School of Biomedical Sciences - Research Publications
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ItemNo Preview AvailableLipopeptide vaccines illustrate the potential role of subtype-crossreactive T cells in the control of highly virulent influenza(WILEY, 2009-07)BACKGROUND: The best form of protection against influenza is high-titred virus-neutralizing antibody specific for the challenge strain. However, this is not always possible to achieve by vaccination due to the need for predicting the emerging virus, whether it be a drift variant of existing human endemic influenza type A subtypes or the next pandemic virus, for incorporation into the vaccine. By activating additional arms of the immune system to provide heterosubtypic immunity, that is immunity active against all viruses of type A influenza regardless of subtype or strain, it should be possible to provide significant benefit in situations where appropriate antibody responses are not achieved. Although current inactivated vaccines are unable to induce heterosubtypic CD8(+) T cell immunity, we have shown that lipopeptides are particularly efficient in this regard. OBJECTIVES: To examine the role of vaccine-induced CD8(+) T cells in altering the course of disease due to highly virulent H1N1 influenza virus in the mouse model. METHODS: The induction of influenza-specific CD8(+) T cells following intranasal inoculation with lipopeptide vaccine was assessed by intracellular cytokine staining (ICS) and the capacity of these cells to reduce viral loads in the lungs and to protect against death after viral challenge was determined. RESULTS AND CONCLUSIONS: We show that CD8(+) T cells are induced by a single intranasal vaccination with lipopeptide, they remain at substantial levels in the lungs and are efficiently boosted upon challenge with virulent virus to provide late control of pulmonary viral loads. Vaccinated mice are not only protected from death but remain active, indicative of less severe disease despite significant weight loss.
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ItemA method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry.(Springer Science and Business Media LLC, 2012-08-20)BACKGROUND: Pulmonary load of Legionella pneumophila in mice is normally determined by counting serial dilutions of bacterial colony forming units (CFU) on agar plates. This process is often tedious and time consuming. We describe a novel, rapid and versatile flow cytometric method that detects bacteria phagocytosed by neutrophils. FINDINGS: Mice were infected with L. pneumophila via intratracheal or intranasal administration. At various times after bacteria inoculation, mouse lungs were harvested and analysed concurrently for bacterial load by colony counting and flow cytometry analysis. The number of L. pneumophila-containing neutrophils correlated strongly with CFU obtained by bacteriological culture. CONCLUSIONS: This technique can be utilised to determine pulmonary bacterial load and may be used in conjunction with other flow cytometric based analyses of the resulting immune response.
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ItemCrystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine(PUBLIC LIBRARY SCIENCE, 2012-06-25)Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608-621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a 'back-to-back' dimer of dimers compared to the 'head-to-head' architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a 'back-to-back' homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study.
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ItemA Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells(PUBLIC LIBRARY SCIENCE, 2011-11-09)Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.
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ItemThe Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm(PORTLAND PRESS LTD, 2011-12)BACKGROUND INFORMATION: Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells. RESULTS: Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes. CONCLUSIONS: These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.
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ItemTiming of Immune Escape Linked to Success or Failure of Vaccination(PUBLIC LIBRARY SCIENCE, 2010-09-16)Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIV(mac251) challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIV(mac251) stock had high levels of viral replication and rapid CTL escape. Unvaccinated naïve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of naïve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.
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ItemPrevention and Treatment of Influenza with Hyperimmune Bovine Colostrum Antibody(PUBLIC LIBRARY SCIENCE, 2010-10-26)BACKGROUND: Despite the availability of specific vaccines and antiviral drugs, influenza continues to impose a heavy toll on human health worldwide. Passive transfer of specific antibody (Ab) may provide a useful means of preventing or treating disease in unvaccinated individuals or those failing to adequately seroconvert, especially now that resistance to antiviral drugs is on the rise. However, preparation of appropriate Ab in large scale, quickly and on a yearly basis is viewed as a significant logistical hurdle for this approach to control seasonal influenza. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bovine colostrum, which contains approximately 500 g of IgG per milking per animal, has been investigated as a source of polyclonal antibody for delivery to the respiratory tract. IgG and F(ab')2 were purified from the hyperimmune colostrum of cows vaccinated with influenza A/Puerto Rico/8/34 (PR8) vaccine and were shown to have high hemagglutination-inhibitory and virus-neutralizing titers. In BALB/c mice, a single administration of either IgG or F(ab')2 could prevent the establishment of infection with a sublethal dose of PR8 virus when given as early as 7 days prior to exposure to virus. Pre-treated mice also survived an otherwise lethal dose of virus, the IgG- but not the F(ab')2-treated mice showing no weight loss. Successful reduction of established infection with this highly virulent virus was also observed with a single treatment 24 hr after virus exposure. CONCLUSIONS/SIGNIFICANCE: These data suggest that a novel and commercially-scalable technique for preparing Ab from hyperimmune bovine colostrum could allow production of a valuable substitute for antiviral drugs to control influenza with the advantage of eliminating the need for daily administration.
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ItemAN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS(ROCKEFELLER UNIV PRESS, 1993-08-01)Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.
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ItemCandesartan Attenuates Diabetic Retinal Vascular Pathology by Restoring Glyoxalase-I Function(AMER DIABETES ASSOC, 2010-12)OBJECTIVE: Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy. RESEARCH DESIGN AND METHODS: Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats. RESULTS: In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO(•)), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO(•). Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO(•), and restored GLO-I. CONCLUSIONS: We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.
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ItemComprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH)(BMC, 2009-07-01)BACKGROUND: In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. RESULTS: We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. CONCLUSION: We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.
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