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Biomedical subjects

D O'Hagan

Publications and source records attributed to D O'Hagan.

At least 19 recordsLinked to original sources
PubMed2001

Human immunodeficiency virus type 1 Gag-specific vaginal immunity and protection after local immunizations with sindbis virus-based replicon particles.

The majority of human immunodeficiency virus (HIV) infections occur through vaginal and rectal transmission. In seeking a safe, nonreplicating gene-delivery vector that can induce mucosal and systemic immune responses and protection, Sindbis virus-based replicon particles expressing HIV-1 Gag (SIN-Gag) were developed. In mice, after nasal or intramuscular immunization with SIN-Gag and vaginal challenge with vaccinia virus (VV) expressing HIV-1 Gag (VV-Gag), CD8(+) T cell-mediated responses were detected locally, in the vaginal mucosa and in the draining iliac lymph nodes (ILNs), and systemically, in the spleen. However, the mice were not protected against VV-Gag replication in the ovaries. In contrast, after vaginal or rectal immunization with SIN-Gag and vaginal challenge with VV-Gag, despite lower local CD8(+) T cell-mediated responses in the vaginal mucosa and ILNs, the mice were protected against VV-Gag replication in the ovaries. Therefore, local immunization with SIN-Gag induced both local mucosal cell-mediated responses and protection.

AIDS Vaccines↗
PubMed2001

Mucosal immunization with HIV-1 gag DNA on cationic microparticles prolongs gene expression and enhances local and systemic immunity.

There is an urgent need to develop vaccines against transmission of HIV through the vaginal and rectal mucosa. In the present study we tested the ability of DNA encoding HIV-1 gag adsorbed onto the surface of cationic polylactide co-glycolide microparticles (PLG-DNA) to induce local and systemic gag-specific immunity following mucosal delivery. We found gag-specific cell- and antibody-mediated responses in local as well as systemic lymphoid tissues following intranasal (IN) immunizations with PLG-DNA but not with naked DNA. IN immunizations with PLG-DNA, but not naked DNA, induced prolonged expression of gag protein in local and systemic lymphoid tissues. These data have important implications for DNA vaccine development.

AIDS Vaccines↗
PubMed2001

Controlled release of recombinant insulin-like growth factor from a novel formulation of polylactide-co-glycolide microparticles.

The purpose of the current study was to develop a controlled-release delivery system for recombinant insulin-like growth factor (rhIGF-I). Polylactide-co-glycolide (PLG) microparticles with entrapped rhIGF-I were prepared by a novel emulsion based solvent evaporation process. Microparticles with two loading levels of rhIGF-I were prepared (4 and 20% w/w). The integrity of released rhIGF-I was characterized by RP-HPLC, SDS-PAGE and a bioactivity assay. In vitro and in vivo release profiles of rhIGF-I from these microparticles were also evaluated. Reproducible batches of microparticles with 4% and 20% w/w loading of rhIGF-I were prepared, with excellent encapsulation efficiency (81 and 85% of total protein respectively entrapped). The protein retained integrity after the microencapsulation process as evaluated by RP-HPLC, SDS-PAGE and bioactivity assay. The in vitro profiles exhibited a significant burst release of rhIGF-I (20-30%), followed by controlled release of protein for up to 28 days. A similar level of burst release was observed in vivo, followed by controlled release of protein for 14-18 days. In addition, there was a surprisingly close correlation between in vitro and in vivo release rates. PLG microparticles with entrapped rhIGF-I are a promising delivery system which may allow rhIGF-I to be used for a broad range of therapeutic indications.

Animals↗
PubMed2001

Isolation of an aldehyde dehydrogenase involved in the oxidation of fluoroacetaldehyde to fluoroacetate in Streptomyces cattleya.

Streptomyces cattleya is unusual in that it produces fluoroacetate and 4-fluorothreonine as secondary metabolites. We now report the isolation of an NAD(+)-dependent fluoroacetaldehyde dehydrogenase from S. cattleya that mediates the oxidation of fluoroacetaldehyde to fluoroacetate. This is the first enzyme to be identified that is directly involved in fluorometabolite biosynthesis. Production of the enzyme begins in late exponential growth and continues into the stationary phase. Measurement of kinetic parameters shows that the enzyme has a high affinity for fluoroacetaldehyde and glycoaldehyde, but not acetaldehyde.

Acetaldehyde↗
PubMed2001

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines.

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

AIDS Vaccines↗
PubMed2000

Cationic microparticles: A potent delivery system for DNA vaccines.

An approach involving the preparation of biodegradable microparticles with a cationic surface was developed to improve the delivery of adsorbed DNA into antigen-presenting cells after i.m. injection. The microparticles released intact and functional DNA over 2 weeks in vitro. In addition, the microparticles induced higher levels of marker gene expression in vivo. After i.m. immunization, the microparticles induced significantly enhanced serum antibody responses in comparison to naked DNA. Moreover, the level of antibodies induced by the microparticles was significantly enhanced by the addition of a vaccine adjuvant, aluminum phosphate. In addition, in contrast to naked DNA, the cationic microparticles induced potent cytotoxic T lymphocyte responses at a low dose.

Animals↗
PubMed2000

Biosynthetic studies on the tropane ring system of the tropane alkaloids from Datura stramonium.

Isotopic labelling experiments have been carried out in Datura stramonium root cultures with the following isotopically labelled precursors; [2H3]- [2-13C, 2H3]-, [1-13C, 18O2]-acetates, 2H2O, [2H3-methyl]-methionine, [2-13C]-phenyllactate, [3-2H]-tropine and [2'-13C, 3-2H]-littorine. The study explored the incorporation of isotope into the tropane ring system of littorine 1 and hyoscyamine 2 and revealed that deuterium from acetate is incorporated only into C-6 and C-7, and not into C-2 and C-4 as previously reported. Oxygen-18 was not retained at a detectable level into the C(3)-O bond from [1-13C, 18O2]-acetate. The intramolecular nature of the rearrangement of littorine 1 to hyoscyamine 2 is revealed again by a labelling study using [2'-13C, 3-2H]-littorine, [2-13C]-phenyllactate and [3-2H]-tropine.

Alkaloids↗
PubMed2000

Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells.

Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescent-labeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of the fluorescence was concentrated in endosomal compartments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was subsequently processed and presented, resulting in stimulation of an H-2kd-restricted, gag-specific T cell hybridoma. Activation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1-20 microg DNA (10-2000 microg PLG) and was sustained up to 5 days after transfection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells. Gene Therapy (2000) 7, 2105-2112.

Adsorption↗
PubMed2000

Genetically detoxified mutants of heat-labile enterotoxin from Escherichia coli are effective adjuvants for induction of cytotoxic T-cell responses against HIV-1 gag-p55.

There is an urgent need for prophylactic and therapeutic vaccines against human immunodeficiency virus (HIV). Mucosal immunization strategies have great potential to elicit both mucosal and systemic cellular immunity required to protect against HIV-induced acquired immune deficiency syndrome (AIDS). However, mucosal immunizations with soluble protein antigens generally require adjuvants. In this study, we tested two mutants of the heat-labile enterotoxin (LT) from Escherichia coli, LTK63: with no measurable ADP-ribosyltransferase activity, and LTR72: with residual ADP-ribosyltransferase activity, as mucosal adjuvants for induction of cytotoxic T lymphocyte (CTL) responses to coadministered HIV gag p55 protein. We found that intranasal (i.n.) immunizations with HIV gag p55 protein coadministered with LTK63 or LTR72 induced systemic CTL responses comparable to that obtained following intramuscular (i. m.) immunizations with the same adjuvants. Moreover, oral coadministration of LTR72, but not LTK63, resulted in local as well as systemic p55-specific CTL responses in mesenteric lymph nodes (MLN) and spleens (SP) of the immunized mice. These data have important implications for current efforts to develop a safe vaccine against HIV.

AIDS Vaccines↗
PubMed2000

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins, subunits, and unrelated adjuvants.

We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 microg. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADP-ribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1beta [IL-1beta] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.

Adenosine Diphosphate Ribose↗
PubMed1999

Intranasal immunization with recombinant gD2 reduces disease severity and mortality following genital challenge with herpes simplex virus type 2 in guinea pigs.

The ability of a genetically detoxified mutant of heat labile enterotoxin (LTK63) to act as a mucosal adjuvant following intranasal immunization with recombinant gD2 has previously been reported in mice [Ugozzoli M, O'Hagan DT, Ott GS. Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses. Immunol 1998;93:563-571.]. In the current studies, these observations were extended to the guinea pig model. Immunized guinea pigs were subsequently challenged intravaginally with HSV-2. Intranasal immunization with gD2 and LTK63 induced a significant reduction in disease severity and a reduction in mortality. However, only intramuscular immunization with a potent adjuvant (MF59) induced protection against the incidence of disease.

Administration, Intranasal↗
PubMed1999

Advances in vaccine adjuvants.

Currently, aluminum salts and MF59 are the only vaccine adjuvants approved for human use. With the development of new-generation vaccines (including recombinant subunit and mucosal vaccines) that are less immunogenic, the search for more potent vaccine adjuvants has intensified. Of the novel compounds recently evaluated in human trials, immunostimulatory molecules such as the lipopolysaccharide derived MPL and the saponin derivative QS21 appear most promising, although doubts have been raised as to their safety in humans. Preclinical work with particulate adjuvants, such as the MF59 microemulsion and lipid-particle immune-stimulating complexes (Iscoms), suggest that these molecules are also potent elicitors of humoral and cellular immune responses. In addition, preclinical data on CpG oligonucleotides appear to be encouraging, particularly with respect to their ability to selectively manipulate immune responses. While all these adjuvants show promise, further work is needed to better define the mechanisms of adjuvant action. Ultimately, the development of more potent adjuvants may allow vaccines to be used as therapeutic, rather than prophylactic, agents.

Adjuvants, Immunologic↗
PubMed1999

Effects of lasalocid on performance of lactating dairy cows.

Sixty lactating dairy cows (30 multiparous and 30 primiparous) were used in a completely randomized block design to determine the effect of lasalocid supplementation on dairy cow performance. Starting wk 2 prepartum and lasting through wk 17 of lactation, cows received one of three experimental diets. The experimental total mixed rations were control (CD), control + 10 mg/kg of lasalocid (CD + 10) and control + 20 mg/kg of lasalocid (CD + 20). The alfalfa-based control diet (40:60; forage:concentrate) was formulated to contain 18% crude protein, 35% nonstructural carbohydrates, 31% neutral detergent fiber, and 6.6% ether extract. Lasalocid supplementation linearly decreased dry matter intake (DMI) without affecting milk production or milk composition. Mean milk production and percentages of fat and protein were 30.0, 30.8, and 28.6; 3.56, 3.51, and 3.63; 3.06, 3.05, and 3.09; respectively for treatments CD, CD + 10, and CD + 20. Lasalocid supplementation decreased milk urea N (MUN) when compared to control cows, and increasing supplementation caused a significant linear decrease in MUN. For the primiparous cows, lasalocid supplementation decreased DMI and MUN while increasing body condition score and feed efficiency. Results from this study indicate that lactating dairy cows and, in particular, primiparous cows may benefit from lasalocid supplementation in terms of more efficient utilization of nutrients for milk production, reduced MUN levels, reduced body condition loss, and higher margin over feed cost.

Animal Feed↗
PubMed1998

The preparation and characterization of polymeric antigen delivery systems for oral administration.

Although polymeric delivery systems are well established for the oral administration of conventional drugs, they have not yet been commercially developed for vaccine delivery. The problems inherent with the oral route of delivery, including low pH, gastric enzymes, rapid transit and poor absorption of large molecules, has made the goal of oral delivery of antigens very challenging. Nevertheless, several polymeric delivery systems for the oral administration of vaccines are currently being evaluated, including microencapsulation in poly(lactide-co-glycolides), alginates, polyanhydrides, starch, polymethacrylates, polyamino acids and enteric coating polymers. These approaches are designed to protect the antigen in the gut, to target the antigen to the gut-associated lymphoid tissue, or to increase the residence time of the antigen in the gut through bioadhesion. Each of these approaches is discussed in relation to antigen encapsulation and integrity, process reproducibility, ease of preparation and encapsulation efficiency. Potential problems associated with the scale-up of these approaches are also briefly addressed. Of particular relevance are the prospects for the application of these formulation processes for commercial development.

Journal Article↗
PubMed1998

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells.

Recombinant adenovirus (AVR) promises to be an efficient vector in gene therapy for neuromuscular diseases, but in preclinical experiments the expression of therapeutic genes is shorter lived in immunocompetent animals than in immunocompromised hosts. Interferons (IFN), which are known to have a role both in early antiviral activity and in late cytotoxic immunoreaction against the virus or transduced cells, may influence the efficiency of gene transfer. In this study we investigated the role of IFNs in determining the efficiency of gene transfer by AVR. AVRs expressing beta-galactosidase (beta-gal) from either a cytomegalovirus (CMV) or a troponin-I promoter were used. Muscle cells were infected by AVR after exposure to various IFNs. The alphaIFN treatment significantly reduced (up to fivefold) the CMV promoter-driven gene expression in muscle cells in vitro and in immature muscles in vivo, while the least effective inhibitor was betaIFN. The decrease in gene expression by IFNs was more pronounced with the CMV-driven transgene than troponin-I promoter-driven one and was due to a decrease in transcript level. Intrinsic IFNs that are triggered by AVR administration can decrease the efficiency of gene transfer in muscle cells. Therefore the use of muscle specific promoters in AVR and/or IFN inhibitory agents will likely improve the prospects of effective gene therapy by AVR.

3T3 Cells↗