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Biomedical subjects

M Little

Publications and source records attributed to M Little.

At least 37 records · Page 2Linked to original sources
PubMed1997

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

Amino Acid Sequence↗
PubMed1996

Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.

Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.

Amino Acid Sequence↗
PubMed1996

RNA binding by the Wilms tumor suppressor zinc finger proteins.

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.

Animals↗
PubMed1996

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.

BACKGROUND: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). OBJECTIVES: pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. STUDY DESIGN AND RESULTS: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. CONCLUSIONS: Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.

Amino Acid Sequence↗
PubMed1996

An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling.

The Wilms' tumour suppressor gene 1 (WT1) (1,2) encodes four C2H2 zinc finger-containing proteins (3) critical for normal mammalian urogenital development (4). Mutations in this gene are observed in the childhood kidney cancer, Wilms' tumour (WT) (5). WT1 can bind specific DNA targets within the promoters of many genes (6-9) and both transcriptional repression and activation domains have been identified (10). On this basis, it has been assumed that regulation of transcription is the basis of WT1 tumour suppressor activity. However, subnuclear localization studies have revealed an association between WT1 proteins and 'speckled bodies' within the nucleus. Degradation of nuclear RNA in cells expressing WT1 abolishes this speckled localization and WT1 co-immunoprecipitates with a number of spliceosomal proteins, suggesting that it may also bind to RNA (11). Using structural rather than sequence comparison, we have now identified an evolutionarily conserved N-terminal RNA recognition motif (RRM) in all known WT1 isoforms similar to that in the constitutive splicing factor U1A. Given the association between WT1 mutations and Wilms' tumours, this study, together with other recent findings, may suggest a novel tumour suppression mechanism.

Amino Acid Sequence↗
PubMed1996

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion.

In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites. To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin. The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies. After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed. The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C). Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable. The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers. This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies. We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium. These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands.

Antibodies↗
PubMed1996

Cell surface display of a single-chain antibody for attaching polypeptides.

To provide an efficient means of coupling proteins, peptides and other suitable moieties to cells, we have constructed a retroviral expression vector for cell surface display of a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-oxazo-line-5-one (phOx). The hapten phOx can be easily conjugated to primary amino and sulfhydryl groups, thus providing points of attachment for the cell surface-bound anti-phOx scFv. This universal cell coupling system could prove to be particularly useful for anchoring monoclonal antibodies for tumor targeting and to present co-stimulatory molecules and other ligands (even mixtures) at the cell surface for gene therapy.

Animals↗
PubMed1996

The enemy within: diarrheal rates among British and Australian troops in Iraq.

British and Australian medical teams working in Northern Iraq in 1991 providing primary care to refugees and the war wounded were subjected to a descriptive retrospective survey, 5 weeks after arriving in Iraq. The aim was to document different rates of diarrhea in British and Australian troops. The British, who were not taking daily doxycycline and did not enforce a plate- and hand-washing routine, experienced higher rates of diarrhea (69% of British troops compared with 36% of Australian troops), which was more severe and of a longer duration (p < 0.001) and resulted in twice as many days being lost (p < 0.001) in spite of the British team being half the size of the Australian contingent, and the region having enteropathogens with a high rate of antibiotic resistance. Vigorous hand- and plate-washing routines along with doxycycline prophylaxis appear to significantly reduce incapacitation from diarrhea in this military setting and have an important implication for operational effectiveness.

Anti-Bacterial Agents↗
PubMed1995

Human antibody libraries in Escherichia coli.

A potentially vast pool of human antibodies with novel specificities for diagnostic and therapeutic purposes can be generated in Escherichia coli. Antibodies to infectious agents have already been isolated by amplifying the heavy and light chain repertoires of donor lymphocytes and they have even been rescued many years after the initial infection from memory cells cultivated in SCID mice. Eventually, however, the creation of extremely large and diverse libraries from the naive antibody repertoire of unactivated B lymphocytes or by gene synthesis using random oligonucleotides for the hypervariable regions could provide a rapid means of obtaining human antibodies to any particular antigen. An important breakthrough for exploiting the potential size and diversity of these libraries has been the development of systems for the surface display of antibodies that are physically linked to their own genes. This allows large numbers of clones to be screened simultaneously and antibodies with affinities of up to 10(8) M-1 have already been obtained using these vectors. It seems quite feasible, therefore, that antibodies with affinities approaching those obtained in the secondary immune response can be obtained by systematically optimizing the strategies for making antibody libraries. Furthermore, it might be possible to establish extremely large antibody repertoires in E. coli by the in vivo recombination of phage and plasmid antibody libraries. The affinity of the selected antibodies could be increased by chain shuffling or random mutagenesis followed by several rounds of selection under increasingly stringent conditions.

Animals↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗