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Biomedical subjects

D R Harper

Publications and source records attributed to D R Harper.

At least 19 recordsLinked to original sources
PubMed2002

Immunization with a DNA vaccine expressing a truncated form of varicella zoster virus glycoprotein E.

The gE glycoprotein of varicella zoster virus (VZV) is involved with cell entry and it is the most abundant glycoprotein produced in VZV-infected cells. It is also the first glycoprotein to be recognized by the immune system and induces neutralizing antibodies and cellular immunity. We have shown previously that immunization with a DNA vaccine encoding full length gE induces high antibody titres in BALB/c mice. In this study, we engineered a truncated form of gE to facilitate secretion of the glycoprotein, which is thought to increase the quantity of antigen available for B cells to mount an immune response. This hypothesis was tested by using inverse PCR mutagenesis (IPCRM) to engineer a mutated form of gE that was secreted from the cell. This construct was then evaluated as a potential DNA vaccine. Following immunization studies, the magnitude of the immune response induced with the mutant form of gE was found to be similar to that induced by membrane bound protein. This finding suggests that, in the case of VZV, a DNA vaccine expressing a secreted protein has no advantage over one expressing a membrane bound protein. However, mice immunized with the truncated form of gE (gED) displayed responses favouring IgG1 (Th2) in comparison with mice immunized with the full length gE construct, which generated an IgG2a (Th1) response. This observation indicates that immunization with a truncated form of a gene may induce immune modulation, a phenomenon that should be taken into account for the design of vaccines.

Animals↗
PubMed2000

Immunization with a DNA expression vector encoding the varicella zoster virus glycoprotein E (gE) gene via intramuscular and subcutaneous routes.

In this study we constructed a plasmid containing the gene encoding varicella-zoster virus transmembrane glycoprotein gE (VZV gE) and evaluated its utility for DNA immunization in mice. Our initial work demonstrates that intramuscular and subcutaneous injection of VZV gE DNA, without the use of costimulatory molecules or other adjuvant materials, results in the generation of antigen-specific antibodies of primarily the IgG2a subclass, indicating that this vaccine can stimulate Th1 type immunity. This is the first report of a prototype DNA vaccine for varicella-zoster virus.

Animals↗
PubMed1999

Nucleic acid immunization: concepts and techniques associated with third generation vaccines.

A radical change in vaccine methodology arrived nine years ago with the advent of nucleic acid immunization. Aspects such as plasmid design, gene selection, the use of immunostimulatory complexes and clinical trials are discussed in this review. Furthermore, concepts and protocols involved in the construction, evaluation and immunization of a DNA vaccine have been examined as new strategies to enhance this technology continues to grow.

Adjuvants, Immunologic↗
PubMed1999

Randomised, double blind placebo controlled trial of pentoxifylline in the treatment of venous leg ulcers.

OBJECTIVE: To determine whether pentoxifylline 400 mg (Trental 400) taken orally three times daily, in addition to ambulatory compression bandages and dressings, improves the healing rate of pure venous ulcers. DESIGN: Randomised, double blind placebo controlled trial, parallel group study of factorial design, permitting the simultaneous evaluation of alternative pharmaceutical, bandaging, and dressings materials. SETTING: Leg ulcer clinics of a teaching and a district general hospital in southern Scotland. PARTICIPANTS: 200 patients with confirmed venous ulcers and in whom other major causal factors were excluded. INTERVENTIONS: Pentoxifylline 400 mg three times daily or placebo. MAIN OUTCOME MEASURE: Complete healing (full epithelialisation) of all ulcers on the trial leg. RESULTS: Complete healing occurred in 65 of the 101 (64%) patients receiving pentoxifylline and 52 of the 99 (53%) patients receiving placebo. CONCLUSIONS: The difference in the healing rates between patients taking pentoxifylline and those taking placebo did not reach statistical significance.

Adult↗
PubMed1999

Perceived health in a randomised trial of treatment for chronic venous ulceration.

STUDY OBJECTIVE: To observe changes in perceived health in patients during a clinical trial of treatments for venous leg ulceration. DESIGN: Randomised prospective factorial trial in patients with venous ulceration. Each patient randomised to a bandage, dressing and a drug. Perceived health assessed at entry and after 24 weeks. SETTING: Outpatient departments and patient's home. PATIENTS: Two hundred patients presenting to two vascular services in Falkirk and Edinburgh with chronic (duration > 2 months) non-healing venous ulceration. STATISTICAL ANALYSIS AND MAIN RESULTS: Analysis using the Nottingham Health Profile revealed that after 24 weeks there were significant improvements in all subscores (p < 0.01) with the exception of social isolation (p = 0.081). Patients with healed ulceration had improved in energy, pain, emotion, sleep and mobility compared with those whose ulceration failed to heal (p < 0.05). Patients randomised to four layer bandaging had significantly better energy (diff = 7.9, 95% CI 0.2, 15.6, p = 0.04) and mobility (diff = 4.5, 95% CI 0.0, 9.0, p = 0.046). This difference could be explained largely by the improved healing of patients randomised to this bandage system (67/97 vs. 50/103, OR = 2.37, 95% CI 1.31, 4.27). CONCLUSIONS: Improvements in perceived health were significantly greater in patients whose ulcers had completely healed. Methods of treatment which offer improved healing for patients with venous leg ulceration are likely to improve patients' perceived health status.

Adult↗
PubMed1999

Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients.

Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI). The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens. PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs. As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples. The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination.

Adult↗
PubMed1998

High-titre, cryostable cell-free varicella zoster virus.

Work with varicella-zoster virus has been seriously hampered by the difficulty of preparing high-titre cell free virus, and by the instability of such virus when frozen and thawed. We have evaluated the use of a range of protocols and demonstrate that relatively high titres of 19,000 plaque forming units per millilitre may be recovered after freezing in PBS-sucrose-glutamate-serum buffer (PSGC). In addition, we have shown that the virus obtained by this method gives similar results in neutralisation and antiviral susceptibility assays to that freshly prepared from infected cells, allowing the use of high titre, titrated virus stocks in such assays.

Antibodies, Monoclonal↗
PubMed1998

Molecular biology of varicella-zoster virus. A review prepared for the UK Advisory Group on Chickenpox.

Varicella-zoster virus (human herpesvirus 3; VZV) is one of eight herpes viruses that routinely infect humans. It is classified as a member of the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. Of the other human herpes viruses it is most closely related to the herpes simplex viruses (also members of the Alphalerpesvirinae). Like all herpes viruses, the virus has a large double-stranded DNA genome within an icosahedral nucleocapsid. This is surrounded by a proteinaceous tegument and a trilaminar membrane derived from host-cell membranes into which the viral glycoproteins are inserted. The structure of the virion is summarized in Fig. 1.

Antiviral Agents↗
PubMed1998

Popliteal vein reflux reduces the healing of chronic venous ulcer.

BACKGROUND: The relationship between deep and superficial venous reflux and healing of venous ulceration by non-operative compression therapy has not been studied previously. METHODS: A total of 155 patients with chronic venous ulcers underwent duplex ultrasonography before treatment with compression bandaging at a hospital-based venous clinic. RESULTS: At 24 weeks, 104 (67 per cent) of ulcers had healed. There was no significant difference in the pattern of either deep or superficial venous reflux between healed and non-healed ulcers except with respect to the popliteal vein. In healed ulcers, 39 scans (38 per cent) indicated competence of the above-knee popliteal vein compared with five (10 per cent) in the non-healing group (P < 0.001, chi 2 test). Similarly, 43 scans (42 per cent) showed below-knee popliteal vein competence in the healed ulcers compared with only five (10 per cent) performed in legs remaining ulcerated (P < 0.001, chi 2 test). CONCLUSION: Popliteal vein incompetence is an indicator of poor response to compression therapy for venous ulceration.

Adult↗
PubMed1998

Subclinical herpes virus reactivation and latency.

Although there has been considerable refinement in our understanding of the processes underlying the establishment and maintenance of latency, important research questions remain. Results from various workers imply that the establishment of latency may be a dynamic process and may offer possible therapeutic targets. The role played by the latency-associated transcripts appear to be important in both establishing latency and in aiding reactivation. Recent work has shown that reactivation in vivo is much more frequent than previously thought and leads principally to subclinical viral shedding. Factors influencing subclinical shedding for most patient groups and the response and limitation of therapy have been clearly determined.

Journal Article↗
PubMed1997

Cloning, expression, and immunogenicity of the assembly protein of varicella-zoster virus and detection of the products of open reading frame 33.

Herpesviruses produce assembly proteins (AP) that act as scaffolding proteins for the assembly of the viral capsids. The products of the assemblin gene, which encodes both maturational protease and AP, have been established for herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (CMV). We cloned an inframe ORF (encoding amino acids 304-605), found within the ORF 33 assemblin gene of VZV, into a yeast expression vector. The 34-kDa AP was expressed as a fusion protein with the particle-forming Ty p1 protein, resulting in high-level production of hybrid AP-virus-like particles (AP-VLPs). When AP-VLPs were injected into mice and rabbits, antibodies were produced that reacted with, but that did not neutralise, native VZV. Three of four inbred strains of mice immunised with AP-VLPs produced a VZV-specific T-cell response. The mouse and rabbit sera reacted with six bands on native VZV by Western blot analysis. The dominant bands were found at 34 and 38 kDa. Bands were also seen at 66, 63, 41, and 31 kDa. The 38-kDa protein may represent the mature AP derived from the 41-kDa precursor AP, itself the release product from the full-length 66-kDa assemblin. The 34-kDa protein probably represents the product of the inframe co-translational gene within ORF 33 encoding amino acids 304-605. The genetic organisation and proteolytic maturation of VZV assemblin are, therefore, analogous to those of other herpesviruses.

Animals↗
PubMed1995

Identification of immunodominant regions and linear B cell epitopes of the gE envelope protein of varicella-zoster virus.

The envelope proteins of varicella-zoster virus (VZV) are highly immunogenic and one of the most abundant is glycoprotein E (gE). However, its immunodominant regions and epitopes have not been identified. In this study, using human sera from individuals with recent varicella or zoster infections, we have localized antigenic sequences of gE using recombinant hybrid Ty-virus-like particles (VLPs) carrying overlapping fragments of the gE protein. gE(1-134)-VLPs (particles carrying amino acids 1-134 of gE) and, to a lesser extent, gE(101-161)-VLPs were found to be the most antigenic when tested by Western blotting and ELISA. Other fragments of gE (spanning residues 161-623) showed weak or no antigenicity. Pepscan analysis of human sera on overlapping synthetic peptides representing residues 1-135 of gE revealed that the most antigenic region was between residues 50 and 135. Three immunodominant sequences (residues 86-105, 116-135, and, to a lesser extent, 56-75) were detected using sera from both varicella and zoster patients. All sera from varicella, but not zoster, patients reacted strongly with an epitope in peptide 66-85. Other epitopes were recognized weakly by some varicella or zoster sera. More sera need to be tested to assess the potential disease specificity of these epitopes. The neutralizing monoclonal antibody (MAb) IF-B9 reacted with residues 71-90; however, another neutralizing MAb, SG1A, which bound to both gE(1-134)-VLPs and gE(101-161)-VLPs did not bind to any peptide. The identification of immunodominant sequences of gE will help toward the development of a subunit VZV vaccine.

Amino Acid Sequence↗
PubMed1995

Varicella-zoster virus assembly protein p32/p36 is present in DNA-containing as well as immature capsids.

Varicella-zoster virus (VZV) produces a group of nucleocapsid proteins (the p32/p36 nucleoprotein complex) which are the VZV analogues of the herpes simplex virus type 1 (HSV-1) and cytomegalovirus (CMV) assembly proteins. There are multiple components in the VZV p32/p36 complex, with major proteins of 32 and 36 kDa and minor proteins of 34 and 38 kDa. In HSV-1 the assembly proteins have been shown to be present in immature (B) capsids, but are removed prior to the formation of mature (C) capsids containing the viral DNA genome. Our work has shown that VZV produces capsids corresponding to the B and C forms. However, in contrast to HSV-1, VZV also produces "B/C" capsids that appear to contain both the assembly proteins and the viral DNA genome. Possible mechanisms for this are discussed. In addition, it was shown that VZV capsids appear to lack the 36 and 38 kDa proteins, and based on this observation we suggest that these may represent unprocessed forms of the assembly protein. In both HSV and CMV, a much larger, cross-reactive protein has been identified as the full-length product of the gene coding for the assembly protein. The homologous VZV gene (ORF 33) theoretically has the capacity to produce a 66 kDa protein. However, no such protein is readily apparent in VZV-infected cells. The presence of an immunoreactive 64 kDa protein was demonstrated in purified VZV capsids which may represent the full-length ORF 33 protein.

Antibodies, Monoclonal↗
PubMed1994

Epidemiology of varicose veins. A review.

Disease of the venous system is a major problem affecting western societies, resulting in considerable morbidity in the population and cost to the health service. In many countries "varicose veins are probably the commonest disorder presenting to general surgeons" and an average of 30% of district nursing time is estimated to be spent caring for patients with venous ulcers. For the patient with varicose veins or leg ulceration, there is often persistent discomfort and disability extending over long periods of time. Despite this, little epidemiological research has been carried out on venous disease, perhaps partly because of society's perception that venous disease is not a major problem and it is not normally a cause death. More recently however, efforts have been made to conduct structured epidemiological studies to identify risk factors and to clarify the geographical variations suggested in the past by anecdotal evidence. This article reviews recent epidemiological studies, discusses the prevalence of varicose veins and presents evidence for and against the differing theories of causation.

Adolescent↗
PubMed1993

Inhibition of varicella-zoster virus replication by an inhibitor of protein myristoylation.

Inhibitors of myristoylation and analogues of myristic acid inhibit the replication of some retroviruses including human immunodeficiency virus, but no studies with other virus families have been reported. We have shown that replication of varicella-zoster virus (VZV) in tissue is inhibited by DL-2-hydroxymyristic acid at concentrations similar to those required for inhibition with acyclovir. Protein synthesis is not inhibited, but protein myristoylation is non-specifically reduced. Despite this lack of specificity, DL-2-hydroxymyristic acid inhibits VZV replication without apparent cytotoxicity. This is in agreement with our earlier suggestion that non-specific inhibitors of myristoylation could have antiviral effects without toxicity to cells due to the stability of cellular myristoylproteins. This supports suggestions that myristoylation inhibitors have potential as antiviral drugs against the many viruses that produce myristoylproteins.

Acyclovir↗