probably responsible for a segment of common respiratory illnesses in children. Their properties, serologic characterization and the evidence bearing upon their relation to disease will . . .

Adeno-associated virus (AAV) is a replication-defective parvovirus that is being developed as a vector for human gene transfer. However, a major obstacle to commonplace usage of AAV vectors is the production of recombinant virions (rAAV) in sufficient quantities for not only human trials, but also for preclinical studies of basic biology, toxicology, and efficacy. Unfortunately, current methods for large-scale production are cumbersome and expensive. We have developed a simplified method for generating rAAV by establishing neomycin-resistant cell lines containing copies of the AAV rep-cap genes and a rAAV vector. After infection with adenovirus, these cell lines were shown to produce infectious rAAV in relatively high titer. This method eliminates the need for exogenous DNA transfection and scale-up procedures are limited only by the normal constraints of growing cells in culture. This paper describes a simplified method for the production of recombinant adeno-associated virus (AAV) that involves the establishment of stable cell lines containing copies of the AAV rep and cap genes and a rAAV vector genome. In this system, rAAV is produced by merely infecting the cell line with adenovirus. Transfection of exogenous DNA is not required, and thus, large-scale production strategies become feasible.

OBJECTIVE: To assess the diagnostic accuracy and prognostic value of functional MRI (fMRI) in determining lateralization and predicting postsurgical language and memory outcomes. METHODS: An 11-member panel evaluated and rated available evidence according to the 2004 American Academy of Neurology process. At least 2 panelists reviewed the full text of 172 articles and selected 37 for data extraction. Case reports, reports with <15 cases, meta-analyses, and editorials were excluded. RESULTS AND RECOMMENDATIONS: The use of fMRI may be considered an option for lateralizing language functions in place of intracarotid amobarbital procedure (IAP) in patients with medial temporal lobe epilepsy (MTLE; Level C), temporal epilepsy in general (Level C), or extratemporal epilepsy (Level C). For patients with temporal neocortical epilepsy or temporal tumors, the evidence is insufficient (Level U). fMRI may be considered to predict postsurgical language deficits after anterior temporal lobe resection (Level C). The use of fMRI may be considered for lateralizing memory functions in place of IAP in patients with MTLE (Level C) but is of unclear utility in other epilepsy types (Level U). fMRI of verbal memory or language encoding should be considered for predicting verbal memory outcome (Level B). fMRI using nonverbal memory encoding may be considered for predicting visuospatial memory outcomes (Level C). Presurgical fMRI could be an adequate alternative to IAP memory testing for predicting verbal memory outcome (Level C). Clinicians should carefully advise patients of the risks and benefits of fMRI vs IAP during discussions concerning choice of specific modality in each case.

IN the first seven reports of this series the development and mode of preparation of an avianized attenuated measles-virus vaccine together with observations on the clinical and immunologic responses induced by this vaccine in susceptible monkeys and children are described. This vaccine will be referred to as "measles vaccine (attenuated)." In a final communication the data obtained by the various investigators collaborating in these studies will be summarized and critically evaluated. Preliminary accounts of the first trial in children have been published.1 , 2 IntroductionNumerous attempts have been made to devise an effective and harmless means for the artificial induction in . . .

GIANT-cell pneumonia, often referred to as Hecht's giant-cell pneumonia, is an interstitial pneumonitis that has so far been observed only in children. It is characterized by the presence of multinuclear giant cells with intranuclear and intracyto-plasmic inclusion bodies.1 2 3 4 5 6 7 8 9 Additional pathological features are a preponderance of mononuclear cells in the infiltrate, squamous metaplasia of the bronchial and bronchiolar epithelium, proliferation of alveolar lining cells and the occurrence in occasional cases of giant cells in organs other than the lungs. The changes characteristically found in the affected lung are illustrated in Figures 1, 2 and 3. The diagnosis of giant-cell pneumonia, which has been . . .

The potential benefits of functional magnetic resonance imaging (MRI) for the investigation of normal development have been limited by difficulties in its use with children. We describe the practical aspects, including failure rates, involved in conducting large-scale functional MRI studies with normal children. Two hundred and nine healthy children between the ages of 5 and 18 years participated in a functional MRI study of language development. Reliable activation maps were obtained across the age range. Younger children had significantly higher failure rates than older children and adolescents. It is concluded that it is feasible to conduct large-scale functional MRI studies of children as young as 5 years old. These findings can be used by other research groups to guide study design and plans for recruitment of young subjects.

The study was designed to compare intravenous ketorolac to rectal acetaminophen for analgesia and bleeding in pediatric patients undergoing tonsillectomy. We studied 50 patients, aged 2-15 yr undergoing tonsillectomy with or without adenoidectomy. In a randomized, prospective double-blind fashion, patients were assigned to receive either ketorolac (1 mg/kg) or rectal acetaminophen (35 mg/kg). Bleeding was evaluated by measuring intraoperative blood loss and noting extra measures required to obtain hemostasis. Bleeding times were also measured before and during surgery. Pain was evaluated using a standard objective pain score for the first 3 h. Persistent pain was treated with morphine, acetaminophen, and codeine and recorded for 24 h. Blood for determination of acetaminophen levels was drawn at 20 and 40 min after the administration of study drugs. Pain scores were not significantly different between the ketorolac and acetaminophen groups. The majority of patients in both groups required additional opioid in the postoperative period. Acetaminophen levels were all less than the therapeutic range. Intraoperative bleeding times were normal in all patients, but blood loss was significantly higher in the ketorolac group (2.67 mL/kg) compared to the acetaminophen group (1.44 mL/kg), P = 0.025. Significantly more measures to achieve hemostasis were required in the ketorolac group (P = 0.012). We conclude that ketorolac is no more effective than high-dose rectal acetaminophen for analgesia in the patient undergoing tonsillectomy. Hemostasis during tonsillectomy was significantly more difficult to achieve in patients receiving ketorolac.

DEVELOPMENTAL DYNAMICS provides a focus for communication among developmental biologists who study the progressive and dynamic emergence of form and function during embryonic development. The journal is an international forum for the exchange of novel and substantive information on mechanisms that control development. We seek manuscripts presenting work done at all levels of biological organization, ranging from the molecular to the organismal, using both animal and plant model systems, and we welcome studies that advance our understanding of the developmental basis of human disease.

The correlation of many HLA-associated autoimmune and genetic diseases with the polymorphic complement C4 genes may be attributed to the presence of disease susceptibility genes in the close proximity of C4. We have cloned and characterized a pair of partially duplicated genes, RP1 and RP2, located 611 base pairs upstream of the human C4A and C4B genes, respectively. The putative RP protein, consisting of 364 amino acid residues, is basic and highly hydrophilic. There is a bipartite nuclear localization signal at residues 114-131 and therefore RP may be a nuclear protein. Northern blot analysis suggested that RP is ubiquitously expressed. The 5' region of the RP1 gene is CpG rich, which is a characteristic of housekeeping genes. The RP1 gene contains nine exons. Located in the fourth intron is a cluster of Alu elements, and a newly defined composite retroposon SVA with a SINE, multiple copies of GC-rich VNTRs and an Alu element altogether enclosed by direct terminal repeats. Members of SVA are also present in the complement C2 gene located about 20 kilobases upstream of RP1 in the HLA and in the cytochrome CYP1A1 gene. Determination of the DNA sequences for RP2 from two different HLA haplotypes revealed identical hybrid sequences which resulted from fusion of RP with the tenascin-like Gene X and truncation of the 5' regions of both genes. Cumulative data suggest that the four tandemly arranged genes RP, complement C4, steroid 21-hydroxylase (CYP21), and Gene X altogether form a modular structure, RCCX. The number of RCCX modules varies from one to three or more in the population. Absence of the truncated genes RP2 and Gene XA have been detected in genomes with single RCCX modules. Duplication of the RCCX modules probably occurred before the speciation of great apes and humans as they contain the same breakpoint region of RP and Gene X gene duplication.

The frequent variations of human complement component C4 gene size and gene numbers, plus the extensive polymorphism of the proteins, render C4 an excellent marker for major histocompatibility complex disease associations. As shown by definitive RFLPs, the tandemly arranged genes RP, C4, CYP21, and TNX are duplicated together as a discrete genetic unit termed the RCCX module. Duplications of the RCCX modules occurred by the addition of genomic fragments containing a long (L) or a short (S) C4 gene, a CYP21A or a CYP21B gene, and the gene fragments TNXA and RP2. Four major RCCX structures with bimodular L-L, bimodular L-S, monomodular L, and monomodular S are present in the Caucasian population. These modules are readily detectable by TaqI RFLPs. The RCCX modular variations appear to be a root cause for the acquisition of deleterious mutations from pseudogenes or gene segments in the RCCX to their corresponding functional genes. In a patient with congenital adrenal hyperplasia, we discovered a TNXB-TNXA recombinant with the deletion of RP2-C4B-CYP21B. Elucidation of the DNA sequence for the recombination breakpoint region and sequence analyses yielded definitive proof for an unequal crossover between TNXA from a bimodular chromosome and TNXB from a monomodular chromosome.

The generation of tissue eosinophilia is governed in part by chemokines; initial investigation has identified three chemokines in the human genome with eosinophil selectivity, referred to as eotaxin-1, -2, and -3. Elucidation of the role of these chemokines is dependent in part upon analysis of murine homologues; however, only one murine homologue, eotaxin-1, has been identified. We now report the characterization of the murine eotaxin-2 cDNA, gene and protein. The eotaxin-2 cDNA contains an open reading frame that encodes for a 119-amino acid protein. The mature protein, which is predicted to contain 93 amino acids, is most homologous to human eotaxin-2 (59.1% identity), but is only 38.9% identical with murine eotaxin-1. Northern blot analysis reveals three predominant mRNA species and highest constitutive expression in the jejunum and spleen. Additionally, allergen challenge in the lung with Aspergillus fumigatus or OVA revealed marked induction of eotaxin-2 mRNA. Furthermore, eotaxin-2 mRNA was strongly induced by both transgenic over-expression of IL-4 in the lung and administration of intranasal IL-4. Analysis of eotaxin-2 mRNA expression in mice transgenic for IL-4 but genetically deficient in STAT-6 revealed that the IL-4-induced expression was STAT-6 dependent. Recombinant eotaxin-2 protein induced dose-dependent chemotactic responses on murine eosinophils at concentrations between 1-1000 ng/ml, whereas no activity was displayed on murine macrophages or neutrophils. Functional analysis of recombinant protein variants revealed a critical role for the amino terminus. Thus, murine eotaxin-2 is a constitutively expressed eosinophil chemokine likely to be involved in homeostatic, allergen-induced, and IL-4-associated immune responses.

We administered high doses of calcitriol (up to 32 micrograms per day) to an infant with malignant osteopetrosis, in an attempt to stimulate bone resorption. The patient was placed on a low-calcium diet to prevent hypercalcemia. Measures of bone turnover increased during calcitriol therapy; hydroxyproline excretion rose from 140 to 1358 micrograms per milligram of creatinine per 24 hours, with parallel increases in the ratio of calcium to creatinine in the urine, urinary gamma-carboxyglutamic acid, serum osteocalcin, and serum alkaline phosphatase. A pretreatment bone-biopsy specimen contained no osteoclasts with ruffled borders, a feature of active osteoclasts. After 11 days of calcitriol, ruffled borders were noted. After three months, numerous osteoclasts with ruffled borders and associated bony disruption were evident. Before therapy, the patient's monocytes were incapable of in vitro bone resorption, but after calcitriol, their resorptive capacity was increased to 3.3 times control levels. These data demonstrate that calcitriol increased bone mineral and matrix turnover in our patient. However, during the three months of calcitriol therapy there was only slight clinical improvement in her severe disease. Early and sustained treatment with calcitriol may be useful in osteopetrosis.

During 29 months of prospective longitudinal study of diarrhea in the home, human rotaviruses (HRVs) infected one or more members in 51% of 65 families, 35 of 126 children (28%) and 16 of 124 adults (13%). Within the 33 affected families, 57% of 62 children and 25% of 65 adults were infected. HRV gastroenteritis peaked at 40/100 person years at ages 12 to 23 months and decreased to 5 episodes/100 person years in adults. Among 25 children 0 through 36 months of age who had HRV infection, 88% were symptomatic. Of the 22 children with symptomatic HRV infection, 1 required hospitalization and 8 were seen by their physician for supportive care. HRVs were found in 12% of 216 stools obtained during gastrointestinal illness, but in only 0.2% of 1238 non-illness stools tested. HRV infections were noted as early as October and as late as April. Of 33 families who were studied for 2 seasons, at least 1 individual in each of 3 families experienced HRV infections in both years, but only one, an adult, shed virus and had symptoms in both seasons.

Gene transfer to muscle holds overt promise for the treatment of inherited myopathies, lysosomal storage disorders, and serum protein deficiencies. In addition, muscle could provide a reservoir for delivery of therapeutic molecules like blood clotting factors, erythropoietin, or insulin. To date, successful gene transfer to muscle has been limited by the inefficiency of the vector delivery systems and the transient nature of gene expression. In this paper, we show that a vector based on recombinant adeno-associated virus (rAAV) can efficiently transduce adult mouse skeletal muscle. Transduced myofibers escape immune elimination and transgene expression is robust beyond 5 months. Importantly, input vector DNA appears to undergo conversion from single-stranded genomes to high-molecular-weight concatameric forms. These data suggest that rAAV might have a significant advantage over many other viral and nonviral gene delivery methods, and holds significant promise as a vector for gene transfer to mature muscle. A recombinant adeno-associated virus (rAAV) vector was shown to transduce mouse skeletal muscle effectively. Transgene expression (β-galactosidase) was evident early after injection and lasted for more than 5 months. Transduced cells were not eliminated by the immune system, but neutralizing antibodies appeared to block a second dose of vector. Analysis of vector DNA after injection showed a shift from low- to high-molecular-weight forms. rAAV vectors appear to have significant advantages over other currently available methods for gene delivery to muscle.

Human rotavirus (HRV) type 1 or 2, adenovirus, or non-cultivatable 27 nm virus-like particles were demonstrated by electron microscopy and/or rotavirus ELISA in fecal samples from 45.5% of 604 gastroenteritis inpatients, 25.0% of 200 gastroenteritis outpatients and 6.0% of 812 control subjects, all sampled at Children's Hospital National Medical Center. Washington, DC. Rotaviruses were the most common pathogens detected as 39% and 22% of gastroenteritis inpatients and outpatients, respectively, shed HRV. About three-fourths of the rotaviruses were type 2, which was prevalent during five successive epidemic years from January, 1974, through June, 1978. HRV type 1 was detected in the last four successive epidemic years and represented nearly half of the HRV infections observed among gastroenteritis inpatients during the year 1977--1978. Both rotavirus serotypes were detected most often in the month of January, when 71% of 123 gastroenteritis inpatients and 62% of 34 gastroenteritis outpatients shed one of these viruses. Uncultivatable adenoviruses were detected significantly more frequently in stools from patients with gastroenteritis (3.9%) than from control subjects (0.6%), suggesting that these viruses played a role in acute enteric disease. The frequency of detection of 27 nm particles was not significantly different in gastroenteritis and control patients. Numerically, HRV infection was detected most often in gastroenteritis inpatients who were 10 through 12 months of age. The group of gastroenteritis inpatients with the highest percentage of HRV infection was 13 through 15 months of age. The excess of type 2 HRV infection relative to type 1 infection was especially large in those aged 7 through 24 months. Lower socioeconomic status or greater crowding appeared to be associated with the occurrence of rotavirus infection earlier in life and earlier in the epidemic year.

IN the previous paper1 isolation of measles virus in 3 cases of giant-cell pneumonia (Hecht) without associated signs and symptoms of measles was described. These findings provide direct evidence in support of the hypothesis long current that this form of pneumonia may be caused by this agent. The present communication presents the results of studies of 4 leukemic children with overt measles complicated by severe pneumonitis (Table 1). The data supplement the evidence already obtained for measles virus in cases of giant-cell pneumonia without clinically apparent measles. They also indicate that the presumptive diagnosis of this condition can be made . . .

PURPOSE: The objectives of this study were to determine if the addition of etoposide, thioguanine, and dexamethasone to daunorubicin and cytarabine (five-drug regimen) during induction would improve remission induction rates and survival of children with acute myeloid leukemia (AML) when compared with the standard regimen of cytarabine and daunorubicin (7 + 3) and whether allogeneic bone marrow transplantation (BMT) or intensive chemotherapy consolidation with or without maintenance would give a superior outcome. PATIENTS AND METHODS: A total of 591 assessable children with AML entered Childrens Cancer Group (CCG) trial 213 between January 1986 and February 1989. The status of patients as of September 1, 1992 forms the basis of this report. The results were compared with previous AML studies. RESULTS: The projected survival rate of all patients at 5 years is 39% (event-free survival [EFS] rate, 31%), which is superior to that of the prior CCG study (P = .01). The induction rate was 79% for 7 + 3 and 76% for the five-drug regimen (not significant). Comparisons of BMT to chemotherapy favored BMT, but these differences do not always reach statistical significance (eg, 5-year disease-free survival [DFS] rate, 46% v 38% [P = .06] with donor available and 54% v 37% [P = .002] if treated according to protocol intent). No benefit for maintenance therapy was found and, in some comparisons, it was inferior to discontinuation of therapy (5-year survival rate, 46% v 68%, P < .01). CONCLUSION: The 5-year EFS rate of patients with AML is 31% and has improved. The five-drug induction regimen is no better than standard induction, BMT appears superior to chemotherapy, and maintenance therapy was not beneficial.