Biomedical subjects

M Little

Publications and source records attributed to M Little.

At least 55 records · Page 3Linked to original sources

PubMed2000

A cell surface-displayed anti-c-myc single-chain antibody: new perspectives for the genetic improvement of cellular tumor vaccines.

We have shown recently that cell surface-bound, single-chain Fv antibodies (scFv) are a powerful tool for the improvement of cellular tumor vaccines. To simplify this approach and to develop a general tool for the generation and improvement of cellular tumor vaccines, we chose an scFv against a peptide from the human proto-oncogene c-myc that could anchor any c-myc-tagged protein to the cell surface. The retroviral vector p50-Mx-neo (pMESV) was used to express scFv on the surface of the human melanoma line SkMel63. The cell-bound anti-c-myc scFv bound specifically to a soluble purified anti-CD28 scFv carrying a c-myc peptide-tag at its C terminus. Proof of principle was determined by incubating human peripheral blood lymphocytes with a mixture of (a) anti-c-myc-transfected SkMel63 cells binding the anti-CD28 scFv and (b) SkMel63 cells transfected with an anti-CD3 scFv. A clear synergistic effect on T-cell activation was observed that was comparable with that obtained in previous studies using SkMel63 cells transfected with the gene for the anti-CD28 scFv. As the cell surface-displayed anti-c-myc scFv can bind any c-myc-tagged protein of interest, this technique facilitates the genetic engineering of cellular vaccines for the therapy of virtually all human neoplasias.

Antibodies, Monoclonal↗

PubMed2000

Conflict of interests, vested interests and health research.

This paper examines conflict of interest as it may arise in the activities of research advisory committees and ethical review committees. It distinguishes between vested interests and true conflict of interest. It also examines the ways in which stakeholdings differ from vested interests and conflicting interests differ from conflicts of interest. It explores the overlapping terrain of corruption and conflict of interest. The paper further examines the ways in which scientists, communities and the subjects of medical research all have legitimate stakeholdings in medical research. Each group thus has differently vested interests in the outcomes of the research. The vested interests of medical scientists are particularly complex because scientists have moral commitments to the welfare of patients that may compete with professional and personal interests in the outcome of research performed on those patients as research subjects. The more these interests diverge, the more opportunity will arise for conflict of interest. These observations have implications for the constitution of research advisory and ethical review committees, and the ways in which their discussions are conducted. Some practical help with protocols of discussion can be gained from philosophical and management writings.

Biomedical Research↗

PubMed2000

Effects of modeled auditory information on a sequential timing task.

The purposes of the present experiment were to determine (a) whether an auditory model enhanced relative or absolute timing, (b) the extent to which the reduced frequency presentation of the auditory model resulted in enhanced retention, and (c) the degree to which executing the timing sequence was independent of the role of the effectors in carrying out the movement sequence. Participants (N = 45) were asked to alternately press two keys on a computer keyboard in an attempt to match the goal intervals presented on the computer monitor. Groups differed in terms of the frequency with which an auditory model (100, 50, or 0%) was presented. The results indicated that the auditory model (100% or 50% groups) enhanced relative timing performance and learning but not the learning of absolute timing. In addition, the 50% group did not appear to become dependent on the auditory model. However, significant decrements in performance were seen for the 100% group when the model was withdrawn. Last, participants were able to execute the timing sequences equally well when they reversed the hand used to execute the timing sequence. This was interpreted as strong evidence for the effector independence (Schmidt, 1975, 1988) and modularity of the timing sequence (Keele, Davidson, & Hayes, 1998).

Female↗

PubMed2000

On surviving cancer.

Explore the source record for details and available documents.

Adaptation, Psychological↗

PubMed2000

Characterization of human lens major intrinsic protein structure.

PURPOSE: To determine the primary covalent structure of human lens major intrinsic protein (MIP) in lenses of varying age. METHODS: MIP was isolated from single human lenses of various ages (7- 86 years) by homogenization of the lenses, followed by centrifugation and urea washes of the membranes. Proteins present in the membrane preparation were reduced, alkylated, and cleaved by CNBr. Peptide fragments were fractionated by reverse-phase high-performance liquid chromatography, and the primary structures of the peptides were determined by tandem mass spectrometry and Edman sequencing. RESULTS: Complete coverage of the human MIP sequence was observed in the form of CNBr fragments. In addition, peptide structures resulting from in vivo heterogeneous N- and C-terminal cleavage were characterized. The amount of intact MIP decreased with lens age; however, the pattern of truncation did not change from 7 to 86 years. The major site of phosphorylation was identified as serine 235. Asparagine residues 246 and 259 were completely deamidated by age 7 years. CONCLUSIONS: The major structural modifications of human lens MIP have been determined. Human MIP is heterogeneously modified in lenses ranging in age from 7 to 86 years of age by N- and C-terminal truncation, phosphorylation, and deamidation, resulting in decreased levels of native intact MIP with age.

Adolescent↗

PubMed2000

Understanding medical ethics.

Moral thinking is embedded within cultures, and we use ethics all the time in our dealings with one another. Many functioning communities tend to share some values that reflect a particular view of the importance of human life in quantity and quality. Rights and duties form an interconnected network of obligations that protect the security of individuals and groups. In health care, the motives and virtues of practitioners are important sources of the determination to provide care for the ill within the limits of resource constraints. Ethics and the law have similarities, but also significant differences that may cause tension between the two systems. Health care is morally grounded, and provides a bulwark against the widespread fear of disease and suffering. The way in which health care is delivered depends on both national wealth and community values. Ethical problems can be seen as dilemmas, in which there are conflicting values. Modern ethical thinking in health is complicated by the need to consider the values and interests of many stakeholders--patients, health care workers, families, politicians, administrators, health bureaucrats and many others. There are ways of ethical thinking that take account of these often countervailing interests. No universally 'right' answers can be specified. The mode and the thoroughness of ethical consideration, and the careful consideration of local community values, will help to assure that we make the best possible decisions for the time and place.

Culture↗

PubMed1999

Generation of a large complex antibody library from multiple donors.

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.

Blood Donors↗

PubMed1999

Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (VHand VL) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3xCD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3(+)and CD19(+)cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19(+)cells in the presence of activated human PBL. Incubated in human serum at 37 degrees C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.

Animals↗

PubMed1999

A bispecific diabody that mediates natural killer cell cytotoxicity against xenotransplantated human Hodgkin's tumors.

CD16/CD30 bispecific monoclonal antibodies can induce remissions of Hodgkin's disease refractory to chemo- and radiotherapy. However, the development of human antimouse immunoglobulin antibodies and allergic reactions precludes repeated applications of the antibody. Moreover, problems of producing and purifying sufficient amounts of material limit the clinical practicability of this novel treatment approach. To overcome these obstacles, we have constructed a bispecific antibody in a diabody form that only employs the variable domains of the CD16/CD30 hybrid hybridoma. The diabody compared favorably with the parent CD16/CD30 bispecific antibody in its ability to activate and target natural killer cells in vitro. Its administration to mice bearing xenografted Hodgkin's lymphoma resulted in a marked regression of tumor growth, thus proving for the first time the capability of a diabody for immune recruitment in vivo. The CD16/CD30 diabody is a novel reagent that should considerably facilitate the immunotherapy of patients with refractory Hodgkin's lymphoma.

Animals↗

PubMed1999

Apoptosis of a human melanoma cell line specifically induced by membrane-bound single-chain antibodies.

CD28 is a key regulatory molecule in T cell responses. Ag-TCR/CD3 interactions without costimulatory signals provided by the binding of B7 ligands to the CD28R appear to be inadequate for an effective T cell activation. Indeed, the absence of B7 on the tumor cell surface is probably one of the factors contributing to the escape of tumors from immunological control and destruction. Therefore, to increase the immunogenicity of tumor cell vaccines, we have expressed anti-CD3 and anti-CD28 single-chain Abs (scFv) separately on the surface of a human melanoma SkMel63 cell line (HLA-A*0201). A mixture of cells expressing anti-CD3 with cells expressing anti-CD28 resulted in a marked activation of allogeneic human PBL in vitro. The apparent induction of a Th1 differentiation pathway was accompanied by the proliferation of MHC-independent NK cells and MHC-dependent CD8+ T cells. PBL that had been cultured together with transfected SkMel63 tumor cells were able to specifically induce apoptosis in untransfected SkMel63 cells. In contrast, three other tumor cell lines expressing HLA-A*0201, including two melanoma cell lines, showed no significant apoptosis. These results provide valuable information for both adoptive immunotherapy and the generation of autologous tumor vaccines.

Animals↗

PubMed1999

Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding.

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B-cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv-18 formed monomers, dimers and small amounts of tetramers; scFv-10 formed dimers and small amounts of tetramers; scFv-1 formed exclusively tetramers; scFv-0 formed exclusively trimers. The affinities of the scFv-10 (diabody) and scFv-1 (tetrabody) were approximately 1.5- and 2.5-fold higher, respectively, than that of the scFv-0 (triabody). The tetrabody displayed a significantly prolonged association with cell-bound antigen (t1/2 cell surface retention at 37 degrees C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B-cell malignancies.

Amino Acid Sequence↗

PubMed1999

T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody.

Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment (scFv) directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects.

Antibodies, Monoclonal↗

PubMed1999

Use of the Pediatric Symptom Checklist to screen for psychosocial problems in pediatric primary care: a national feasibility study.

BACKGROUND: Routine use of a brief psychosocial screening instrument has been proposed as a means of improving recognition, management, and referral of children's psychosocial morbidity in primary care. OBJECTIVE: To assess the feasibility of routine psychosocial screening using the Pediatric Symptom Checklist (PSC) in pediatrics by using a brief version of the checklist in a large sample representative of the full range of pediatric practice settings in the United States and Canada. We evaluated large-scale screening and the performance of the PSC in detecting psychosocial problems by (1) determining whether the prevalence of psychosocial dysfunction identified by the PSC was consistent with findings in previous, smaller samples; (2) assessing whether the prevalence of positive PSC screening scores varied by population subgroups; and (3) determining whether the PSC was completed by a significant proportion of parents from all subgroups and settings. PATIENTS AND METHODS: Twenty-one thousand sixty-five children between the ages of 4 and 15 years were seen in 2 large primary care networks: the Ambulatory Sentinel Practice Network and the Pediatric Research in Office Settings network, involving 395 pediatric and family practice clinicians in 44 states, Puerto Rico, and 4 Canadian provinces. Parents were asked to complete a brief questionnaire that included demographic information, history of mental health services, the 35-item PSC, and the number of pediatric visits within the past 6 months. RESULTS: The overall prevalence rates of psychosocial dysfunction as measured by the PSC in school-aged and preschool-aged pediatric outpatients (13% and 10%, respectively) were nearly identical to the rates that had been reported in several smaller samples (12%-14% among school-aged children and 7%-14% among preschoolers). Consistent with previous findings, children from low-income families were twice as likely to be scored as dysfunctional on the PSC than were children from higher-income families. Similarly, children from single-parent as opposed to those from 2-parent families and children with a past history of mental health services showed an elevated risk of psychosocial impairment. The current study was the first to demonstrate a 50% increase in risk of impairment for male children. The overall rate of completed forms was 97%, well within an acceptable range, and at least 94% of the parents in each sociodemographic subgroup completed the PSC form. CONCLUSIONS: Use of the PSC offers an approach to the recognition of psychosocial dysfunction that is sufficiently consistent across groups and locales to become part of comprehensive pediatric care in virtually all outpatient settings. In addition to its clinical utility, the consistency and widespread acceptability of the PSC make it well suited for the next generation of pediatric mental health services research, which can address whether earlier recognition of and intervention for psychosocial problems in pediatrics will lead to cost-effective outcomes.

Adolescent↗

PubMed1999

WT1: what has the last decade told us?

When positionally cloned in late 1989, it was anticipated that mutations within the Wilms' tumour suppressor gene (WT1) would prove responsible for this common solid kidney cancer of childhood. Characterisation of the WT1 expression pattern and of the structure of the encoded protein isoforms and their mode of action has now spanned almost a decade. WT1 proteins act as nucleic acid-binding zinc finger-containing transcription factors involved in both transactivation and repression. These activities are facilitated and constrained by interactions with other proteins. Expression analyses and knockout mice indicate that WT1 protein plays a critical role in normal kidney and gonad development. Specific constitutional WT1 mutations results in several urogenital anomaly syndromes. While only 10% of sporadic Wilms' tumours do display WT1 mutation, WT1 is mutated in other cancers, including acute myeloid leukaemia. Much is still to be determined in WT1 biology. The next decade will see at least three streams of attention. The first two, elucidation of the role of WT1 in RNA metabolism and the characterisation of further protein partners, may together explain the distinct tissue-specific functions of WT1. Finally, further research into the role of WT1 in haematopoiesis will improve our understanding of WT1 in leukaemia.

Animals↗

PubMed1999

The antigen binding domain of non-idiotypic human anti-F(ab')2 autoantibodies: study of their interaction with IgG hinge region epitopes.

In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab')2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.

Amino Acid Sequence↗

PubMed1999

Assisted suicide, suffering and the meaning of a life.

The ethical problems surrounding voluntary assisted suicide remain formidable, and are unlikely to be resolved in pluralist societies. An examination of historical attitudes to suicide suggests that modernity has inherited a formidable complex of religious and moral attitudes to suicide, whether assisted or not. Advocates usually invoke the ending of intolerable suffering as one justification for euthanasia of this kind. This does not provide an adequate justification by itself, because there are (at least theoretically) methods which would relieve suffering without causing the physical death of the suffering person. Carried to extremes, these methods would finish the life worth living, but leave a being which was technically alive. Such acts, however, would provide no moral escape, since they would create beings without meaning. Arguments seeking to justify ending the lives of others need some grounding in concepts of the meaning of a life. The euthanasia discourse therefore needs to take at least some account of the meaning we construct for our lives and the lives of others.

Attitude to Death↗

PubMed1999

Research, ethics and conflicts of interest.

In this paper, I have tried to develop a critique of committee procedures and conflict of interest within research advisory committees and ethical review committees (ERCs). There are specific features of conflict of interest in medical research. Scientists, communities and the subjects of research all have legitimate stakeholdings. The interests of medical scientists are particularly complex, since they are justified by the moral and physical welfare of their research subjects, while the reputations and incomes of scientists depend on the success of their science. Tensions of this kind must at times produce conflict of interest. It is important to recognise that conflicts of interest may unwittingly lead to manipulation of research subjects and their lay representatives on research committees. It is equally important to recognise distinctions between the legal and moral aspects of conflict of interest. Some practical suggestions are made which may go some way towards resolving these difficulties. They indicate what might be needed to ensure the validity of ethical discourse, and to reduce the risks associated with conflict of interest.

Advisory Committees↗

PubMed1999

Generation of recombinant antibodies.

Recombinant antibody technology is opening new perspectives for the development of novel therapeutic and diagnostic agents. In this review we focus on advances in the generation of both genetically engineered humanized and fully human monoclonal antibodies. Methods for their production in different expression systems are also discussed.

Animals↗