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W H Langridge

Publications and source records attributed to W H Langridge.

At least 19 recordsLinked to original sources
PubMed2001

A plant-based multicomponent vaccine protects mice from enteric diseases.

Cholera toxin (CT) B and A2 subunit complementary DNAs (cDNAs) were fused to a rotavirus enterotoxin and enterotoxigenic Escherichia coli fimbrial antigen genes and transferred into potato. Immunoblot and enzyme-linked immunosorbent assay (ELISA) results indicated that the fusion antigens were synthesized in transformed tuber tissues and assembled into cholera holotoxin-like structures that retained enterocyte-binding affinity. Orally immunized mice generated detectable levels of serum and intestinal antibodies against the pathogen antigens. Elevated levels of interleukin 2 (IL2) and interferon gamma (INFgamma) detected in immunogen-challenged spleen cells from the immunized mice indicated the presence of a strong Th1 immune response to the three plant-synthesized antigens. This result was supported by flow cytometry analysis of immunized mouse spleen cells that showed a significant increase in CD4+ lymphocyte numbers. Diarrhea symptoms were reduced in severity and duration in passively immunized mouse neonates following rotavirus challenge. The results suggest that food plants can function as vaccines for simultaneous protection against infectious virus and bacterial diseases.

Animals↗
PubMed2000

Expression of full-length bioactive antimicrobial human lactoferrin in potato plants.

A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEK-DEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods. The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter. The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods. Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis. The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF. Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein. Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues. This is the first report of synthesis of full length, biologically active hLF in edible plants.

Agrobacterium tumefaciens↗
PubMed2000

Edible vaccines.

Explore the source record for details and available documents.

Autoimmune Diseases↗
PubMed1999

Improvements in human health through production of human milk proteins in transgenic food plants.

Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.

Adult↗
PubMed1999

Food plant-delivered cholera toxin B subunit for vaccination and immunotolerization.

Developments in recombinant DNA technology have enabled molecular biologists to introduce a variety of novel genes into plant species for specific purposes. From crop improvement to vaccine antigen and antibody production, plants are attractive bioreactors for production of recombinant proteins, as their eukaryotic nature often permits appropriate post-translational modification of recombinant proteins to retain native biological activity. The autotrophic growth of plants requires only soil minerals, water, nitrogen, sunlight energy and carbon dioxide for the synthesis of constituent proteins. Furthermore, production of biologically active proteins in food plants provides the advantage of direct delivery through consumption of edible transformed plant tissues. The production of cholera toxin B subunit in potato plants and applications for prevention of infectious and autoimmune disease are explained in this contribution.

Animals↗
PubMed1999

Antitumor effect of vaccinia virus in glioma model.

The ability of certain viruses to lyse cancer cells suggests that they may have potential as oncolytic agents. We investigated the effect of vaccinia virus (VV) and its recombinant derivatives (recVV2, rVV-p53) on growth of C6 rat glioma cells that form fast growing tumors in athymic nude mice. VV effectively infected C6 cells in vitro, inducing high level of foreign gene expression. Most of C6 cells infected in vitro with rVV-p53 expressing the tumor suppressor p53 protein showed apoptosis specific morphological changes in DAPI-stained nuclei and DNA fragmentation pattern on gel electrophoresis; infection with VV induced low level of cell apoptosis. In an ex vivo experiment, VV-infected C6 cells were implanted s.c. in athymic nude mice and tumor development was monitored. In contrast to the control PBS group, most of mice implanted with infected cells remained tumor free until the end of the observation period. In an in vivo experiment, injection of VV or rVV-p53 after the C6 cells had been implanted in nude mice induced effective inhibition of tumor growth in comparison with control PBS groups. The oncolytic effect was greater with rVV-p53, apparently due to overexpressed p53 and p53-mediated cell apoptosis. In study of virus virulence we did not observe disease symptoms in athymic mice infected with a high dose of VV. Experimental results indicate that vaccinia virus itself and vaccinia-mediated delivery of therapeutic genes represent novel potential strategies for tumor therapy.

Animals↗
PubMed1998

Molecular characterization of a stably transformed Bombyx mori cell line: identification of alternative transcriptional initiation sites of the A3 cytoplasmic actin gene.

We have isolated a stably transformed Bormbyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase, under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. By using this cell line we have identified alternative transcriptional initiation sites for the A3 actin gene. One of these start sites is located approximately 35 bp upstream from the previously determined transcription initiation site. The two mRNA start sites are utilized with a similar efficiencies in the BmN cell line. In addition, we detected transcripts that initiated in the first intron of the A3 actin gene. These transcripts may be synthesised under control of an alternative promoter. The stably transformed B. mori cell line used in this study was also extensively characterized. Integration of the plasmid molecules into the host genome was demonstrated by Southern and in situ hybridization analyses. Establishment and characterization of stably transformed insect cell lines, like the one described here, represents an important step in the development of nonlytic insect expression systems.

Acetyltransferases↗
PubMed1998

Efficacy of a food plant-based oral cholera toxin B subunit vaccine.

Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for GMI-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor GMI-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.

Animals↗
PubMed1998

A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes.

Oral administration of disease-specific autoantigens can prevent or delay the onset of autoimmune disease symptoms. We have generated transgenic potato plants that synthesize human insulin, a major insulin-dependent diabetes mellitus autoantigen, at levels up to 0.05% of total soluble protein. To direct delivery of plant-synthesized insulin to the gut-associated lymphoid tissues, insulin was linked to the C-terminus of the cholera toxin B subunit (CTB). Transgenic potato tubers produced 0.1% of total soluble protein as the pentameric CTB-insulin fusion, which retained GM1-ganglioside binding affinity and native antigenicity of both CTB and insulin. Nonobese diabetic mice fed transformed potato tuber tissues containing microgram amounts of the CTB-insulin fusion protein showed a substantial reduction in pancreatic islet inflammation (insulitis), and a delay in the progression of clinical diabetes. Feeding transgenic potato tissues producing insulin or CTB protein alone did not provide a significant reduction in insulitis or diabetic symptoms. The experimental results indicate that food plants are feasible production and delivery systems for immunotolerization against this T cell-mediated autoimmune disease.

Animals↗
PubMed1997

Expression of the human milk protein beta-casein in transgenic potato plants.

A 1177 bp cDNA fragment encoding the human milk protein beta-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase (mas1',2') promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human beta-casein cDNA. The presence of human beta-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human beta-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human beta-casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human beta-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as beta-casein. The above experiments demonstrate the expression of human milk beta-casein as part of an edible food plant. These findings open the way for reconstitution of human milk in edible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children.

Caseins↗
PubMed1997

Expression of cholera toxin B subunit oligomers in transgenic potato plants.

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.

Agrobacterium tumefaciens↗
PubMed1996

Detection of baculovirus gene expression in insect cells and larvae by low light video image analysis.

Recombinant baculovirus isolates BmNPVluc and AcNPVluc (Kopylova-Sviridova et al., 1990) expressing the luc gene in Bombyx mori N-4, and in Sf 9 and Trichoplusia ni 368 cells, respectively, were studied. Luc gene expression driven by baculovirus regulatory elements was detected by enzyme and photometric assays. The expression of recombinant AcNPVluc and BmNPVluc genes in infected larvae of the cabbage looper, T. ni and the tomato hornworm Manduca sexta was analyzed by low-light video image methods. Expression of the luc gene was detected at high levels in both the lepidopteran cells and in third to fifth instar T. ni larvae. However, no light emission was detected in M. sexta caterpillars. High levels of light emission were detected in T. ni larvae when occlusion bodies containing both wild type and recombinant virus were fed to larvae. The results of these experiments demonstrate that video image analysis can be used to monitor the progression of baculovirus infection in susceptible insect cells and larvae. Bioluminescence in recombinant virus infected larvae can be used to determine virus host range, to monitor latent virus infection in insect cells and to assess the spread of recombinant viruses in the environment. Video image analysis was found to be a sensitive method for rapid detection and semiquantitative measurement of luc gene expression in baculovirus infected cells and for monitoring virus infection in larval tissues.

Animals↗
PubMed1989

Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones.

Temporal and spacial distribution of mannopine synthase (mas) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase luxA and luxB as reporter genes. Luciferase activity was determined by luminometry in vitro and visualized by computer-enhanced single-photon video imaging in vivo. The activity of the mas dual promoters increased basipetally in developing plants and was wound-inducible in leaf and stem tissue. Hormone bioassays with isolated plant tissues and tumors deficient in the transferred DNA (T-DNA)-encoded genes iaaM, iaaH, and ipt indicated that activity of the mas dual promoters is regulated by auxin and enhanced by cytokinin in both differentiated and tumorous plant cells.

Journal Article↗
PubMed1987

Expression and assembly of functional bacterial luciferase in plants.

The luxA and luxB structural genes of Vibrio harveyi luciferase [alkanal,reduced FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] were introduced into a plant expression vector and transferred into tobacco and carrot cells by Agrobacterium-mediated or direct DNA transformation. Simultaneous expression of the luxA and luxB genes was monitored by protein immunoblot analysis. Luciferase-mediated light emission provided evidence for the assembly of the two protein subunits into a functional dimeric enzyme in plant protoplasts, in transformed calli, and in leaves of transformed plants. Bacterial luciferase may provide a useful marker-gene system for the quantitative assay of coordinate gene expression in transgenic plants.

Journal Article↗