Search PubMed⌕ Search

Biomedical subjects

D M Shaw

Publications and source records attributed to D M Shaw.

At least 19 recordsLinked to original sources
PubMed2000

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Amino Acid Sequence↗
PubMed2000

Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli.

The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression. Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.

Animals↗
PubMed1999

Instruments for oral disease-intervention strategies: recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis.

Lactobacillus strains possess properties that make them attractive candidates as vehicles for oral administration of therapeutics. In this report we describe the construction and analysis of recombinant Lactobacillus casei applicable in oral vaccination against an infectious disease (tetanus) and in oral tolerance induction for intervention in an autoimmune disease, multiple sclerosis. Recombinant L. casei which express surface-anchored tetanus toxin fragment C (TTFC) were generated. Quantitative analysis by flow cytometry demonstrated a high level of cell wall-bound expression of TTFC and immunogenicity was demonstrated by parenteral immunization with whole cell extracts of the recombinants. A series of expression vectors was constructed to secrete human myelin basic protein (hMBP) or hMBP as a fusion protein with beta-glucuronidase from Escherichia coli. These heterologous products produced by L. casei were detected in the growth medium and parenteral immunization with this medium evoked antibodies against hMBP, confirming that secretion indeed had occurred. Based on the different localization of the heterologous proteins, lactobacilli expressing surface-anchored TTFC are ideally suited for the induction of antibody responses, whereas lactobacilli that secrete myelin proteins can be used for the induction of peripheral T-cell tolerance. In conclusion, the specific technology described here allows the construction of a wide array of safe live recombinant lactobacilli which may prove to be useful in oral intervention strategies for the prevention of infectious diseases or treatment of autoimmune diseases.

Administration, Oral↗
PubMed1998

Assessing individual family members' constructions of family problems.

Much contemporary family therapy theory and practice takes into account clients' cognitive constructions of their family problems. Recent calls for therapists to elicit and work with clients' causal explanations and narratives parallel accumulating evidence in the social-clinical literature about the predictive importance of attributions in family relationships. In this article, we introduce the Constructions of Problems Scale (CPS), provide preliminary evidence of its reliability and validity, and suggest ways in which it can be used clinically to reveal new areas for questioning and to generate new ideas. The CPS is a brief questionnaire that can be used to create a profile of each individual family member's private constructions. To complete the CPS, each family member writes a free-form narrative of the presenting problem and then rates his or her perceptions of the contributing causes. The CPS profiles can be used to compare the perspectives of different family members and to assess cognitive constructions at different points in treatment. We discuss its potential for these and other clinical uses.

Adult↗
PubMed1997

Peptide mimics of a conformationally constrained protective epitopes of respiratory syncytial virus fusion protein.

AIMS: To identify peptides that mimic (mimotopesi conformational and protective epitopes of RSV fusion protein and to assess their efficacy as immunogens and potential vaccines. MATERIAL AND METHODS: An 8-mer solid-phase (TG resin) library was screened with a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads, reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysed by amino acid replacement using the SPOTs method. RESULTS: Mabs were not able to bind to the free and soluble peptides, nor did these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to produce de-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproduce this conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versions of the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance. Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibited the binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80-640); and (4) reduce significantly the viral load in the lungs of mice challenged with RSV (P < 0.01). CONCLUSIONS: This report demonstrates for the first time that: (1) a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with these mimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novel concept for the development of a vaccine against RSV.

Animals↗
PubMed1997

Fibrotic healing of adult and late gestation fetal wounds correlates with increased hyaluronidase activity and removal of hyaluronan.

The lack of scarring and fibrosis in healing fetal skin wounds may relate to a prolonged presence of hyaluronan (HA). It has been suggested that fetal wounds may lack hyaluronidase, but the hyaluronidase levels in fetal wounds remain unknown. The size of HA influences its biological action, especially in relation to angiogenesis, which is also reduced in fetal wound healing. The present study determined the levels and size of HA, as well as hyaluronidase levels, in fetal and adult lamb wounds. Wire mesh cylinders, or polyvinyl acetate sponges, were placed subcutaneously in fetal lambs at 75, 100 or 120 days gestation. Wound fluid and wound tissue were harvested 3, 7 or 14 days later. Samples were digested with papain and both HA and hyaluronidase activity were determined in a competitive ELISA assay. Size distribution of HA was estimated using a Sephacryl S1000 column and fractions were collected for HA determination. Adult wound fluid HA remained low (4-5 micrograms/ml) over the 14 days. Fetal fluids were similar on day 3, but increased to 15-25 micrograms/ml by day 7. In 75/100-day wounds, HA remained elevated at 14 days, but in 120-day fluids decreased to levels similar to adult fluid. The HA in all fluids was polydisperse with a main peak at 200 kDa. Hyaluronidase levels were detected in all samples, reaching a peak 7 days post-wounding. In adult wound fluids hyaluronidase was much higher than the fetal wound fluids. These data suggest that lower hyaluronidase levels in fetal wounds may underlie the different pattern of HA deposition seen in fetal wounds.

Amniotic Fluid↗
PubMed1995

The induction of respiratory syncytial virus-specific cytotoxic T-cell responses following immunization with a synthetic peptide containing a fusion peptide linked to a cytotoxic T lymphocyte epitope.

Previously published work has shown that a cytotoxic T-cell epitope (CTL) representing residues 82-90 of the M2 protein of respiratory syncytial virus (RSV) is the target for a protective response against the virus. In this report, we demonstrate that a synthetic peptide representing residues 81-95, when covalently linked to a fusion peptide derived from the conserved N-terminal 19 residues of the F1 protein of measles virus efficiently primes RSV-specific CTLs in vivo following immunization without adjuvant.

Amino Acid Sequence↗
PubMed1993

Influence of the T-helper epitope on the titre and affinity of antibodies to B-cell epitopes after co-immunization.

We have assessed the influence of different T-helper cell epitopes on the level and affinity of antibody to B-cell epitopes induced following co-immunization with free peptides mimicking epitopes from measles and respiratory syncytial virus envelope proteins. The responses obtained following co-immunization have been compared to those obtained following immunization with chimeric synthetic peptide immunogens in which the epitopes were covalently coupled. The results show that covalent linkage of the B- and T-cell epitopes is not necessary for the generation of T-cell dependent antibody responses to non-immunogenic B-cell epitopes. In addition the induction of memory B-cells required adjuvant but subsequent stimulation of these memory cells did not. The responses obtained were non-MHC restricted since co-immunization resulted in the production of antibody responses to B-cell epitopes in a panel of five inbred mouse strains but there were differences in the ability of different T-cell epitopes to provide help for antibody production to the same B-cell epitope. The affinity of antibodies to the B-cell epitopes induced following immunization with chimeric T:B peptides was higher than that obtained following co-immunization. These results indicate the value of co-immunization for the induction of antibody responses to B-cell epitopes across MHC differences and suggest that this strategy may be of value in the development of synthetic peptide vaccines. However, modifications of the approach need to be developed to ensure the production of antibody of the highest possible affinity.

Amino Acid Sequence↗
PubMed1990

The management of patients with an intrinsic supratentorial brain tumour.

The management of patients presenting with supratentorial glioma between 1978 and 1986 is reviewed. Complete follow-up in 517 cases was obtained. One hundred and fifty eight patients were not submitted to any form of surgery, 299 patients were biopsied and 60 patients underwent craniotomy and internal decompression. The no surgery group contained a higher proportion of patients with poor prognostic indicators than either the biopsy or craniotomy groups. The craniotomy group consisted of patients with better prognostic indicators than the biopsy group, in particular, younger age and more favourable site, type and grade of tumour. This was reflected in the difference in outcome between the groups. Median survival was 14 months in the craniotomy group, four months in the biopsy group and 2.2 months in the no surgery group. The outcome in patients with histologically proven malignant gliomas was best in those patients who received radiotherapy. The craniotomy group had a median survival of 18.5 months, a two year survival of 48% and a five year survival of 9%. The median survival following radiotherapy of those patients with proven malignant gliomas who had a biopsy was 9.5 months with a two year survival of 16% and a five year survival of 2%. These results compare favourably with studies which have adopted a more aggressive approach, suggesting that outcome is determined as much by patient selection using favourable prognostic indicators as by the treatment itself. The need for prospective trials of the management of unselected consecutive glioma patients randomizing them to conservative and radical treatment groups in order to define the role of both conventional therapy and radical therapy is discussed.

Adolescent↗
PubMed1989

Plasma amino acids in patients with senile dementia and in subjects with Down's syndrome at an age vulnerable to Alzheimer changes.

Plasma concentration of tryptophan, tyrosine, leucine and thiamine were reduced in senile dementia of Alzheimer type. In Down's syndrome, where Alzheimer-type histology appears consistently at an early age, there was a definite type of abnormality, raised concentrations of isoleucine, leucine, phenylalanine and cystine. Because of the competition between amino acids for transfer into the brain, either or both of these types of change could lie in the aetiological chain underlying the development of Alzheimer pathology. Alternatively, these patterns could reflect the requirements of aberrant central/peripheral protein turnover.

Adult↗
PubMed1989

Outcome in unipolar affective disorder after stereotactic tractotomy.

An evaluation of 15 patients treated by subcaudate stereotactic tractotomy (SST) for treatment-resistant unipolar affective disorder was made for frequency and severity of recurrence of illness. One-third had died by the time of assessment, but none by suicide. Two-thirds of the sample experienced a reduction in the severity of depressive episodes after the operation; only one-third reported a reduction in frequency of episodes.

Adult↗
PubMed1988

Pharmacological management of treatment-resistant depression.

Treatment-resistant depression might be considered as the failure of two families of antidepressant therapy given sequentially at sufficient dosage, for an adequate length of time and with continuous compliance. Patients with treatment-resistant depression should be assessed as though they were new referrals, and factors which may be contributing should be dealt with whenever possible. A sequence of therapies based on a tricyclic antidepressant and a monoamine oxidase inhibitor, each combined with putative adjuvant therapies, is proposed as a working model pending further studies and developments.

Antidepressive Agents↗