Showing papers by "Boston Children's Hospital published in 1997"

Heart rate variability: Origins, methods, and interpretive caveats

Abstract: Components of heart rate variability have attracted considerable attention in psychology and medicine and have become important dependent measures in psychophysiology and behavioral medicine. Quantification and interpretation of heart rate variability, however, remain complex issues and are fraught with pitfalls. The present report (a) examines the physiological origins and mechanisms of heart rate variability, (b) considers quantitative approaches to measurement, and (c) highlights important caveats in the interpretation of heart rate variability. Summary guidelines for research in this area are outlined, and suggestions and prospects for future developments are considered.

Atomic structure of the ectodomain from HIV-1 gp41

Abstract: Fusion of viral and cellular membranes by the envelope glycoprotein gp120/gp41 effects entry of HIV-1 into the cell. The precursor, gp160, is cleaved post-translationally into gp120 and gp41 which remain non-covalently associated. Binding to both CD4 and a co-receptor leads to the conformational changes in gp120/gp41 needed for membrane fusion. We used X-ray crystallography to determine the structure of the protease-resistant part of a gp41 ectodomain solubilized with a trimeric GCN4 coiled coil in place of the amino-terminal fusion peptide. The core of the molecule is found to be an extended, triple-stranded alpha-helical coiled coil with the amino terminus at its tip. A carboxy-terminal alpha-helix packs in the reverse direction against the outside of the coiled coil, placing the amino and carboxy termini near each other at one end of the long rod. These features, and the existence of a similar reversal of chain direction in the fusion pH-induced conformation of influenza virus HA2 and in the transmembrane subunit of Moloney murine leukaemia virus (Fig. 1a-d), suggest a common mechanism for initiating fusion.

Tensegrity: the architectural basis of cellular mechanotransduction

Abstract: Physical forces of gravity, hemodynamic stresses, and movement play a critical role in tissue development. Yet, little is known about how cells convert these mechanical signals into a chemical response. This review attempts to place the potential molecular mediators of mechanotransduction (e.g. stretch-sensitive ion channels, signaling molecules, cytoskeleton, integrins) within the context of the structural complexity of living cells. The model presented relies on recent experimental findings, which suggests that cells use tensegrity architecture for their organization. Tensegrity predicts that cells are hard-wired to respond immediately to mechanical stresses transmitted over cell surface receptors that physically couple the cytoskeleton to extracellular matrix (e.g. integrins) or to other cells (cadherins, selectins, CAMs). Many signal transducing molecules that are activated by cell binding to growth factors and extracellular matrix associate with cytoskeletal scaffolds within focal adhesion complexes. Mechanical signals, therefore, may be integrated with other environmental signals and transduced into a biochemical response through force-dependent changes in scaffold geometry or molecular mechanics. Tensegrity also provides a mechanism to focus mechanical energy on molecular transducers and to orchestrate and tune the cellular response.

Three-dimensional structure of the tyrosine kinase c-Src

Abstract: The structure of a large fragment of the c-Src tyrosine kinase, comprising the regulatory and kinase domains and the carboxy-terminal tall, has been determined at 1.7 A resolution in a closed, inactive state. Interactions among domains, stabilized by binding of the phosphorylated tail to the SH2 domain, lock the molecule in a conformation that simultaneously disrupts the kinase active site and sequesters the binding surfaces of the SH2 and SH3 domains. The structure shows how appropriate cellular signals, or transforming mutations in v-Src, could break these interactions to produce an open, active kinase.

Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines

Abstract: The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential1–3. Telomerase is an enzyme that restores telomeric DNA sequences4, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo5. Telomerase activity has been detected in 50–100% of tumors of different types, but not in most normal adult somatic tissues6,7. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date7. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization8–11 presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres)11. Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.

Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS.

Abstract: The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability CTL escape may play an important role in the pathogenesis of HIV infection

Preliminary definition of improvement in juvenile arthritis

Abstract: Objective. To identify a core set of outcome variables for the assessment of children with juvenile arthritis (JA), to use the core set to develop a definition of improvement to determine whether individual patients demonstrate clinically important improvement, and to promote this definition as a single efficacy measure in JA clinical trials. Methods. A core set of outcome variables was established using a combination of statistical and consensus formation techniques. Variables in the core set consisted of 1) physician global assessment of disease activity; 2) parent/patient assessment of overall well-being; 3) functional ability; 4) number of joints with active arthritis; 5) number of joints with limited range of motion; and 6) erythrocyte sedimentation rate. To establish a definition of improvement using this core set, 21 pediatric rheumatologists from 14 countries met, and, using consensus formation techniques, scored each of 72 patient profiles as improved or not improved. Using the physicians' consensus as the gold standard, the chi-square, sensitivity, and specificity were calculated for each of 240 possible definitions of improvement. Definitions with sensitivity or specificity of <80% were eliminated. The ability of the remaining definitions to discriminate between the effects of active agent and those of placebo, using actual trial data, was then observed. Each definition was also ranked for face validity, and the sum of the ranks was then multiplied by the kappa statistic. Results. The definition of improvement with the highest final score was as follows: at least 30% improvement from baseline in 3 of any 6 variables in the core set, with no more than 1 of the remaining variables worsening by >30%. The second highest scoring definition was closely related to the first; the third highest was similar to the Paulus criteria used in adult rheumatoid arthritis trials, except with different variables. This indicates convergent validity of the process used. Conclusion. We propose a definition of improvement for JA. Use of a uniform definition will help standardize the conduct and reporting of clinical trials, and should help practitioners decide if a child with JA has responded adequately to therapy. We are in the process of prospectively validating this definition and several others that scored highly.

CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia

Abstract: Several members of the chemokine receptor family are used together with CD4 for HIV-1 entry into target cells1–6. T cell line-tropic (T-tropic) HIV-1 viruses use the chemokine receptor CXCR4 as a co-receptor1, whereas macrophage-tropic (M-tropic) primary viruses use CCR5 (refs 2–6). Individuals with defective CCR5 alleles exhibit resistance to HIV-1 infection7,8, suggesting that CCR5 has an important role in vivo in HIV-1 replication. A subset of primary viruses can use CCR3 as well as CCR5 as a co-receptor5,6, but the in vivo contribution of CCR3 to HIV-1 infection and pathogenesis is unknown. HIV-1 infects the central nervous system (CNS) and causes the dementia associated with AIDS9. Here we report that the major target cells for HIV-1 infection in the CNS, the microglia9–11, express both CCR3 and CCR5. The CCR3 ligand, eotaxin, and an anti-CCR3 antibody inhibited HIV-1 infection of microglia, as did MIP-1β, which is a CCR5 ligand. Our results suggest that both CCR3 and CCR5 promote efficient infection of the CNS by HIV-1.

Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome

Abstract: Growth retardation resulting in short stature is a major concern for parents and due to its great variety of causes, a complex diagnostic challenge for clinicians. A major locus involved in linear growth has been implicated within the pseudoautosomal region (PAR1) of the human sex chromosomes. We have determined an interval of 170 kb of DNA within PAR1 which was deleted in 36 individuals with short stature and different rearrangements on Xp22 or Yp11.3. This deletion was not detected in any of the relatives with normal stature or in a further 30 individuals with rearrangements on Xp22 or Yp11.3 with normal height. We have isolated a homeobox-containing gene (SHOX) from this region, which has at least two alternatively spliced forms, encoding proteins with different patterns of expression. We also identified one functionally significant SHOX mutation by screening 91 individuals with idiopathic short stature. Our data suggest an involvement of SHOX in idiopathic growth retardation and in the short stature phenotype of Turner syndrome patients.

Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III

Abstract: Analysis of patients with inherited hypokalaemic alkalosis resulting from salt-wasting has proved fertile ground for identification of essential elements of renal salt homeostasis and blood-pressure regulation. We now demonstrate linkage of this phenotype to a segment of chromosome 1 containing the gene encoding a renal chloride channel, CLCNKB. Examination of this gene reveals loss-of-function mutations that impair renal chloride reabsorption in the thick ascending limb of Henle's loop. Mutations in seventeen kindreds have been identified, and they include large deletions and nonsense and missense mutations. Some of the deletions are shown to have arisen by unequal crossing over between CLCNKB and the nearby related gene, CLCNKA. Patients who harbour CLCNKB mutations are characterized by hypokalaemic alkalosis with salt-wasting, low blood pressure, normal magnesium and hyper- or normocalciuria; they define a distinct subset of patients with Bartter's syndrome in whom nephrocalcinosis is absent. These findings demonstrate the critical role of CLCNKB in renal salt reabsorption and blood-pressure homeostasis, and demonstrate the potential role of specific CLCNKB antagonists as diuretic antihypertensive agents.

Measuring quality of life

Abstract: Progress in the assessment of quality of life (QoL) measures for children and their value in clinical practice are considered. The most parsimonious approach to assessment includes modular assessment of generic and disease-specific QoL. Differences occur between child and parent proxy ratings, so it is recommended that ratings from multiple observers are made where possible. QoL measures may be of value in clinical practice, but the inadequate availability of the resources necessary to analyse results and provide the required support is a major barrier. Although the assessment of QoL in children remains challenging, its value is increasingly recognised by both researchers and clinicians.

Otitis Media in 2253 Pittsburgh-Area Infants: Prevalence and Risk Factors During the First Two Years of Life

Abstract: Objective. As part of a long-term study of possible effects of early-life otitis media on speech, language, cognitive, and psychosocial development, we set out to delineate the occurrence and course of otitis media during the first 2 years of life in a sociodemographically diverse population of infants, and to identify related risk factors. Methods. We enrolled healthy infants by age 2 months who presented for primary care at one of two urban hospitals or one of two small town/rural and four suburban private pediatric practices. We intensively monitored the infants9 middle-ear status by pneumatic otoscopy, supplemented by tympanometry, throughout their first 2 years of life; we monitored the validity of the otoscopic observations on an ongoing basis; and we treated infants for otitis media according to specified guidelines. Results. We followed 2253 infants until age 2 years. The proportions developing ≥1 episode of middle-ear effusion (MEE) between age 61 days (the starting point for data analysis) and ages 6, 12, and 24 months, respectively, were 47.8%, 78.9%, and 91.1%. Overall, the mean cumulative proportion of days with MEE was 20.4% in the first year of life and 16.6% in the second year of life. Tympanostomy-tube placement was performed on 1.8% and 4.2% of the infants during the first and second years of life, respectively. By every measure, the occurrence of MEE was highest among urban infants and lowest among suburban infants; these differences were greatest in the earliest months of life. Overall, unadjusted mean cumulative proportions of days with MEE were higher among boys than girls, higher among black than white infants, and higher among Medicaid than private health insurance enrollees. Cumulative proportions of days with MEE varied directly with the number of smokers in the household and with the number of other children to whom infants were exposed, whether at home or in day care, and varied inversely with birth weight, maternal age, level of maternal education, a socioeconomic index, and duration of breastfeeding. After adjustment, using multivariate analysis, the only variables that each remained independently and significantly related to the cumulative proportion of days with MEE were: during the first year of life, study site grouping, sex, the socioeconomic index, breastfeeding for ≥4 months, the number of smokers in the household, and an index rating the degree of exposure to other children at home or in day care; and during the second year of life, sex, the socioeconomic index, and the child exposure index. The duration of breastfeeding and the degree of exposure to tobacco smoke contributed little to the explained variance; most was attributable to differences in the socioeconomic index and the child exposure index. Conclusions. Contrary to findings in many previous reports, the prevalence of otitis media during the first 2 years of life among lower-socioeconomic-status black infants appears to be as high as, if not higher than among lower-socioeconomic-status white infants, and certainly higher than among middle-class white infants. Among middle-class white infants the prevalence may also be higher than commonly assumed. The most important sociodemographic risk factors for otitis media appear to be low socioeconomic status and repeated exposure to large numbers of other children, whether at home or in day care.

A lineage‐selective knockout establishes the critical role of transcription factor GATA‐1 in megakaryocyte growth and platelet development

Abstract: Transcription factor GATA-1 is essential for red blood cell maturation and, therefore, for survival of developing mouse embryos. GATA-1 is also expressed in megakaryocytes, mast cells, eosinophils, multipotential hematopoietic progenitors and Sertoli cells of the testis, where its functions have been elusive. Indeed, interpretation of gene function in conventional knockout mice is often limited by embryonic lethality or absence of mature cells of interest, creating the need for alternate methods to assess gene function in selected cell lineages. Emerging strategies for conditional gene inactivation through site-specific recombinases rely on the availability of mouse strains with high fidelity of transgene expression and efficient, tissue-restricted DNA excision. In an alternate approach, we modified sequences upstream of the GATA-1 locus in embryonic stem cells, including a DNase I-hypersensitive region. This resulted in generation of mice with selective loss of megakaryocyte GATA-1 expression, yet sufficient erythroid cell levels to avoid lethal anemia. The mutant mice have markedly reduced platelet numbers, associated with deregulated megakaryocyte proliferation and severely impaired cytoplasmic maturation. These findings reveal a critical role for GATA-1 in megakaryocyte growth regulation and platelet biogenesis, and illustrate how targeted mutation of cis-elements can generate lineage-specific knockout mice.

Plasma Leptin Levels in Healthy Children and Adolescents: Dependence on Body Mass Index, Body Fat Mass, Gender, Pubertal Stage, and Testosterone

Abstract: Leptin, the product of the ob gene, is thought to play a key role in the regulation of body fat mass. Beyond this function, it appears to be an integral component of various hypothalamo-pituitary-endocrine feedback loops. Because childhood and puberty are periods of major metabolic and endocrine changes, leptin levels and various hormonal parameters were investigated in a large cohort of healthy children and adolescents (312 males, 401 females, age 5.8–19.9 yr). For this purpose, a specific and sensitive RIA was developed that allowed the accurate measurement of low leptin levels in young lean children. With this assay, leptin proved to be a comparatively stable protein under common conditions of blood sampling and storage. Leptin levels increased in girls with age (r = 0.47, P < 0.0001), but decreased in boys (r =− 0.34, P < 0.0001). An analysis according to pubertal stage showed a steady increase in girls between 2.51 μg/L (median) at Tanner stage 1 to 6.24 μg/L at Tanner stage 5. In boys, leptin levels...

Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor.

Abstract: Increased vascular permeability and excessive neovascularization are the hallmarks of endothelial dysfunction, which can lead to diabetic macular edema and proliferative diabetic retinopathy in the eye. Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization and a known vasopermeability factor in nonocular tissues. In these studies, we demonstrate that intravitreal injection of VEGF rapidly activates protein kinase C (PKC) in the retina at concentrations observed clinically, inducing membrane translocation of PKC isoforms alpha, betaII, and delta and >threefold increases in retinal vasopermeability in vivo. The effect of VEGF on retinal vascular permeability appears to be mediated predominantly by the beta-isoform of PKC with >95% inhibition of VEGF-induced permeability by intravitreal or oral administration of a PKC beta-isoform-selective inhibitor that did not inhibit histamine-mediated effects. These studies represent the first direct demonstration that VEGF can increase intraocular vascular permeability through activation of PKC in vivo and suggest that oral pharmacological therapies involving PKC beta-isoform-selective inhibitors may prove efficacious for the treatment of VEGF-associated ocular disorders such as diabetic retinopathy.

Early Clinical and Laboratory Indicators of Acute Dengue Illness

Abstract: A prospective observational study was conducted to identify early indicators of acute dengue virus infection. Children with fever for <72 h without obvious cause were studied at hospitals in Bangkok and Kamphaeng Phet, Thailand, until resolution of fever. Of 172 evaluable subjects (91% of enrollees), 60 (35%) had dengue, including 32 with dengue fever (DF) and 28 with dengue hemorrhagic fever (DHF). At enrollment, children with dengue were more likely than children with other febrile illnesses (OFI) to report anorexia, nausea, and vomiting and to have a positive tourniquet test, and they had lower total white blood cell counts, absolute neutrophil and absolute monocyte counts, and higher plasma alanine and aspartate (AST) aminotransferase levels than children with OFI. Plasma AST levels were higher in children who developed DHF than in those with DF. These data identify simple clinical and laboratory parameters that help to identify children with DF or DHF.

Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear.

Abstract: This study evaluates the feasibility of growing tissue-engineered cartilage in the shape of a human ear using chondrocytes seeded onto a synthetic biodegradable polymer fashioned in the shape of a 3-year-old child's auricle. A polymer template was formed in the shape of a human auricle using a nonwoven mesh of polyglycolic acid molded after being immersed in a 1% solution of polylactic acid. Each polyglycolic acid-polylactic acid template was seeded with chondrocytes isolated from bovine articular cartilage and then implanted into subcutaneous pockets on the dorsa of 10 athymic mice. The three-dimensional structure was well maintained after removal of an external stent that had been applied for 4 weeks. Specimens harvested 12 weeks after implantation and subjected to gross morphologic and histologic analysis demonstrated new cartilage formation. The overall geometry of the experimental specimens closely resembled the complex structure of the child's auricle. These findings demonstrate that polyglycolic acid-polylactic acid constructs can be fabricated in a very intricate configuration and seeded with chondrocytes to generate new cartilage that would be useful in plastic and reconstructive surgery.

Autism or atypical autism in maternally but not paternally derived proximal 15q duplication.

Abstract: Duplications of proximal 15q have been found in individuals with autistic disorder (AD) and varying degrees of mental retardation Often these abnormalities take the form of a supernumerary inverted duplicated chromosome 15, more properly described as an isodicentric chromosome 15, or idic(15) However, intrachromosomal duplications also have been reported In a few cases, unaffected mothers, as well as their affected children, carry the same duplications During the course of the genotyping of trios of affected probands with AD and their parents, at the positional candidate locus D15S122, an intrachromosomal duplication of proximal 15q was detected by microsatellite analysis in a phenotypically normal mother Microsatellite and methylation analyses of the pedigree in the following report show that, among three children, the two with autism or atypical autism have maternal inheritance of a 15q11-q13 duplication whereas the third child, who is unaffected, did not inherit this duplication Their mother's 15q11-q13 duplication arose de novo from her father's chromosomes 15 This finding documents, for the first time, the significance of parental origin for duplications of 15q11-q13 In this family, paternal inheritance leads to a normal phenotype, and maternal inheritance leads to autism or atypical autism

Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes.

Abstract: The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the beta subunit of tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six beta-tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa beta-tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific beta-tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.

Vascular development: cellular and molecular regulation.

Abstract: The vascular system forms through a combination of vasculogenesis and angiogenesis. In vasculogenesis, vessels form de novo via the assembly of endothelial precursors called angioblasts, whereas in angiogenesis new vessels arise by migration and proliferation of endothelial cells from preexisting vessels. Although the two processes are distinct in some respects, recent evidence suggests that they share a number of regulatory mechanisms. The identification of a number of defined growth factors, observations of genetically manipulated mice, and the recognition of the importance of cell-cell interactions have greatly expanded our understanding of the regulation of vascularization. The paracrine actions of a variety of polypeptide growth factors, including platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor-beta, and the angiopoietins, appear to be orchestrated in a complex sequence of steps that lead to the development of the adult vascular system. Thus, communicati...