Biomedical subjects

M Mackett

Publications and source records attributed to M Mackett.

At least 19 recordsLinked to original sources

PubMed2001

Transferrin receptor-mediated gene transfer to the corneal endothelium.

BACKGROUND: The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. METHODS: Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. RESULTS: Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eight-fold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. CONCLUSIONS: Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.

Animals↗

PubMed2001

Comparative analysis of the expression of the Epstein-Barr virus (EBV) anti-apoptotic gene BHRF1 in nasopharyngeal carcinoma and EBV-related lymphoid diseases.

Epstein-Barr virus (EBV) has been identified in a wide range of neoplastic and non-neoplastic disorders. The EBV open reading frame BHRF1 encodes a protein with partial sequence and functional homology to the anti-apoptotic onco-protein Bcl-2 and may therefore have a role in the proliferation of EBV positive cells. We have developed a rat monoclonal antibody against pBHRF1, which can detect BHRF1 in paraffin sections. While a number of mutant versions of BHRF1 were recognised, the monoclonal did not detect the BHRF1 homologue encoded by Herpesvirus papio or two mutants with deletions in the BH2 region. This novel rat monoclonal antibody (6A9) was used to examine tissue sections from 39 cases of non-keratinising undifferentiated nasopharyngeal carcinoma (NPC), 6 cases of metastatic NPC, 7 cases of EBV-positive NPC with squamous differentiation from Chinese patients, 15 cases of EBV-positive post-transplant lymphoproliferative disorder (PTLD), 6 EBV-containing lymphoblastoid cell lines, and 2 cases of oral hairy leukoplakia (OHL). In 11 cases of undifferentiated NPC, RT-PCR data were available for comparison with the immunohistochemistry. Both cases of OHL and two cases of LCL were positive for BHRF1 but none of the PTLD showed positive staining. All cases of undifferentiated NPC were positive for Bcl-2 but only one BHRF1 positive cell was identified in 1 of 39 cases of primary undifferentiated NPC. The 6A9 antibody produced less background staining and no nuclear positivity compared with the commercially available mouse monoclonal 5B11. It is concluded that BHRF1 can not be detected by immunohistochemistry in NPC and therefore it appears not to play a significant anti-apoptotic role in the progression of this EBV-associated tumour. The 6A9 monoclonal appears to be superior to 5B11 for the detection of pBHRF1 in tissue sections.

Amino Acid Sequence↗

PubMed2000

Heparin and heparan sulfate bind interleukin-10 and modulate its activity.

Glycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-beta, IL-7, and interferon-gamma. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin-agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, K(d), of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10-induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC(50) values of 100 to 500 microg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC(50) of 2,000 to 5,000 microg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10- induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10-mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity. (Blood. 2000;96:1879-1888)

Amino Acid Sequence↗

PubMed2000

Epidemiology of herpesvirus papio infection in a large captive baboon colony: similarities to Epstein-Barr virus infection in humans.

The epidemiology of herpesvirus papio, a lymphocryptovirus similar to Epstein-Barr virus (EBV), was studied in a captive colony of >1900 baboons. Herpesvirus papio IgG antibody titers were measured by IFA. In total, 438 specimens from 296 baboons were assessed, including 116 serial specimens from 52 juveniles and 6 infants studied monthly for 1 year following birth and at age 18 months. Maternally derived antibody reached a nadir at 4 months of age. About 75% of animals at 12 months of age and >95% of animals after age 24 months demonstrated serologic evidence of herpesvirus papio infection. After age 3 years, the geometric mean titer was 1:60-75. The epidemiology of herpesvirus papio infection in baboons closely parallels that of EBV infection in humans. An animal model of lymphocryptovirus infection will facilitate investigations of human lymphocryptovirus biology.

Animals↗

PubMed2000

Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.

There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin-2 and gamma-interferon were assayed using a co-culture of immune splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.

Acyltransferases↗

PubMed2000

Herpesvirus papio encodes a functional homologue of the Epstein-Barr virus apoptosis suppressor, BHRF1.

The human tumour virus Epstein-Barr virus (EBV) encodes a 17 kDa protein, BHRF1, which is a member of the BCL:-2 family and has been shown to suppress apoptosis. The role of this gene in the life-cycle of EBV has not been fully elucidated. In order to identify motifs conserved in herpesviruses and possibly shed light on its function we isolated a BHRF1 homologue from herpesvirus papio (cercopithecine herpesvirus-12) a closely related gammaherpesvirus of baboons. The gene, hvpBHRF1, also encodes a 17 kDa protein which shares 64% identity and 79% similarity with EBV BHRF1 at the amino acid level. In biological assays, hvpBHRF1 and BHRF1 conferred similar levels of protection on human keratinocytes induced to apoptose with cis-platin.

Amino Acid Sequence↗

PubMed1999

Protection against herpes B virus infection in rabbits with a recombinant vaccinia virus expressing glycoprotein D.

Herpes B virus infects naturally monkeys of the macaque genus in whom it can cause recurrent oral and genital lesions. However, when the virus infects humans it causes a neurological illness with a high case fatality rate. Successful treatment is possible but this depends on diagnosis prior to the onset of respiratory arrest, and fatalities over the last 10 years have been the result of late or no diagnostic data on which to base anti-viral intervention. An effective vaccine would be an ideal way to combat the risk of herpes B virus disease in humans working with potentially infected monkeys or their tissues. A recombinant vaccinia virus expressing herpes B virus glycoprotein D (gD) was constructed and rabbits inoculated with the chimeric virus were tested for immunoglobulin responses to herpes B virus by virus neutralisation, ELISA and Western blot analyses. Anti-gD humoral responses were detected in all vaccinated animals by ELISA and Western blot but neutralising antibody was not detected prior to challenge with herpes B virus. Non-vaccinated rabbits died within 8 days of challenge while 10/11 vaccinated animals were protected against herpes B virus disease. No antibodies to herpes B virus proteins other than gD were detectable in surviving animals, suggesting minimal herpes B virus replication post challenge. Autopsies were carried out on 4/10 rabbits which had remained healthy at 31 days post challenge and the dorsal root ganglia adjacent to the inoculation site were removed. Attempts to detect herpes B virus DNA by PCR followed by hybridisation proved negative suggesting protection against latent herpes B virus infection.

Animals↗

PubMed1999

Recombinant vaccinia viruses protect against Clostridium perfringens alpha-toxin.

Recombinant vaccinia viruses that expressed the nontoxic C-domain of Clostridium perfringens alpha-toxin were constructed. The J2R (thymidine kinase [TK] gene) and B13R (serpin 2 [SPI-2] gene) loci were used as insertion sites for the clostridial DNA, and expression of the foreign protein was measured in each case. A double recombinant that encoded the alpha-toxin truncate at the B13R locus and the protective antigen of Bacillus anthracis at the J2R locus was also constructed. Although differences in expression of the alpha-toxin C-domain were recorded, all of the vaccinia recombinants protected mice against a lethal challenge with alpha-toxin demonstrating that a recombinant vaccinia virus can be used to provide protection against a toxin challenge that is known to be solely antibody mediated.

Animals↗

PubMed1998

Immunization of common marmosets with Epstein-Barr virus (EBV) envelope glycoprotein gp340: effect on viral shedding following EBV challenge.

Epstein-Barr virus (EBV), the cause of infectious mononucleosis, is involved in the pathogenesis of several human cancers, the highest frequency of association being found in undifferentiated nasopharyngeal carcinoma and endemic Burkitt's lymphoma. The development of animal models in which potential vaccines can be tested is important. EBV infection of the common marmoset, using the M81 strain originally derived from a patient with nasopharyngeal carcinoma, induces a carrier state in this animal. Persistent infection is characterized by the production of antibodies to viral antigens, and the secretion of EBV DNA into buccal fluids. Following immunization with envelope glycoprotein gp340 derived from a bovine papilloma virus expression vector, prior to EBV infection, viral DNA was detected significantly less frequently in the buccal fluids of immunized, than of nonimmunized, infected animals, indicating that although the carrier state had not been abolished, it had been altered. A reduction in virus load was also observed when offspring of seronegative, and on occasion seropositive, parents were immunized neonatally, before EBV challenge.

Animals↗

PubMed1997

Preventive effect of IgG from EBV-seropositive donors on the development of human lympho-proliferative disease in SCID mice.

The effect of weekly treatments with various gammaglobulin preparations on the development of human B-cell tumors was studied in severe combined immunodeficient (SCID) mice. SCID mice were injected i.p. with human peripheral blood mononuclear cells (PBMCs) from an Epstein-Barr virus (EBV)-seropositive healthy blood donor. Repopulated SCID mice were divided into 7 treatment groups receiving either PBS, 2 commercial gammaglobulin preparations, purified IgG prepared from pooled plasma from EBV-seronegative or -seropositive blood donors, a rabbit anti-serum against EBV envelope glycoprotein gp340 or interferon (IFN)-alpha. All treatments started 1 day after injection of PBMC and continued for 8 weeks. In the PBS-treated control group, 85% of mice developed tumors in the abdominal cavity, mostly with liver metastasis within 150 days. Tumor formation was prevented by treatment with the 2 commercial gammaglobulin preparations as well as by purified IgG from EBV-seropositive donors. In contrast, purified IgG from EBV-seronegative donors, rabbit anti-gp340 anti-serum or IFN-alpha had no effect. Our results indicate that the effect of gammaglobulin is due to the presence of specific antibodies against EBV antigens. Further experiments showed that both the time of onset and the duration of treatment, as well as the dose of Ig, are important factors for prevention of tumor formation. Studies aiming at identification of target antigens for antibodies which prevent lymphoma development may be clinically relevant for prevention and possibly treatment of lympho-proliferative disease in severely immuno-compromised patients.

Abdominal Neoplasms↗

PubMed1997

BHRF1, a viral homologue of the Bcl-2 oncogene, is conserved at both the sequence and functional level in different Epstein-Barr virus isolates.

BHRF 1, a component of the restricted early antigen (EA) complex of the Epstein-Barr virus (EBV) lytic cycle, encodes a 17 kDa putative transmembrane protein with both sequence and functional homology to the Bcl-2 proto-oncogene. To determine whether there was any sequence variation over the BHRF1 open reading frame (ORF), 15 EBV isolates from different geographical regions and from both healthy donors and patients with EBV-associated diseases were sequenced. A small number of base changes which resulted in amino acid substitutions in the BHRF1 protein were found relative to the prototype B95.8 EBV sequence and these were predominantly clustered near the amino terminus of the BHRF1 protein outside conserved domains identified in the Bcl-2 homologues. In transient transfection assays none of the mutations in the BHRF1 ORF from eight different EBV isolates had a significant effect on BHRF1 protein localization compared to the B95.8 BHRF1 protein. However, transient expression of the adenovirus 12 19K protein or Bcl-2 resulted in localization patterns distinct from that observed with BHRF1 protein. Whilst all eight EBV isolates and E1B-19K gave comparable levels of protection to the DNA-damaging agent cis-platin, Bcl-2 did not afford significant protection. Thus, despite several amino acid changes in the BHRF1 ORF of some of the EBV isolates studied, the ability of the protein to protect against cis-platin induced apoptosis is conserved. The highly conserved nature of BHRF1 amongst different EBV isolates at both the sequence and functional level supports the proposed important role of BHRF1 in delaying cell death, thereby maximizing the production of progeny virus and facilitating the establishment of virus persistence.

Cloning, Molecular↗

PubMed1997

Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.

Animals↗

PubMed1997

The processing, transport and heterologous expression of Epstein-Barr virus gp110.

Epstein-Barr virus (EBV) glycoprotein gp110 has substantial structural and sequence homology with herpes simplex virus (HSV) gB and gBs of other alpha- and betaherpesviruses but unlike HSV gB localizes differently in infected cells and is absent from virions. To facilitate the analysis of EBV gp110, antisera were raised to fragments of gp110 expressed in a bacterial system. They recognized a protein of the predicted size in recombinant bacterial lysates, in lymphoblastoid cells and in recombinant vaccinia virus-gp110 infected cells. gp110 from all sources possessed a high-mannose type of N-glycosylation implying that gp110 has not passed through the Golgi. Immunofluorescence and immuno-electron microscopy confirmed this conclusion and demonstrated that, in contrast to HSV gB, the majority of immunoreactive gp110 was present at the nuclear membrane or endoplasmic reticulum (ER) but not at the cell membrane. Unexpectedly, a truncated version of gp110 lacking the hydrophobic C-terminal region, despite forming dimers analogous to HSV dimers, was transported in a similar manner to full-length gp110. Two chimeric proteins constructed by replacing the N- and C-terminal domains of gp110 with corresponding regions of gp340/220 were also transported to the nuclear membrane/ER. These data suggest that unlike HSV gB both the N- and C-terminal portions of EBV gp110 contain independent signals sufficient to direct the molecule to the ER/nuclear membrane. Specific transport of gammaherpesvirus gB homologues to the nuclear membrane, from where herpesviruses bud, suggests that they may be involved in the egress of virus from the nucleus.

Biological Transport↗

PubMed1996

Env-specific CTL predominate in cats protected from feline immunodeficiency virus infection by vaccination.

Animal models of HIV-1 have a key role to play in elucidation of the cellular mechanisms responsible for protective immunity. Vaccination of domestic cats with whole inactivated feline immunodeficiency virus (FIV) elicits virus-neutralizing Abs and virus-specific CTL in the peripheral blood and lymphoid organs and affords protection from homologous virus challenge. In the present study we confirm the induction of virus-specific CTL following immunization with whole inactivated FIV vaccine and demonstrate that cats are protected for up to 1 yr following vaccination. Long term protection in vaccinated cats correlates with both higher levels of FIV Env-specific CTL in the peripheral blood following vaccination and the presence of FIV Env-specific memory CD8+ CTL in the lymph nodes, which persist for up to 1 yr following challenge in the absence of detectable virus. The CTL responses observed in vaccinated protected cats differ qualitatively from those in FIV-infected cats. The latter cats either do not generate a memory CTL response or exhibit a Gag-specific memory CTL response. These results show that the protective immunity observed in whole inactivated virus-vaccinated cats is associated with the induction of high levels of Env-specific CTL activity.

Animals↗

PubMed1996

Murine gammaherpesvirus-68 encodes homologues of thymidine kinase and glycoprotein H: sequence, expression, and characterization of pyrimidine kinase activity.

We have sequenced a 4.5-kb fragment of DNA spanning the junction of the BamHI D and E fragments of murine gammaherpesvirus-68 (MHV-68). This sequence was found to code for two major open reading frames (orfs) of 1934 and 2192 bp which showed significant homology to the thymidine kinase (TK) and glycoprotein H (gH) sequences of other gammaherpesviruses. Upstream from the TK gene another orf was found which showed amino acid sequence homology to the HSV1 UL24 gene. Analysis of the 1934-bp orf revealed the presence of all six of the recognized sites that are conserved between herpesvirus TKs although, uniquely among sequenced herpesvirus TK enzymes, MHV-68 lacks the consensus nucleotide binding site GXXGXGK, the second glycine being replaced by alanine. The MHV-68 TK has a predicted M(r) of 68,443, while the gH is predicted to have a M(r) of 82,890. Northern blot analysis showed an early TK message of 2.6 kb and a late gH-specific message of 2.5 kb. Both TK and gH probes detected a 4.3-kb late message, implying that this late message spans gH and TK. The TK coding sequence was expressed using an in vitro transcription translation system and was shown to encode functional TK activity.

Amino Acid Sequence↗

PubMed1996

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Amino Acid Sequence↗

PubMed1996

Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.

Amino Acid Sequence↗

PubMed1995

The Epstein-Barr virus encoded cytokine viral interleukin-10 enhances transformation of human B lymphocytes.

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in their growth transformation and establishment of immortalised lymphoblastoid cell lines. The virus was recently found to encode a homologue of the pleitropic cytokine interleukin-10 (IL-10), which has wide ranging effects on the immune system. We have investigated the effect of this virally encoded growth factor on the ability of EBV to immortalize B lymphocytes from tonsils and from adult and neonatal blood. Recombinant viral interleukin-10 (vIL-10) was found to increase dramatically the growth transformation of B cells from all three populations infected with either the highly transforming type 1 strain B95-8 or the less efficient type 2 strain BL16. This striking enhancement of transforming ability in the presence of viral IL-10 may be in part due to increased viability of the B cells during infection and decreased levels of interferon-gamma, a cytokine known to inhibit EBV transformation. Thus viral IL-10 influences a number of cell types of the immune system to allow the enhanced outgrowth of EBV transformed cells.

Adult↗