Paternal irradiation and childhood leukaemia.
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Child↗
Biomedical subjects
M Little
Publications and source records attributed to M Little.
Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv).
Multivalent and multispecific antibodies with defined stoichiometry could provide valuable tools for biological and medical research and for the diagnosis and therapy of cancer. We have therefore fused single chain antibodies (scFv) with core-streptavidin. This chimeric protein, expressed by the vector pSTE-215 (plasmid for streptavidin-tagged expression), can form tetrameric complexes, binds antigen and contains the biotin binding site which may be used for further complex formation. An additional cysteine was inserted near the carboxy terminus to facilitate the construction of covalently linked bifunctional molecules. The scFv fusion protein could be purified by affinity chromatography using biotin analogues. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA and Western blots when stained with biotinylated horseradish peroxidase.
Amino Acid Sequence↗
The Pediatric Symptom Checklist. Support for a role in a managed care environment.
OBJECTIVES: To gather data based on studies of the Pediatric Symptom Checklist, identify risk factors associated with high levels of dysfunction in primary care pediatric settings, and explore the relationship between common risk factors and psychosocial problems identified by pediatricians. DESIGN: Retrospective review and cross-sectional, case-referent survey. SETTING: Subjects were selected from three primary care pediatric clinics in Massachusetts: a private practice in a predominantly white, middle-class suburb, an urban health maintenance organization clinic, and an inner-city clinic. PARTICIPANTS: Of 423 outpatients aged 6 to 12 years screened for psychosocial problems, 72 children and their families were seen for in-depth structured and clinical interviews (24 from each site). INTERVENTIONS: None. MEASUREMENTS/MAIN RESULTS: Children with a single parent and/or those who were economically disadvantaged were significantly more likely to show psychosocial impairment. The specificity of the Pediatric Symptom Checklist was 100% in samples with a lower socioeconomic status compared with 68% in middle-class samples, and sensitivity was 95% in middle-class samples compared with 80% in lower-class samples. Pediatricians identified psychosocial problems in eight of 15 children with a history of familial mental illness or substance abuse and seven of eight children with a history of physical or sexual abuse, but only six of 17 cases from single-parent families and four of 11 cases from poor families. CONCLUSIONS: Pediatricians should be sensitive to psychosocial dysfunction especially in single-parent and low-income families. Use of the Pediatric Symptom Checklist for psychosocial screening in a managed health care delivery system could target capitated resources efficiently by providing early identification and secondary prevention of psychosocial morbidity.
Affective Symptoms↗
Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines.
Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.
Amino Acid Sequence↗
Placement outcomes of 206 severely maltreated children in the Boston Juvenile Court system: a 7.5-year follow-up study.
This study examines placement outcomes of 206 severely maltreated children 7.5 years after arraignment in Boston Juvenile Court (BJC) on Care and Protection Petitions. Sixty-seven percent (n = 138) of the sample had been permanently removed from their parents and 33% (n = 68) had their cases dismissed in the BJC. At time of this follow-up, 21% of the full sample (n = 44) were still in temporary custody awaiting permanent placement. In addition, 4% (n = 8) of children had "drifted" back to their abusive/neglectful parents despite prior permanent removal. The average time children in this sample spent in probate proceedings (awaiting permanent placement) had increased substantially to 2.1 years since the last overview study of this sample 4 years ago. The rate of court referral for incidences of reabuse (a C&P filing), or delinquency was significantly lower among children who had been permanently placed (p < .003). Rates of court-referral for reabuse charges were the same (16%) for children who were in temporary custody at the time of follow-up and children who had been dismissed back to the parent for whom the original C&P had been filed. Results are discussed in light of the urgent need to restructure time limits in juvenile court proceedings, integrate adequate tracking of child abuse and neglect cases through and across court and agency boundaries, and the use standardized assessments of abused and neglected children as a tool in the adjudication process.
Adolescent↗
Methylation and p16: suppressing the suppressor.
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5-Methylcytosine↗
DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations.
Constitutional point mutations in the zinc finger (ZF) region of the Wilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS). Patients with this syndrome display renal failure, Wilms' tumour (WT) and pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directly interact with the DNA target; (b) substitution of amino acids involved in zinc complexing; and (c) nonsense mutations leading to the removal of at least two zinc fingers. We have expressed the WT1 zinc fingers as glutathione-S-transferase fusion proteins, with the lysine-threonine-serine (KTS) alternate splice between ZF3 and ZF4 either present or absent. WT1 fusion constructs with all three classes of DDS mutation were also created. Wild-type and mutant fusion proteins were assayed for their DNA-binding affinity using four previously identified WT1 DNA targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; and +P5, a genomic fragment isolated by its affinity for WT1 + KTS. WT1-KTS bound all four targets, but WT1 + KTS only bound +P5. All three classes of DDS mutation investigated, with or without KTS, abolished binding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect through disturbed isoform dosage balance.
Abnormalities, Multiple↗
Screening for psychosocial dysfunction in pediatric inpatients.
Screening pediatric inpatients for psychosocial dysfunction offers physicians an opportunity to identify emotional and behavioral problems that might otherwise go unrecognized. In this study, the Pediatric Symptom Checklist (PSC), a brief, parent-completed questionnaire, which has been validated in a variety of outpatient settings, was used to screen 98 pediatric inpatients. Results indicated that the PSC can be easily administered in a busy inpatient setting and is well-tolerated by both house staff and patients' parents as a routine part of the admissions process. The percentage of children who screened positive with the PSC in this inpatient setting was similar to the percentages generated by using the PSC in outpatient settings. Routine use of the PSC in inpatient settings serves to heighten house staff awareness of psychosocial concerns and facilitate parent-physician discussion of pediatric mental health issues.
Adolescent↗
A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity.
Tumour necrosis factor (TNF) is an important mediator of immune and inflammatory responses and has been recognized as a major pathogenic factor in several autoimmune and inflammatory diseases. TNF receptor TR60 plays a critical role in signalling the pathogenic activities of TNF. We here describe molecular cloning and bacterial production of a single-chain antibody (scFv H398) directed against TR60 which possesses antagonistic activity. VH and VL encoding sequences were isolated by PCR from the murine hybridoma cell line H398, cloned into a scFv expression vector and expressed in Escherichia coli. The recombinant antibody (Ab) fragment was found as an active soluble protein in the periplasm but also formed inclusion bodies. Re-folded scFv H398 purified from inclusion bodies was shown to be functional and stable at 37 degrees C with a half-life of 50 h. Comparison of the antigen binding characteristics of scFv with the parental enzymatically produced Fab H398 revealed that both Ab fragments have the same epitope specificity and an identical antigen binding affinity of 1.5 nM. In an in vitro assay it was demonstrated that scFv H398 is an efficient inhibitor of TNF mediated cytotoxicity with an IC50 of 22 nM, which is comparable to the antagonistic activity of natural Fab H398 with an IC50 of 12 nM. As scFv H398 possesses the high affinity TR60 binding and receptor antagonistic activity of the parental Ab H398 but is expected to be less antigenic in man, it provides a valuable tool for the development of novel therapeutic reagents against TNF mediated diseases.
Animals↗
Health, education, and the virtues of efficiency.
Managerialism has been seen in recent times as an answer to problems which fall well outside the boundaries of commercial enterprise. This trend can be seen in both education at university level and health. But there are major risks in transporting the techniques and values of the market into domains which serve non-commercial ends. Many of the most desired achievement of both health and education cannot be costed in monetary terms, and many of the activities that can be costed cannot produce profits. Education and health both have aims that are essentially moral, and the intrusion of managerialism undermines the morality and excuses departures from the central functions of both disciplines. This is not to say that efficiency and cost-cutting may not serve the good of education and health, but they cannot replace functions such as teaching, learning, and healing. There is a cogent argument for redefining the functions and structures of management in order to serve the knowledge workers whose role in health and education is indispensable, rather than to devolve increasing responsibility for management to them. The freedom of knowledge workers to practise their professions, to teach, to do research, and to reflect should be increased, rather than diminished progressively by institutional demands for more and more commitment the minutiae of management.
Efficiency, Organizational↗
Single-chain antibody streptavidin fusions: tetrameric bifunctional scFv-complexes with biotin binding activity and enhanced affinity to antigen.
To increase the avidity of single-chain antibodies (scFv) for their antigen, we have fused them to core-streptavidin. The chimeric protein, expressed by the vector pSTE (plasmid for streptavidin-tagged expression) from Escherichia coli, can form tetrameric complexes, binds its antigen and contains four biotin binding sites per tetrameric complex. An additional cysteine inserted near the carboxy terminus further stabilised the complex. The scFv fusion protein tetramers could be enriched by affinity chromatography using the biotin analog 2-iminobiotin from periplasmic inclusion bodies after refolding. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA when stained with biotinylated horseradish peroxidase. The affinity of the scFv-antibody complex was substantially increased by avidity effects due to the tetrameric structure. The biotin binding sites may be used for coupling other antibodies and molecules to form bispecific and bifunctional reagents.
Amino Acid Sequence↗
Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers.
To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.
Amino Acid Sequence↗
Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages.
Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.
Animals↗
Another kind of flying doctor.
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Air Ambulances↗
Performance of screening tools for mental health problems in chronically ill children.
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Child↗
Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies.
A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli. Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster. ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E. coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents. In a final step, the components were separated by size exclusion chromatography. All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.
Amino Acid Sequence↗
Surface display of antibodies.
To screen antibody libraries that contain many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This can be achieved by displaying antibodies on the surface of microorganisms containing the antibody's gene, analogous to the expression of the IgM antigen receptor on the surface of unactivated B-lymphocytes. Specific clones can then be selected using immobilized antigens. The minor coat protein of filamentous phages, pIII, which initiates the infection of E.coli by binding to their F-pili, and the major coat protein, pVIII, have been used as carriers for displaying antibodies on the phage surface. Recombinant antibodies have also been targeted to the cell surface of bacteria by fusing them with outer membrane components derived from lipoproteins, OmpA and an IgA protease. However, only the pIII system has been routinely used for screening antibody libraries. Here we describe the various antibody surface display systems and the screening of antibody libraries generated from the gene repertoire of lymphocytes and by gene synthesis. Finally, we have made a short comparison of the bacterial production of Fabs versus single chain antibodies (scFv).
Journal Article↗
Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR.
A method for the facile simultaneous mutagenesis of complementary-determining regions (CDRs) in a single chain antibody (scFv) is described. Overlapping sets of oligonucleotides containing random sequences within the CDRs corresponding to the heavy chain variable region (VH) jointed to a linker peptide (J) and the light chain variable region (VL) were extended under PCR conditions to full-length genes. These gene products were then further amplified using short PCR primers containing complementary overlaps between the 3' and 5' ends of the VH-J and VL genes respectively. In a final step, the VH-J and VL gene products were mixed and assembled into scFv DNA products by overlap extension under standard PCR conditions. Sequence analyses indicated that the method is basically successful. However, some deletions were observed, which probably reflects difficulties in the automatic synthesis of long degenerate oligonucleotides.
Base Sequence↗