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Biomedical subjects

M Q Wei

Publications and source records attributed to M Q Wei.

16 recordsLinked to original sources
PubMed2001

Lentiviral vector-mediated tyrosinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR' CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4+ and CD8+ cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

Animals↗
PubMed2001

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle.

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Bali cattle (Bos javanicus), which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5'- and 3'-long terminal repeats (LTRs), 0.4kb of truncated gag and 1.1kb of 3'-env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences, including gag, pol, vif, tat and rev, but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped, disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2)x10(6)CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as well. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus.

Animals↗
PubMed2001

Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency.

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.

Animals↗
PubMed2001

A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors.

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50,000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10,000 g for 2 h at 4 degrees C. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3,000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10,000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

Animals↗
PubMed1999

Antiapoptotic marker Bcl-X(L), expression on Reed-Sternberg cells of Hodgkin's disease using a novel monoclonal marker, YTH-2H12.

Inhibitors of apoptosis may regulate tissue differentiation and promote cell survival in neoplasia. A new apoptosis inhibitor of the bcl-2 gene family, bcl-X(L), was recently found in some types human neoplasia but not in normal tissue. We investigated bcl-X(L) expression in 419 cases of normal and neoplastic lymphoid lesions using immunohistochemistry with the monoclonal antibody bcl-X(L) (YTH-2H12). Ninety-four percent (141/150) of classic Hodgkin's disease (HD) were positive for bcl-X(L) with strong intensity in most Reed-Sternberg (RS) cells. Forty-eight percent (38/80) of nodular lymphocyte predominance (LPHD) were positive. In the non-Hodgkin's lymphomas (NHL), bcl-X(L) was expressed in a low percentage of cases (< 20%), with the exception of follicle center lymphoma, grade III/III (78%). All reactive hyperplastic lesions were negative for bcl-X(L). RS cells, which expressed bcl-X(L), were not labeled by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found RS cells expressing bcl-X(L) were absent of DNA fragmentation (apoptosis). Our data provide evidence that bcl-X(L) is abnormally expressed in the RS cells of HD and some types of NHL raising speculation that inhibition of apoptosis may be important in the pathogenesis of lymphoma, specifically HD. In addition, the previously reported correlation between bcl-X(L) and Epstein-Barr virus expression in HD was not supported by this study.

Animals↗
PubMed1999

Sustained gene expression in transplanted skin fibroblasts in rats.

Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted. We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.

Adenosine Deaminase↗
PubMed1999

Silicone gel-filled breast and testicular implant capsules: a histologic and immunophenotypic study.

The immunophenotypic characteristics of silicone gel-filled breast and testicular implant capsules have not been well described. Therefore, we studied 17 paraffin-embedded tissue sections from 9 breast implant patients and 1 testicular implant patient to assess the type and extent of inflammatory responses present. Immunohistochemical analyses were performed on paraffin-embedded tissue sections for expression of CD20, CD45RO, betaF1, CD68, CD44, kappa and A immunoglobulin light chains, and bcl-XL (a member of the bcl-2 family of proteins involved in apoptosis). The most common histologic features included prominent T-cell and foamy macrophage reactions with foreign body giant cells and granulomas in a dense fibrovascular connective tissue. Foci of polyclonal plasma cells and acute inflammatory cells were variably present. In one case, there was reactive germinal center formation, a novel finding. A "pseudosynovium" at the implant capsule interface was present in the majority of cases as previously described; it showed reactivity with CD68. Thin strands of highly refractile, nonpolarizable material, consistent with silicone, were regularly noted in intra- and extracellular locations. The immunohistochemical results included reactivity of the majority of lymphocytes with CD45RO and/or betaF1 (confirming an anamnestic reactive T-cell phenotype), and reactivity of the macrophages, giant cells, and "pseudosynovium" with the macrophage/histiocyte marker, CD68. The reactive germinal centers were positive for CD20. Reactivity for CD44, an activation and intracellular adhesion marker, was frequently observed in the foamy macrophages and foreign body giant cells and has not been previously reported. The plasma cells demonstrated polyclonal immunoglobulin light-chain reactivity, consistent with a reactive process. These findings suggest that silicone implants induce chronic inflammatory responses in many adjacent capsules, which consist of anamnestically responding T cells, reactive B-lymphocytes, and macrophages.

Adult↗
PubMed1996

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.

3T3 Cells↗
PubMed1992

Typing of methicillin-resistant Staphylococcus aureus with IS256.

A simple one-step procedure is described for specifically amplifying and labelling insertion element IS256 which is associated with the gentamicin-resistance transposon Tn4001. The product has been used to probe DNA digests of methicillin-resistant Staphylococcus aureus. The resulting restriction fragment length polymorphisms were found to be able to distinguish isolates which were indistinguishable by other typing methods. The probe also hybridised with methicillin-resistant Staphylococcus aureus which were isolated before the emergence of gentamicin resistance, demonstrating its usefulness in typing species other than those that are gentamicin-resistant.

Bacterial Typing Techniques↗
PubMed1992

Conjugative trimethoprim resistance in Staphylococcus aureus.

A multiply resistant Staphylococcus aureus isolate, WBG7410, harbours plasmids of 38, 26, 2.8, 2.4 and 1.9 kb and transfers trimethoprim and kanamycin resistance at high frequencies by conjugation. The transconjugants contained the 38-kb plasmid, pWBG707, and the 2.8-kb plasmid. Plasmid pWBG707 was shown to encode trimethoprim resistance, was conjugative and mobilised at high frequencies the 2.8-kb plasmid which presumably encodes kanamycin resistance. Plasmid pWBG707 was isolated mostly in the open circular form and analysis with EcoRI restriction endonuclease suggests that pWBG707 is a new conjugative plasmid distinct from the other conjugative plasmids reported in S. aureus.

Conjugation, Genetic↗
PubMed1992

Typing of methicillin-resistant Staphylococcus aureus with an M13 repeat probe.

A bacteriophage M13 tandem repeat has been used to probe EcoRI digested genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA). The patterns generated were found to be useful in typing MRSA and generally confirmed the relationships that had previously been recognized in other studies based on antimicrobial resistance and plasmid profiles. The epidemic MRSA of London hospitals (EMRSA) and the majority of the epidemic MRSA of eastern Australian hospitals (EA MRSA) gave the same pattern. However, two isolates previously classified as EA MRSA gave a different pattern and a third another pattern. One isolate from Dublin, two isolates from Nuneaton and two isolates from Singapore gave the same pattern as the two EA MRSA. With the exception of the early or classic MRSA all the other isolates examined gave their own distinctive patterns. With one exception the classic MRSA belonged to a separate group. The exception was of particular interest because it gave the same pattern as the majority of the EA MRSA. This suggests that there may be an evolutionary relationship between some of the classic MRSA and the EMRSA of London and the EA MRSA of Australia.

Bacterial Typing Techniques↗
PubMed1992

Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus.

Strains of methicillin-resistant Staphylococcus aureus (MRSA) from Australia and the UK were compared by digesting their chromosomal DNA with the low-frequency-cutting restriction enzyme SmaI and separating the restriction fragment length polymorphisms (RFLPs) by contour-clamped homogeneous electric field (CHEF) electrophoresis. The numbers of restriction fragments produced were in the range 14-17 and the sizes of the bands were 7-700 kb. Generally, the results confirmed previous conclusions based on antimicrobial resistance and plasmid profiles. The earlier MRSA isolates were different from more recent isolates, and the epidemic MRSA from eastern Australia (EA MRSA) was the same as the epidemic MRSA (EMRSA) found in London hospitals. However, contrary to previous results, the EA MRSA did not constitute a homogeneous group. The results showed that comparison of RFLPs by CHEF electrophoresis is a useful technique for studying the epidemiology of MRSA.

Australia↗
PubMed1992

Typing of Australian methicillin-resistant Staphylococcus aureus strains by pulsed field gel electrophoresis.

Twenty-six clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected from six Australian hospitals by a National Staphylococcal Study Group were examined by analysis of restriction fragment length polymorphisms (RFLPs) of chromosomal DNA with pulsed field gel electrophoresis. Digestion with the restriction endonuclease SmaI produced 13-17 bands of 7-700 kb. The digestion patterns were easily distinguished and isolates could be classified into 17 groups based on their RFLPs. Isolates giving a pattern associated with one group were from four hospitals in four different states. In another group, the isolates responsible were from three hospitals in two states and in a further group, the isolates were derived from two hospitals in different states. The remaining groups comprised only one member each. The method has promise for typing and studying the epidemiology of MRSA.

Australia↗
PubMedDate not supplied

Novel bovine lentiviral vectors based on Jembrana disease virus.

BACKGROUND: Safety is a concern that must be addressed prior to any clinical use of human immunodeficiency virus (HIV)-based lentiviral vectors in human patients. Unfortunately, efforts to examine the biosafety of the vectors in preclinical animal models are hampered due to the lack of animal models for HIV infection. We have developed new lentiviral vectors based on the recently characterised Jembrana Disease Virus (JDV), which infects a specific species of cattle naturally in Bali, Indonesia. METHODS: Sequences from the JDV genome were amplified by splicing overlap extension polymerase chain reaction (PCR) for the construction of transfer vectors as well as a packaging construct. Co-transfection of these two plasmids into 293T cells with a third encoding a G glycoprotein of vesicular stomatitis virus produced pseudotyped, disabled, replication defective JDV vector particles. Viral titre was obtained by transducing the cells with the supernatant harvested from transfectants and determining the number of cells expressing the transgene. PCR and Southern blotting were used to detect the presence of potential replication-competent viruses as well as transgene integration. RESULTS: Bicistronic JDV vectors encoding the green fluorescent protein (GFP) and the neomycin phosphotransferase were harvested with a titre range of 0.4-1.2 x 10(6) colony forming units/ml from vector-producing cells and were further concentrated by ultracentrifugation to the high titre of approximately 10(7) CFU/ml. Vectors encoding GFP were shown to transduce and integrate efficiently into the chromosomes of a range of primary and transformed cells of different origins in different differentiation status, including growth-arrested cells, with an efficiency of 25-75%. Exhaustive testing with a marker gene transfer assay in combination with a reverse transcriptase assay and PCR amplification of samples of serially passaged, transduced cells showed that no detectable amount of replication competent lentivirus (RCL) was produced. CONCLUSIONS: We showed the feasibility of the development of gene transfer vectors based on a non-primate bovine lentivirus, which will provide the opportunity for examination of the efficacy and biosafety of lentiviral vector-mediated gene transfer in vivo in animal models. JDV-based vectors may be applicable and more readily acceptable than those from HIV for human gene therapy.

Animals↗
PubMedDate not supplied

VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the level of ganciclovir needed to cause suicide cell death.

BACKGROUND: The inefficiency of herpes simplex virus thymidine kinase (TK) gene transfer and toxicity of ganciclovir (GCV) at high concentrations in vivo limits the use of this suicide gene therapy approach for the treatment of cancers in clinical settings. To overcome the problem, we have sought evidence of amplification of cytotoxicity by co-transfer of the TK gene fused with the gene encoding HSV-1 structural protein VP22 which has a remarkable ability for intercellular trafficking. METHODS: The expression of the fusion proteins from the chimeric VP22-TK or VP22-EGFP genes was shown by Western blot and VP22 promoted TK or EGFP intercellular trafficking by an indirect immunofluorescent assay. The cytotoxicity was demonstrated by a colorimetric cell proliferation assay followed by an assessment of the bystander effect on admixtures of transfected with non-transfected naive cells. RESULTS: Our results show the expression of the VP22 fusion proteins and their spread to varying numbers of bystander cells (up to 30, observed in viable cells with VP22-EGFP as well as after methanol fixation), confirming that VP22 assisted intercellular trafficking of the fusion proteins. This VP22 promoted TK spreading resulted in killing by 2.5 microg/ml GCV of virtually all cells in cultures that had been transfected at an efficiency of only 27.5%. In contrast, fewer than 80% of cells were killed when transfected with 'tk alone' at the same efficiency. The cell killing effect was exponentially dependent on GCV concentration in cells transfected with 'tk alone' at GCV concentrations between 0.25 and 0.5 microg/ml, but not those transfected with VP22-TK, probably due to the continuously variable, high sensitivity of about 50% of cells. Even at low concentration of GCV (0.2 microg/ml), the enhancement of cell killing by VP22 was four-fold higher in cells transfected with VP22-TK than in cells transfected with 'tk alone'. CONCLUSIONS: VP22 enhanced intercellular trafficking of TK and amplified the TK/GCV killing effect, especially in the lower range of GCV concentrations. This offers a new strategy to enhance the effectiveness of suicide gene therapy for the treatment of cancers.

Animals↗