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M Little

Publications and source records attributed to M Little.

At least 73 records · Page 4Linked to original sources
PubMed1999

Selection of an anti-CD20, single-chain antibody by phage ELISA on fixed cells.

Cloning the correct genes that code for antibody-variable domains from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from a myeloma cell line. For the rapid functional evaluation of recombinant antibody fragments against cell-surface antigens, we established an efficient expression and screening system using phagemid antibodies and fixed cells. VL and VH-polymerase chain reaction (PCR) products, amplified from hybridoma cDNA, were cloned into the phagemid vector pSEX81. After transduction into E. coli and phage rescue, clones were tested for antigen binding using a phage-enzyme-linked immunosorbent assay (ELISA) procedure with whole cells fixed to ELISA wells. This procedure facilitated the successful cloning of a functional anti-CD20, single-chain antibody from hybridoma cDNA. The CD20 B-lymphocyte surface antigen expressed by B-cell lymphomas is an attractive target for cancer treatment using immunoconjugates or bi-specific antibodies.

Antibodies, Monoclonal↗
PubMed1998

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

Animals↗
PubMed1998

Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells.

For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector containing a dicistronic operon for co-secretion of V(H)3-V(L)19 and V(H)19-V(L)3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatography (IMAC) from the periplasmic extract and culture medium. Flow cytometry experiments revealed specific interactions of the diabody with both CD3 and CD19 positive cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37 degrees C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepared by the quadroma technique.

Amino Acid Sequence↗
PubMed1998

Epitopes fused to F-pilin are incorporated into functional recombinant pili.

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

Amino Acid Sequence↗
PubMed1998

Supporting child psychiatric services using current managed care approaches: you can't get there from here.

For-profit behavioral health care companies have transformed the way mental health services are provided for children. Using marketplace approaches, companies have "carved out" mental health services for many patients receiving care from pediatricians. This report details specific approaches used by these firms to maximize profits, minimize the role of child and adolescent psychiatrists, and limit clinical services. Understanding for-profit carveouts will help primary care pediatricians appreciate the likely consequences of such reimbursement incentives for the care of children and their families.

Adolescent↗
PubMed1998

Assignments of meaning in epidemiology.

Epidemiology works in a public domain, gathering the results of surveys and trials into forms of knowledge which are made available to many stakeholders. Health policy makers, lawyers, the media, medical technology companies, and those who use and deliver health services all have legitimate interests in epidemiology. There is unfortunately no common language in which each of these stakeholders can express their interest in the outcomes of epidemiological studies. The largest and most important gap exists between those who use computational data and those who use cultural and linguistic models to generate their explanations. Methods have been described, however, which allow the identification of all legitimate stakeholders before epidemiological studies are undertaken. Identifying the stakeholders, however, will serve no purpose unless there is a prior commitment by epidemiologists to respect both reductionist and narrative accounts of truth.

Attitude of Health Personnel↗
PubMed1998

Liminality: a major category of the experience of cancer illness.

Narrative analysis is well established as a means of examining the subjective experience of those who suffer chronic illness and cancer. In a study of perceptions of the outcomes of treatment of cancer of the colon, we have been struck by the consistency with which patients record three particular observations of their subjective experience: (1) the immediate impact of the cancer diagnosis and a persisting identification as a cancer patient, regardless of the time since treatment and of the presence or absence of persistent or recurrent disease; (2) a state of variable alienation from social familiars, expressed as an inability to communicate the nature of the experience of the illness, its diagnosis and treatment; and (3) a persistent sense of boundedness, an awareness of limits to space, empowerment and available time. These subjectivities were experienced in varying degree by all patients in our study. Individual responses to these experiences were complex and variable. The experiences are best understood under the rubric of a category we call "liminality". We believe that all cancer patients enter and experience liminality as a process which begins with the first manifestations of their malignancy. An initial acute phase of liminality is marked by disorientation, a sense of loss and of loss of control, and a sense of uncertainty. An adaptive, enduring phase of suspended liminality supervenes, in which each patient constructs and reconstructs meaning for their experience by means of narrative. This phase persists, probably for the rest of the cancer patient's life. The experience of liminality is firmly grounded in the changing and experiencing body that houses both the disease and the self. Insights into the nature of the experience can be gained from the Existentialist philosophers and from the history of attitudes to death. Understanding liminality helps us to understand what it is that patients with cancer (and other serious illnesses) seek from the system to which they turn for help. Its explication should therefore be important for those who provide health care, those who educate health care workers and those concerned to study and use outcomes as administrative and policy making instruments.

Adult↗
PubMed1998

Relationship between hunger and psychosocial functioning in low-income American children.

OBJECTIVE: Using large-scale surveys from nine states, the Community Childhood Hunger Identification Project (CCHIP) estimates that 8% of American children under the age of 12 years experience hunger each year. CCHIP operationalizes child hunger as multiple experiences of parent-reported food insufficiency due to constrained resources. The current study examined the relationship between food insufficiency and school-age, low-income children's psychosocial functioning. The study also assessed the interinformant (parent versus child) reliability and time-to-time reliability of the CCHIP measure. METHOD: Two hundred four school-age children and their parents from four inner-city public schools were interviewed using parent, teacher, and clinician report measures of psychosocial functioning. Ninety-six children and their parents were reinterviewed 4 months later. RESULTS: Hungry and at-risk for hunger children were twice as likely as not-hungry children to be classified as having impaired functioning by parent and child report. Teachers reported higher levels of hyperactivity, absenteeism, and tardiness among hungry/at-risk children than not-hungry children. Parent and child reports of hunger were significantly related to each other, and time-to-time reliability of the CCHIP measure was acceptable. CONCLUSIONS: Results of this study suggest that intermittent experiences of food insufficiency and hunger as measured by CCHIP are associated with poor behavioral and academic functioning in low-income children. The current study also supports the validity and reliability of the CCHIP measure for assessing hunger in children.

Adolescent↗
PubMed1998

Resource constraints and moral pressures: can we still afford ourselves?

We have reached a phase of diminishing returns in medicine. Increasing costs produce smaller and smaller incremental benefits in health status. Medical scientists continue to work within the ideology of the Enlightenment, whereby advances in knowledge will eventually lead to control of health and welfare. The enormous costs of this ideology have led to two new ideologies: those of economic rationalism and managerialism. At the public level, the Western liberal emphasis on the value of individual life is generally held to justify the amount of public money spent on health. Those who frame health policy are influenced to some extent by this ideal, but we cannot continue to develop costly interventions without constraint. To overcome this impasse, we might accept that economic rationalism provided a proper base for health care; or we might redefine disease so that more people were excluded from treatment programmes; or we might agree to limit medical research in costly areas; we might change our ethical thinking to emphasize classical utilitarianism; or we might undertake systematic studies of community values and opinions to find out what people really want from their health and welfare services. There are serious ethical problems with each of these solutions, except for the last: the idea of modifying services to take note of community values. Testing community values is difficult, but there are ways of doing it, and there have been some exercises in which the process has been undertaken with some success. The recent Constitutional convention suggests that it may even be possible in Australia.

Cost Control↗
PubMed1998

Hunger in children in the United States: potential behavioral and emotional correlates.

OBJECTIVE: Results from a recent series of surveys from 9 states and the District of Columbia by the Community Childhood Hunger Identification Project (CCHIP) provide an estimate that 4 million American children experience prolonged periodic food insufficiency and hunger each year, 8% of the children under the age of 12 in this country. The same studies show that an additional 10 million children are at risk for hunger. The current study examined the relationship between hunger as defined by the CCHIP measure (food insufficiency attributable to constrained resources) and variables reflecting the psychosocial functioning of low-income, school-aged children. METHODS: The study group included 328 parents and children from a CCHIP study of families with at least 1 child under the age of 12 years living in the city of Pittsburgh and the surrounding Allegheny County. A two-stage area probability sampling design with standard cluster techniques was used. All parents whose child was between the ages of 6 and 12 years at the time of interview were asked to complete a Pediatric Symptom Checklist, a brief parent-report questionnaire that assesses children's emotional and behavioral symptoms. Hunger status was defined by parent responses to the standard 8 food-insufficiency questions from the CCHIP survey that are used to classify households and children as "hungry," "at-risk for hunger," or "not hungry." RESULTS: In an area probability sample of low-income families, those defined as hungry on the CCHIP measure were significantly more likely to have clinical levels of psychosocial dysfunction on the Pediatric Symptom Checklist than children defined as at-risk for hunger or not hungry. Analysis of individual items and factor scores on the Pediatric Symptom Checklist showed that virtually all behavioral, emotional, and academic problems were more prevalent in hungry children, but that aggression and anxiety had the strongest degree of association with experiences of hunger. CONCLUSION: Children from families that report multiple experiences of food insufficiency and hunger are more likely to show behavioral, emotional, and academic problems on a standardized measure of psychosocial dysfunction than children from the same low-income communities whose families do not report experiences of hunger. Although causality cannot be determined from a cross-sectional design, the strength of these findings suggests the importance of greater awareness on the part of health care providers and public health officials of the role of food insufficiency and hunger in the lives of poor children.

Affective Symptoms↗
PubMed1997

Construction of a semisynthetic antibody library using trinucleotide oligos.

A semisynthetic antibody library composed of single chain Fv fragments (scFv) was constructed by replacing the heavy chain CDR3 region of a human scFv by a random sequence of eight amino acids using trinucleotide codons. After cloning into a phage display vector, an antibody library was generated with a complexity of 8 x 10(8) independent clones. The library was screened for binders to dinitrophenol, fluorescein isothiocyanate and 3-nitro-4-hydroxy-5-iodophenylacetic acid. scFv antibodies that specifically bound the antigen were obtained in each case.

Antibodies↗
PubMed1997

Antibodies to steroids from a small human naive IgM library.

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4 x 10(7)) of single chain antibodies (scFv). The affinities of both the anti-estradiol and antiprogesterone scFv were approximately 10(8) M(-1). Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

Bacteriophages↗
PubMed1997

Two N-terminal self-association domains are required for the dominant negative transcriptional activity of WT1 Denys-Drash mutant proteins.

Patients with Denys-Drash syndrome (DDS) have been shown to be constitutionally heterozygous for mutations of the WT1 gene. Almost all DDS mutations inactivate or remove the DNA-binding zinc finger region of WT1 and the resulting mutant proteins appear to act in a dominant negative manner. This may occur via WT1 self-association, which has been shown to involve the first 180 amino acids. By creating a series of N-terminal deletions, we have further investigated WT1 self-association using a yeast di-hybrid system and an in vitro protein binding assay. Our results suggest that there are two distinct domains within the N-terminal region facilitating self-association, residing from amino acids 1-45 and 157-253. Co-transfection of WT1 with progressively shorter N-terminal constructs demonstrates that both of these sites are required for a dominant negative activity as assessed by activation of a reporter construct.

Base Sequence↗
PubMed1997

The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody.

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments.

Amino Acid Sequence↗
PubMed1997

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures.

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15-25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80-150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20 degrees C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.

Escherichia coli↗