Biomedical subjects

S Gillam

Publications and source records attributed to S Gillam.

At least 55 records · Page 3Linked to original sources

PubMed1995

Expression and characterization of secreted forms of rubella virus E2 glycoprotein in insect cells.

Two different forms of rubella virus E2 glycoproteins were expressed in insect cells: intact wild-type E2 and a soluble form of E2 (E2 delta Tm) glycoprotein, in which the C-terminal membrane-anchor domain was deleted. E2 delta Tm behaved as a secretory protein and was secreted abundantly (5 mg/liter) from insect cells. In contrast to wild-type E2 (36 kDa), E2 delta Tm was secreted into the media and was detected as two species (33 and 30 kDa). Lectin binding assays in conjunction with glycosidase analyses revealed that both intracellular wild-type E2 and E2 delta Tm contained only N-linked glycans, while the two secreted forms of E2 delta Tm were found to differ in their glycosylation, with the 30-kDa form having only N-linked glycans while the 33-kDa species had both N-linked and O-linked glycans. The secreted E2 delta Tm species were purified by precipitation between 20 and 40% saturation with (NH4)2SO4 and retained full antigenicity. The levels of antibodies elicited in mice immunized with purified E2 delta Tm showed that the immunogenicity of secreted E2 delta Tm compared favorably to that of natural virion E2.

Animals↗

PubMed1995

Brefeldin A and monensin arrest cell surface expression of membrane glycoproteins and release of rubella virus.

The maturation of rubella virus (RV) glycoproteins E2 and E1 was examined by using brefeldin A (BFA) and monensin. BFA, which induces the rapid redistribution of Golgi enzymes residing in the Golgi complex into the endoplasmic reticulum (ER), was used to locate the intracellular site for the modification of carbohydrate side-chains on RV E1 and E2 proteins. The monovalent ionophore monensin, which inhibits intracellular transport of proteins through the ER-Golgi complex, was used to block the transport of E1 and E2 glycoproteins through the Golgi complex. BFA and monensin effectively blocked the cell surface expression of RV E2 and E1 proteins, secretion of an anchor-free form of E2 and budding of RV from the plasma membrane. For O-linked glycosylation, addition of N-acetylgalactosamine and galactose to E2 protein was found to take place in the medial to the trans Golgi. A dramatic change in the intracellular distribution of RV structural proteins was observed when transfected COS cells were treated with BFA or monensin, although the proteolytic processing of RV structural protein precursor was not affected. In the presence of BFA or monensin, virus release from infected Vero cells was only 0.1% of the intracellular virus, and the intracellular virus titre decreased as well. Our results suggest that O-linked glycosylation on the E2 protein occurred in the post-ER region and the transport of RV structural proteins to the Golgi complex and post-Golgi compartment may be a rate-limiting step in RV assembly and budding.

Animals↗

PubMed1995

The carrot, the stick and the general practitioner: how have changes in financial incentives affected health promotion activity in general practice?

BACKGROUND: Financial incentives for increasing health promotion activity in primary care, introduced with the 1990 contract for general practitioners, were amended in 1993 and are now focused on cardiovascular disease. Payments for health promotion clinics were abolished and target payments were introduced. AIM: The study aimed to evaluate the effect of the change, in June 1993, in financial incentives for health promotion activity in primary care on the distribution of health promotion payments in two family health services authorities. METHOD: A retrospective study was undertaken in which data from two family health services authorities were used to determine the annual level of health promotion payments per 1000 practice population before and after the contractual amendment. Health promotion clinic payment data were analysed for 78 practices in Bedfordshire Family Health Services Authority and 85 practices in Kensington, Chelsea and Westminster Family Health Services Authority. Changes in health promotion payments were calculated and related to two measures of relative need: all cause standardized mortality ratios, for patients aged 74 years or less, of the electoral ward in which the practice is located; and the Jarman underprivileged area score. High relative need was defined as a standardized mortality ratio of over 100 or more than 25% of the practice population living in electoral wards with a Jarman score of over 30. RESULTS: Health promotion payments were more evenly distributed after the change in June 1993 than before between the two family health services authorities and between general practices. Single-handed practices were carrying out more clinics in 1992 than multi-partner practices and consequently were one of the greatest financial losers as a result of the change. In addition, practices located in electoral wards with high relative needs lost proportionally more than those in electoral wards with lower needs. CONCLUSION: Changes in the general practitioner health promotion contract have created new financial winners and losers. It now appears that health promotion payments are more evenly distributed but that the distribution is unrelated to need or treatment given. More evidence on the effectiveness of health promotion interventions is required before policies aimed at promoting better health through primary care can be fully evaluated.

England↗

PubMed1994

Assembly of rubella virus structural proteins into virus-like particles in transfected cells.

We have developed a stably transfected CHO cell line (CHO24S) that expresses the three structural proteins of rubella virus (RV). RV proteins C (capsid), E2, and E1 are secreted from CHO24S cells in the form of RV-like particles (RLPs) which form by budding into the cisterna of the Golgi complex. RLPs resemble RV virions in their size and morphology and have an identical buoyant density when purified on sucrose gradients. Release of RLPs into the medium was found to be dependent upon the E1 cytoplasmic tail since deletion or substitution of this domain with the same region from vesicular stomatitis virus G protein abrogated release of RV proteins from transfected cells. These results indicate that the RV 40S genomic RNA is not required for efficient particle assembly. Therefore, RLPs may serve as a convenient source of RV antigen for use in diagnostic assays and as an alternative to live attenuated vaccine strains.

Animals↗

PubMed1994

Mutational analysis of the arginine residues in the E2-E1 junction region on the proteolytic processing of the polyprotein precursor of rubella virus.

Endoproteolytic cleavage of precursors is a key step in biosynthesis of functional proteins. The structural proteins of rubella virus are initially translated as a precursor polyprotein in the order NH2-C-E2-E1-COOH and are cleaved by host signal peptidase to yield three structural proteins. Between regions corresponding to E2 and E1 in the precursor is a region of seven amino acid residues (R-R-A-C-R-R-R) that contains a motif for stop-transfer or a possible target for trypsin-like protease cleavage. Using site-directed mutagenesis, these arginine residues, as well as the signal peptide cleavage site at the N-terminus of E1, have been mutated individually or in combination. Results from in vitro transcription/translation analysis indicated that the mutated E2E1 precursor polyproteins were translocated into the microsome and glycosylated. Expression of mutated precursor polyproteins in COS cells revealed that the cleavage of E2E1 polyprotein precursor was impaired when the signal peptide cleavage site alone or both arginine clusters were altered, whereas partial cleavage was observed in the mutants in which either one of the two arginine clusters was modified. Our data suggest that although the arginine clusters do not function as a basic protease cleavage site, they contribute to maintain the proper configuration of that region for access of cellular signal peptidase.

Amino Acid Sequence↗

PubMed1994

Expression and characterization of a soluble rubella virus E1 envelope protein.

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1 delta Tm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1 delta Tm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1 delta Tm by the insect cells. The secreted E1 delta Tm protein was purified from serum-free medium by one-step immunochromatography. The purified E1 delta Tm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.

Animals↗

PubMed1994

Expression of soluble forms of rubella virus glycoproteins in mammalian cells.

Rubella virus (RV) virions contain two envelope glycoproteins, E1 and E2. Removal of hydrophobic regions in their carboxyl termini by genetic engineering caused them to be secreted rather than maintained in cell membranes of transfected COS cells. Truncated E2 was secreted in the absence of E1, whereas E1 lacking its transmembrane domain required coexpression of E2 for export from the cell. Secreted E2 was found to contain both O-linked and N-linked complex glycans, whereas secreted E1 retained virus neutralization and hemagglutination epitopes, suggesting the possibility of using soluble RV antigens as subunit vaccines and for serodiagnostic purposes. Stable Chinese hamster ovary cell lines secreting RV E1 were constructed for large scale preparation of recombinant E1.

Animals↗

PubMed1994

Immunogenicity study of a synthetic T-cell epitope of rubella virus capsid protein recognized by human T cells in different strains of mice.

The immunogenicity of a human immunodominant T-cell epitope C9 (residues 205-233) of rubella virus capsid protein was studied in three strains of mice using C9 lipopeptide. This peptide induced strong T-cell responses in all three strains of mice. The minimal T-cell epitope C9B (residues 205-216) recognized by human T cells with HLA-DR4 phenotype did not specifically stimulate proliferation of T cells from mice in vitro. T cells specific for C9 from immunized mice were shown to be CD4+, in agreement with results of similar studies in RV-seropositive humans.

Amino Acid Sequence↗

PubMed1994

Expression and characterization of virus-like particles containing rubella virus structural proteins.

Rubella virus (RV) virions contain two envelope glycoproteins (E1 and E2) and a capsid protein (C). Noninfectious RV-like particles (VLPs) containing three structural proteins were expressed in a BHK cell line (BHK-24S) by using an inducible promoter. These VLPs were found to resemble RV virons in terms of their size, their morphology, and some biological activities. In immunoblotting studies, VLPs were found to bind similarly to native RV virions with 10 of a panel of 12 RV-specific murine monoclonal antibodies. Immunization of mice with VLPs induced specific antibody responses against RV structural proteins as well as virus-neutralizing and hemagglutination-inhibiting antibodies. After immunization of mice with VLPs, in vitro challenge of isolated lymphocytes with inactivated RV and individual RV structural proteins stimulated proliferation. Our data suggest the possibility of using VLPs as immunogens for serodiagnostic assays and RV vaccines.

Animals↗

PubMed1994

What makes general practitioners do child health surveillance?

The contribution of different general practitioner characteristics, views, and experiences to the likelihood of their providing child health surveillance (CHS) was determined and their perceived training needs discovered. Family health service authority administrative data on the study population was combined with a postal questionnaire survey. Subjects were all general practitioners in three district health authorities in the North West Thames region. There were striking differences between districts in the proportion of practitioners undertaking CHS. General practitioners with paediatric training were three times more likely to do CHS. Women doctors were twice as likely to do CHS as men. The personal views of general practitioners were significantly associated with whether or not they undertook CHS. The CHS fee did not appear to be the major motivating factor. There was considerable demand for further training. The proportion of general practitioners undertaking CHS is likely to increase with the proportion of women and vocationally trained doctors. More local training is wanted, both by general practitioners already doing CHS and by those who would like to do it. Health authorities need to ensure that such training is convenient and continuing.

Adult↗

PubMed1993

Mapping T-cell epitopes of rubella virus structural proteins E1, E2, and C recognized by T-cell lines and clones derived from infected and immunized populations.

To design a safe and effective synthetic peptide vaccine against rubella virus (RV) infection, it is necessary to identify immunodominant T-cell epitopes of RV structural proteins. To define such epitopes, 49 overlapping synthetic peptides (17-34 residues in length) corresponding to more than 95% of the amino acid sequence of RV virion proteins E1 (23 peptides) and C (11 peptides) and all of E2 (15 peptides) were synthesized and tested for their capacities to induce proliferative responses of rubella-specific T-cell lines and T-cell clones derived from 4 study groups (5 women infected with RV in pregnancy, 5 patients with congenital rubella syndrome, 5 seropositive healthy donors, and 5 RV vaccine recipients). The most frequently recognized epitopes were E1-21 (residues 358-377) with 11/20 responders, E2-4 (residues 54-74) with 6/20 responders, and C11 (residues 255-280) with 11/20 responders, respectively. E1-10 (residues 174-193), E1-16 (residues 272-291) and E1-18 (residues 307-326) were responded to strongly by corresponding T-cell clones, and were recognized by 4 or 5 T-cell lines. T-cell lines derived from three congenital rubella syndrome patients did not respond to any of the synthetic peptides. The results showed that more T-cell epitopes were present in E1 (19/23) and C (10/11) than in E2 (8/15). The identification of T cell sites recognized frequently by RV-infected or -immunized populations could provide the basis for selecting candidate T-cell epitopes for the development of an effective synthetic vaccine against rubella.

Adult↗

PubMed1993

Human T- and B-cell epitopes of E1 glycoprotein of rubella virus.

The identification of T- and B-cell sites recognized frequently by human populations could provide the basis for selecting the candidate T- and B-cell epitopes for the development of an effective synthetic vaccine against rubella. Rubella virus E1 glycoprotein has been shown to be the predominant antigen to which the majority of human populations develop lymphocyte proliferative and antibody responses. To define the T- and B-cell epitopes of E1 glycoprotein of rubella virus, 23 overlapping synthetic peptides corresponding to more than 90% of the amino acid sequence of E1 were synthesized and tested for their capacities to induce proliferative and antibody responses of 10 seropositive individuals. The most frequently recognized T-cell epitopes were EP19 (residues 324-343), with 7 of 10 responders, and both EP12 (residues 207-226) and EP17 (residues 289-308), with 6 of 10 responders, respectively. Two immunodominant linear B-cell epitopes were mapped to residues 157 to 176 (EP9, 8/10) and 374 to 390 (EP22, 6/10) by using peptide-specific enzyme linked immunosorbent assay.

Amino Acid Sequence↗

PubMed1993

Cooperation or conflict over child health surveillance? Views of key actors.

OBJECTIVE: To describe the views of general practitioners, health visitors, and clinical medical officers on child health surveillance, recent changes, perceptions of each other's roles, and attitudes to audit. DESIGN: Postal questionnaire survey. SETTING: Three health districts in North West Thames health region. SUBJECTS: All 602 general practitioners, 272 health visitors, and 42 clinical medical officers in these districts. MAIN MEASURES: Attitudes to and perceptions of child health surveillance and audit. Questionnaires were completed by 440 general practitioners (response rate 73%), 164 health visitors (60%), and 39 clinical medical officers (93%). RESULTS: Attitudes to child health surveillance were less positive among general practitioners than health visitors or clinical medical officers. Few respondents agreed that child health surveillance was a cost effective use of general practitioners' time (general practitioners 28%, 113/407; health visitors 28%, 40/145; clinical medical officers 39%, 15/39) and most thought that health visitors should carry out more of the doctors' examinations (68%, 262/387; 65%, 89/136; 66%, 25/38). General practitioners thought that clinical medical officers were less supportive than other relevant groups of their doing more child health surveillance. Most (72%, 105/146) health visitors thought that the 1990 contract had reduced parental choice of where to attend for child health surveillance. General practitioners were less enthusiastic than health visitors about most forms of clinical audit. CONCLUSIONS: Despite reservations about the impact of recent changes all groups were willing to explore innovative ways of delivering child health surveillance. IMPLICATIONS: There is scope for health visitors to increase their responsibilities and for more varied relationships between general practitioners and community child health doctors.

Attitude of Health Personnel↗

PubMed1992

Characterization of the specificity and genetic restriction of human CD4+ cytotoxic T cell clones reactive to capsid antigen of rubella virus.

Using 11 overlapping synthetic peptides covering more than 95% of the amino acid sequence of capsid protein of rubella virus, 7 CD4+ T cell clones (R10, R11, R18, A2, A10, A11, and A12) isolated from 2 rubella seropositive donors reacted strongly to rubella capsid peptides C6 (residues 119-152), C9 (residues 205-233), or C11 (residues 255-280), respectively, in both proliferation and cytotoxicity assay. Truncated peptides C6E (residues 125-139), C9B (residues 205-216), and C11E (residues 260-272) were shown to be involved directly to the T cell determinants of C6, C9, and C11, respectively. Genetic restriction of these T cell clones was analyzed by using human cell lines with various HLA-DR phenotypes as targets and/or antigen-presenting cells in cytotoxicity assay and/or proliferation assays. The results indicated that the recognition of peptide C6 by T cell clones (R11 and R18) was associated with DRw9 molecule, while the HLA restriction element of the responses of other T cell clones (A2 and A11, A10, and A12) that reacted with peptide C9 or C11 was DR4 molecule. However, there may be a cross-recognition by the T cell clone (A12) between DR1 and DR4 subtypes.

Amino Acid Sequence↗

PubMed1992

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus.

Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of E1 deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1(193) to E1(269)) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues E1(214) to E1(240), while two VN epitopes defined by MAb 21B9H and MAb 16A10E mapped to amino acid residues E1(214) to E1(233) and E1(219) to E1(233), respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.

Amino Acid Sequence↗

PubMed1992

The influence of N-linked glycosylation on the antigenicity and immunogenicity of rubella virus E1 glycoprotein.

Rubella virus E1 glycoprotein contains three functional N-linked glycosylation sites. The role of N-linked glycosylation on the antigenicity and immunogenicity of E1 glycoprotein was studied using vaccinia recombinants expressing E1 glycosylation mutants. Expressed E1 glycosylation mutant proteins were recognized by a panel of E1-specific monoclonal antibodies in radioimmunoprecipitation, immunofluorescence, and immunoblotting, indicating that carbohydrate side chains on E1 are not involved in the constitution of epitopes recognized by these monoclonal antibodies. This observation was further supported by the fact that removal of oligosaccharides on E1 by glycosidase digestion did not significantly change the antigenicity of E1. All the glycosylation mutants were capable of eliciting anti-RV E1 antibodies. The single glycosylation mutants (G1, G2, and G3), but not the double mutant (G23) or the triple mutant (G123), were found to be capable of inducing virus neutralizing antibodies. Among the single glycosylation mutants, only G2 and G3 were active in producing hemagglutination inhibition antibodies in mice. Our findings suggest that although carbohydrate on E1 is not directly involved in the antigenic structures of E1, it is important in maintaining proper protein folding and stable conformation for expression of immunological epitopes on E1.

Animals↗

PubMed1992

Evaluating the drug dependency unit.

An evaluation of the St Mary's Drug Dependency Unit is described. Unit staff were interviewed and a retrospective analysis of 156 records was performed. The advent of HIV infection has led to an emphasis on public health priorities in the treatment of drug misuse. However, DDU staff retain a highly individualistic approach to their work. Staff aiming at abstinence within treatment may compromise important advice on harm minimization. The mean delay between first contact and entering treatment was 10 days; 42% of clients failed to complete the initial assessment. The need to extend access to the service is not easy to reconcile with the increasingly specialised support role for such units implied in recent strategy documents. Little is known of the effectiveness of different services for drug misusers. In this study, only 6% of clients were known to have achieved abstinence at one year. The efficiency of the DDU could be improved by closer definition of the unit's goals and target population, regular progress reviews, shortening the assessment procedure and the computerisation of data collection.

Adult↗

PubMed1992

Cellular and humoral immune responses to rubella virus structural proteins E1, E2, and C.

Better understanding of cell-mediated immune responses to rubella virus would provide the basis for the development of safe and effective vaccines against rubella and would aid in analysis of the pathophysiology of congenital rubella syndrome. We have expressed individual rubella virus structural proteins, E1, E2 and C, via vaccinia virus recombinants. Using the expressed recombinant proteins as antigens, we were able to demonstrate antigen-specific lymphocyte proliferative responses in control individuals and individuals with congenital rubella syndrome. Among the two human groups studied, E1 glycoprotein proved to be a better immunogen than E2 or C. For the control individuals, significant differences in proliferative responses to the structural proteins E1, E2, and C were observed. These differences were not significant in individuals with congenital rubella syndrome. In parallel to the lymphoproliferative responses, immunoglobulin G responses were also found directed mainly to the E1 glycoprotein. These results suggest that E1 may be the most important rubella virus antigen to study in determining the domains required for constructing subunit vaccines against rubella.

Adult↗