ORCID Profile
0000-0002-2162-6497
Current Organisations
University of South Australia Adelaide South Australia AU
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University of South Australia
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Publisher: MyJove Corporation
Date: 29-01-2010
DOI: 10.3791/1752
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.PLEFA.2016.01.006
Abstract: Expression of elevated levels of Indoleamine 2,3-dioxygenase (IDO) is well established as a mechanism of cancer induced immunosuppression. Pharmacological inhibition of IDO activity is thus a promising alternative in the treatment of cancer. Previously we demonstrated that cyclooxygenase derived metabolites of arachidonic acid inhibited the interferon-gamma mediated induction of IDO in both THP-1 cells and human monocytes. Here we identified that of the five primary prostanoids produced by COX-1/COX-2, only PGD2 displayed significant repressor activity. PGD2 inhibited IDO activity with an IC50 of 7.2µM in THP-1 cells and 5.2µM in monocytes. PGD2 caused a significant decrease in both IDO mRNA and protein. Using receptor specific agonists, PGD2 was found to act via the DP1 receptor, while the CRTH2 receptor was not involved. A DP1 antagonist significantly reduced the activity of PGD2, while CRTH2 agonists were ineffective. PGD2 increased intracellular cAMP levels and exogenous N(6)-cAMP was also found to be highly inhibitory. The effects of PGD2 via cAMP were blocked by Rp-cAMP indicating involvement of PKA. PGD2 also stimulated CREB phosphorylation, a PKA dependent transcription factor. This is the first report demonstrating that PGD2, a prostanoid typically associated with allergy, can inhibit IDO activity via the DP1/cAMP/PKA/CREB pathway. Our findings suggest that PGD2 and its derivatives may form the basis of novel repressors of IFNγ-mediated IDO expression.
Publisher: Australasian Society for Computers in Learning in Tertiary Education
Date: 04-11-2022
DOI: 10.14742/AJET.7878
Abstract: The gradual shift to online modes of learning in higher education institutions over the past 2 decades accelerated drastically on a global scale between 2020 and 2022. Students and educators, who have initially grappled with the shift, have now become accustomed to online teaching however, there are concerns about the quality of learning that has resulted. To enable a sustainable and effective online pedagogy, educators may need to learn about fostering higher-order thinking skills, which can be challenging even for experienced educators. To conceptualise effective online pedagogy, the community of inquiry (CoI) framework emphasises cognitive presence (CP), which focuses on the higher-order thinking process. The CoI is the most widely researched framework in online pedagogy, yet contemporary CoI literature lacks collective evidence of factors that influence CP. This scoping review of the CoI literature explores the factors that influence the higher-order thinking that is indicative of CP. Inclusion criteria included evidence of CP in online learning contexts and published between January 2000 and March 2022, providing a total of 121 studies. Results suggest that teaching presence, structure of learning activities and student characteristics all influence CP. Implications for practice or policy: Higher education students enrolled in online courses should be taught how to learn effectively in an online mode. Online course educators must embed learning tasks that foster self-regulation and higher-order skills in students. Online course design should include authentic tasks for students to apply new knowledge to real-life scenarios. Educators must be offered le professional development activities to build their skills in online pedagogy. Institutions should encourage translation of online educational research to practice.
Publisher: Hindawi Limited
Date: 2006
DOI: 10.1002/HUMU.20412
Publisher: Wiley
Date: 27-02-2007
DOI: 10.1002/ART.22432
Abstract: Neutrophils and tumor necrosis factor (TNF) play important roles in the pathogenesis of rheumatoid arthritis (RA). Modulation of TNF receptors (TNFRs) may contribute to the regulation of tissue damage, and n-6 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) can increase the expression of TNFRI and TNFRII on neutrophils. Because the n-3 PUFAs are antiinflammatory in RA, we examined whether, as a novel mechanism of action, n-3 PUFAs can antagonize the AA-induced increase in TNFR expression. Human neutrophils were treated with PUFAs and examined for changes in surface expression of TNFRs by flow cytometry. Translocation of protein kinase C (PKC) and activation of ERK-1/2 MAPK were determined by Western blotting. Intracellular calcium mobilization was measured in Fura 2-loaded cells by luminescence spectrometry. Pretreatment of neutrophils with nanomolar levels of n-3 PUFAs, eicosapentaenoic acid, or docosahexaenoic acid led to a marked inhibition of the AA-induced up-regulation of TNFRs I and II. Such pretreatment, however, did not prevent AA from stimulating the activities of PKC and ERK-1/2, which is required for the actions of AA or its ability to mobilize Ca(2+). Nevertheless, treatment with n-3 PUFAs caused the stimulation of serine proteases that could cleave the TNFRs. These findings suggest a mechanism by which the n-3 PUFAs inhibit the inflammatory response in RA, by regulating the ability of AA to increase TNFR expression. These results help fill the gaps in our knowledge regarding the mechanisms of action of n-3 PUFAs, thus allowing us to make specific recommendations for the use of n-3 PUFAs in the regulation of inflammatory diseases.
Publisher: Portland Press Ltd.
Date: 11-11-2019
DOI: 10.1042/BCJ20190245
Abstract: Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.
Publisher: MDPI AG
Date: 28-03-2022
DOI: 10.3390/IJMS23073689
Abstract: Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all s les. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.
Publisher: Hashemite University
Date: 12-2021
DOI: 10.54319/JJBS/140401
Publisher: Informa UK Limited
Date: 03-02-2022
Publisher: Wiley
Date: 03-11-2022
DOI: 10.1111/IMCB.12598
Abstract: This article covers the career pathway of a research-trained scientist to an immunology educator in a university setting.
Publisher: The American Association of Immunologists
Date: 09-2003
DOI: 10.4049/JIMMUNOL.171.5.2616
Abstract: Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10–20 min) and dose-dependent (0.5–30 μM) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the ω3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A2. The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.
Publisher: Elsevier BV
Date: 05-2019
Publisher: Springer Science and Business Media LLC
Date: 24-09-2018
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.FSI.2019.07.018
Abstract: The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.
Publisher: Elsevier BV
Date: 08-2020
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.CELLSIG.2016.06.007
Abstract: Sphingosine kinase (SK) 1 and 2 are lipid kinases that catalyse the formation of sphingosine 1-phosphate (S1P), a potent signalling molecule with a wide array of cellular effects. SK1 and 2 have been shown to be up-regulated in tumours and their genetic ablation or inhibition has been shown to slow tumour growth as well as sensitise cancer cells to chemotherapeutics. The SKs have been extensively studied, with a plethora of inhibitors developed that target the sphingosine-binding pocket of the enzyme, some with nanomolar affinities. Recently, inhibitors targeting the ATP pocket of SK have also been described. Here we discuss the development of these new small molecule SK inhibitors, summarise the recent discovery of off-targets effects of many current SK inhibitors, and provide an overview of the usefulness of these inhibitors as in vitro tools and therapeutic agents.
Publisher: American Physiological Society
Date: 06-2020
Abstract: Flow cytometry detects and measures the physical and chemical characteristics of cells or particles. In medical laboratories, flow cytometers are used to quantify changes in cell populations associated with disease states, such as AIDS. While a powerful technique, it is challenging to teach the principles of flow cytometry to undergraduate students. One approach is to have students process and analyze a patient s le. However, this is not possible when the patient has an infectious disease. Here we report a two-stage approach to address this challenge. Magnetic beads were used to manipulate leukocytes cell populations in healthy blood to mimic the phenotype of eight immune disease conditions. The cells were then stained against cell surface markers for cell populations and analyzed by flow cytometry. The second stage focused on teaching flow cytometry over 2 wk. Week 1 involved a lecture, followed by a laboratory session where students learned how to stain a blood s le. In week 2, students worked in a computer pool to analyze the previously generated data and determine the immunological status of a control and patient s le. Using this approach, all students achieved 100% correct diagnosis of both control and patient s les. Student feedback via a questionnaire was overwhelmingly positive, and student perceived knowledge of flow cytometry increased after the session significantly. We effectively mimicked several disease states, eliminating the need to source patient s les, yet still teaching undergraduate students the principles of flow cytometry.
Publisher: MyJove Corporation
Date: 29-03-2010
DOI: 10.3791/1923
Publisher: The American Association of Immunologists
Date: 2005
DOI: 10.4049/JIMMUNOL.174.1.233
Abstract: We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (β-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other novel PUFA consisting of β-oxa, β-thia, and γ-thia compounds. All three types displayed anti-inflammatory activity. Each of the unsaturated β-oxa fatty acids showed similar inhibition of PHA-PMA-induced T cell proliferation with a parallel inhibition of TNF-β production. However, β-oxa 25:6n-3 and β-oxa 21:4n-3 displayed lower inhibitory action on IFN-γ production. Surprisingly, β-oxa 23:4n-6 and β-oxa 21:3n-6 had marginal effect on IL-2 production. Thus, structural variation can generate selectivity for different immunological parameters. The β-thia compounds 23:4n-6, 21:3n-6, and 21:3n-3 were highly effective in inhibiting all immunological responses. Of the two γ-thia PUFA tested, γ-thia 24:4n-6 was a strong inhibitor of all responses apart from IL-2, but γ-thia 22:3n-6 had very little inhibitory effect. Two of the most active compounds, β-thia 23:4n-6 and β-thia 21:3n-6, were studied in more detail and shown to have an IC50 of 1–2 μM under optimal conditions. Thus, these PUFA retain the immunosuppressive properties of the n-3 PUFAs, 20:5n-3 and 22:6n-3, but not the neutrophil-stimulating properties. Their action on T lymphocytes is independent of cyclooxygenase or lipoxygenase activity, and they act at a postreceptor-binding level by inhibiting the activation of protein kinase C and ERK1/ERK2 kinases.
Publisher: Hindawi Limited
Date: 03-01-2012
DOI: 10.1002/HUMU.22003
Abstract: Chronic granulomatous disease (CGD) is mainly caused by mutations in X-linked CYBB that encodes gp91. We have identified two novel mutations in CYBB resulting in the rare X91(+)-CGD variant, c.1500T>G (p.Asp500Glu) in two male siblings and c.1463C>A (p.Ala488Asp) in an unrelated male. Zymosan and/or PMA (Phorbol 12-myristate 13-acetate)-induced recruitment of p47(phox) and p67(phox) to the membrane fraction was normal for both mutants. Cell-free assays using recombinant wild-type and the mutant proteins revealed that these mutants were not activated by NADPH (nicotinamide adenine dinucleotide phosphate). Interestingly, the Ala488Asp mutant was activated by NADPH in the presence of glutathione. These data suggest that the mutations prevented NADPH from binding to gp91(phox) and the requirement of a negative charge at residue 500 in gp91(phox) for NADPH oxidase assembly, in contrast to a previously described Asp500Gly change. These mutations and the effect of glutathione provide a unique insight into disease pathogenesis and potential therapy in variant X91(+)-CGD.
Publisher: Elsevier BV
Date: 11-2004
Publisher: Portland Press Ltd.
Date: 03-2003
DOI: 10.1042/BJ20021122
Abstract: The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKCβI, ∊, θ and ζ. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.
Publisher: Springer Science and Business Media LLC
Date: 2008
DOI: 10.1186/AR2426
Publisher: The American Association of Immunologists
Date: 10-2001
DOI: 10.4049/JIMMUNOL.167.7.3980
Abstract: A novel polyunsaturated fatty acid (PUFA), β-oxa 21:3n-3, containing an oxygen atom in the β position, was chemically synthesized, and found to have more selective biological activity than the n-3 PUFA, docosahexaenoic acid (22:6n-3) on cells of the immune system. Although β-oxa 21:3n-3 was very poor compared with 22:6n-3 at stimulating oxygen radical production in neutrophils, it was more effective at inhibiting human T lymphocyte proliferation (IC50 of 1.9 vs 5.2 μM, respectively). β-Oxa 21:3n-3 also inhibited the production of TNF-β, IFN-γ, and IL-2 by purified human T lymphocytes stimulated with PHA plus PMA, anti-CD3 plus anti-CD28 mAbs, or PMA plus A23187. Metabolism of β-oxa 21:3n-3 via the cyclooxygenase and lipoxygenase pathways was not required for its inhibitory effects. Consistent with its ability to suppress T lymphocyte function, β-oxa 21:3n-3 significantly inhibited the delayed-type hypersensitivity response and carrageenan-induced paw edema in mice. In T lymphocytes, β-oxa 21:3n-3 inhibited the agonist-stimulated translocation of protein kinase C-βI and -ε, but not -α, -βII, or -θ to a particulate fraction, and also inhibited the activation of the extracellular signal-regulated protein kinase, but not c-Jun NH2-terminal kinase and p38. In contrast, 22:6n-3 had no effects on these protein kinase C isozymes. The increase in antiinflammatory activity and loss of unwanted bioaction through the generation of a novel synthetic 22:6n-3 analogue provides evidence for a novel strategy in the development of anti-inflammatory agents by chemically engineering PUFA.
Publisher: American Chemical Society (ACS)
Date: 08-01-2014
DOI: 10.1021/NP400704B
Abstract: Dodonaea polyandra is a medicinal plant used traditionally by the Kuuku I'yu (Northern Kaanju) indigenous people of Cape York Peninsula, Australia. The most potent of the diterpenoids previously identified from this plant, polyandric acid A (1), has been examined for inhibition of pro-inflammatory cytokine production and other inflammatory mediators using well-established acute and chronic mouse ear edema models and in vitro cellular models. Topical application of 1 significantly inhibited interleukin-1β production in mouse ear tissue in an acute model. In a chronic skin inflammation model, a marked reduction in ear thickness, associated with significant reduction in myeloperoxidase accumulation, was observed. Treatment of primary neonatal human keratinocytes with 1 followed by activation with phorbol ester/ionomycin showed a significant reduction in IL-6 secretion. The present study provides evidence that the anti-inflammatory properties of 1 are due to inhibition of pro-inflammatory cytokines associated with skin inflammation and may be useful in applications for skin inflammatory conditions including psoriasis and dermatitis.
Publisher: UNS Solo
Date: 14-06-2020
Abstract: Abstract. Hastuti SD, Barton MD, Pyecroft SB, Costabile M. 2020. Assay optimization for measuring the alternate complement pathway activity in Asian seabass (Lates calcarifer). Bio ersitas 21: 3034-3040. Complement proteins are one component of innate immunity present in fish. The measurement of complement activity in fish can be used to monitor the health status of fish. This is particularly important in Asian seabass (Lates calcarifer) aquaculture, where disease can impact on productivity. We have found an optimal condition assay for measuring the alternate complement pathway (ACP) activity of Asian seabass which includes Magnesium chloride (MgCl2) concentration, buffer pH, incubation temperature and incubation time. The assay was optimized using pooled serum of Asian seabass, diluted in Magnesium ethylenediamine tetraacetic acid gelatine veronal buffer (Mg-EDTA-GVB) and added with Rabbit red blood cells (RRBC) suspension. Subsequently, the suspension was incubated and centrifuged. The supernatant was removed and transferred to a well plate and the optical density (OD) was measured at 540 nm. The optimal condition obtained included a 7.5 mM MgCl2, pH optimum of 7.5, 25°C incubation temperature, and a 30 minutes incubation period. The presently developed assay was robust, rapid, and reliable to be used in monitoring the health status of Asian seabass in aquaculture farms. It can be used as guidance in further immunological studies on this fish.
Publisher: American Society for Microbiology
Date: 31-08-2022
Abstract: Determining the antibiotic sensitivity of disease-causing microorganisms is a fundamental process in a clinical microbiology laboratory. With the continued use of antibiotics, the emergence of antibiotic resistance has become a significant health issue.
Publisher: Elsevier BV
Date: 07-1998
Publisher: Springer Science and Business Media LLC
Date: 21-08-2022
DOI: 10.1007/S00726-021-03069-6
Abstract: Glycation is a non-enzymatic reaction that occurs between the free amino group of proteins and reducing sugars and/or lipids, leading to the formation of advanced glycation end products (AGEs). The reaction also produces reactive oxygen species that have detrimental effects on cellular and extracellular proteins. Aminoguanidine is a known inhibitor of AGEs, and some fatty acids are known to have a beneficial role in vivo by reducing inflammation and oxidative stress. However, the role of fatty acids on AGE formation has not been thoroughly reported. We investigated the role of a range of fatty acids in the formation of AGEs and their reactive intermediates using an in vitro BSA-dicarbonyl model. The model assessed a time-dependent (0-72 h) and dicarbonyl concentration (0-2 mM) -dependent studies for the optimal formation of AGEs. A 72 h time point was found to be optimal for the reaction of BSA with either methylglyoxal (MGO) or glyoxal (GO) to generate AGE-BSA complexes. When arachidonic, eicosapentaenoic or docosahexaenoic acids were included in the reaction, a significant decrease in protein-bound fluorescent AGEs was seen compared to the respective controls. In contrast, saturated and 18 carbon polyunsaturated fatty acids showed no significant activity. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed saturated fatty acids significantly decreased the production of N
Publisher: IGI Global
Date: 10-01-2020
DOI: 10.4018/978-1-7998-2212-7.CH015
Abstract: One approach used in teaching scientific principles is laboratory practical classes. However, it can be challenging to teach concepts prior to their introduction in lectures. Academic teaching staff that wish to use alternative approaches to bridge this gap and, in turn, enhance student learning, often require help from their local Educational Developers (EDs). This chapter outlines the process of identifying a problem and then developing, implementing, and evaluating an online interactive simulation to teach enzyme kinetics to undergraduate students at the University of South Australia (UniSA). The challenges faced by the academic and ED in developing the simulation are covered. By the end of the chapter, the reader (academic or ED) will have a better appreciation of the challenges faced in developing a new teaching approach as well as the strategies that can be used to address these challenges.
Publisher: American Physiological Society
Date: 06-2021
Abstract: Hemolytic disease of the newborn (HDN) is a potentially fatal condition caused by a Rhesus (Rh) antigen incompatibility between a mother and fetus. As a result, determining the Rh status of expectant parents is a routine clinical assessment. Both the physiological and immunological basis of this condition are taught to undergraduate students. At the University of South Australia, some undergraduate immunology students find this topic challenging. The author designed, implemented, and assessed the impact of an interactive simulation to facilitate student learning of HDN. The students were actively engaged in determining the blood grouping and Rh status of an expectant mother and father and then determining the possibility of developing HDN. The simulation was found to take only 15 min to complete yet led to a significant increase in student performance in an end of semester exam question. Student perceived understanding was found to significantly improve following the introduction of the simulation, even though the content had been covered in a formal lecture. Student feedback was highly positive of this learning approach. In conclusion, short, interactive simulations can be used effectively to enhance student learning of challenging concepts.
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.JVIROMET.2017.10.022
Abstract: Inhibition of viral replication by icIgA antibodies has only been observed with in vitro studies using epithelial cell lines in transwell cultures. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across polarized cells. Polyclonal guinea pig antisera against purified influenza A virus and mouse antisera prepared against Influenza A/H3N2 hemagglutinin (HA The above findings in clinical specimens would contribute strongly to our understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps of mucosal viral infections. A rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co-localized influenza virus and icIgA - would contribute to further mucosal immunity studies and inform the design of more effective vaccines against influenza and other viral infections transmitted via the mucosal route e.g. respiratory syncytial virus, rotavirus.
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.PLEFA.2012.08.001
Abstract: Using human acute monocytic leukaemic THP-1 cells and human primary monocytes, this study examined the ability of arachidonic acid (AA) to modulate the activity of the IFNγ signalling cascade and its downstream effector indoleamine 2,3-dioxygenase (IDO). We established that AA inhibited IDO enzyme activity with an IC(50) of 20 μM in THP-1 cells and 12 μM in monocytes, and this was due to reduced expression of INDO1 mRNA and reduced level of IDO protein. Further mechanistic analysis revealed that AA interfered with the transcriptional function of the IFNγ signalling pathway by reducing phosphorylation of signal transducer and activator of transcription (STAT1) on tyrosine 701. The importance of AA metabolism via the COX and LOX pathways was investigated using inhibitors. Indomethacin, but not nordihydroguaiaretic acid, prevented the AA-mediated inhibition of STAT1 phosphorylation and thereby IDO enzymatic activity in THP-1 cells and monocytes. This is the first study to demonstrate that AA inhibits the IFNγ/STAT/IDO pathway, and this function is mediated by COX1/2 produced metabolites of AA. We now have evidence demonstrating that the AA metabolites, prostaglandins A(2) and D(2,) were highly inhibitory towards the IFNγ pathway, while prostaglandin E(2) had no effect. Together, these results indicate that the fatty acid AA has the potential to modulate the immunosuppressive activity of IDO and may form the basis of novel inhibitory compounds.
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.PLEFA.2017.06.010
Abstract: Indoleamine 2,3-dioxygenase-1 (IDO-1) catalyses the first and rate-limiting step in the metabolism of L-tryptophan. Degradation of L-Trp leads to the production of several immunosuppressive metabolites, including N-formyl kynurenine and kynurenine (Kyn). Apart from a normal physiological role, IDO-1 has also been identified to play a crucial role in immune suppression and tumour induced tolerance. Indeed, many primary tumours express high levels of IDO-1 compared to normal cells of the same stroma. IDO-1 is accepted as being an inducible negative regulator of T cell viability, proliferation and activation. As such, IDO-1 has become a target of intense interest for pharmacological inhibition, for the treatment of cancer. We have previously demonstrated that AA and the prostaglandin metabolite, PGD
Publisher: Wiley
Date: 10-01-2023
DOI: 10.1111/SJI.13253
Abstract: Virus neutralization at respiratory mucosal surfaces is important in the prevention of infection. Mucosal immunity is mediated mainly by extracellular secretory immunoglobulin A (sIgA) and its role has been well studied. However, the protective role of intracellular specific IgA (icIgA) is less well defined. Initially, in vitro studies using epithelial cell lines with surface expressed polymeric immunoglobulin receptor (pIgR) in transwell culture chambers have shown that icIgA can neutralize influenza, parainfluenza, HIV, rotavirus and measles viruses. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across the polarized cell. Co‐localization of specific icIgA with influenza virus in patients' (virus culture positive) respiratory epithelial cells using well‐characterized antisera was initially reported in 2018. This review provides a summary of in vitro studies with icIgA on colocalization and neutralization of the above five viruses. Two other highly significant respiratory infectious agents with severe global impacts viz. SARS‐2 virus (CoViD pandemic) and the intracellular bacterium— Mycobacterium tuberculosis —are discussed. Further studies will provide more detailed understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps with a specific focus on mucosal infections. This will inform the design of more effective vaccines against infectious agents transmitted via the mucosal route.
Publisher: Wiley
Date: 08-11-2006
DOI: 10.1111/J.1365-2141.2006.06386.X
Abstract: Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti-viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferon-gamma (IFN-gamma) secretion and capture assay to isolate ADV-specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus-specific T cells for cytomegalovirus or Epstein-Barr virus, the ADV response was dominated by CD4(+) T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN-gamma expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV-pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross-reactivity across ADV species a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV-specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection.
Publisher: The American Association of Immunologists
Date: 05-2019
DOI: 10.4049/JIMMUNOL.202.SUPP.61.5
Abstract: An automatic pipette is a key piece of laboratory equipment. The ability to accurately and reproducibly deliver any volume is a critical skill to master for all undergraduate students in the laboratory sciences. Over many years we have noted that students struggle in learning several facets of correct pipette usage. These include, choosing the correct pipette for a given volume, correctly setting the pipette and correctly using the plunger to aspirate and dispense the liquid. Students also struggle with unit conversions between ml and μl and vice versa. Lastly, students fail to retain these skills and information in following semesters of teaching. In an attempt to help students with converting between units (μl and ml) as well as choosing the most appropriate pipette for a given volume, I developed an interactive simulation which covered all these major aspects. Using a Gilson pipette as an ex le, the simulation covered how to set the pipette for a number of defined volumes. An Android/iOS app of the simulation has also been developed which can be used during laboratory classes. In 2017, we polled first year undergraduate students about their level of experience, knowledge of pipettes and level of comfort in converting between units and we then explained the simulation to them. The feedback on their skills and the software will be discussed. We also tested the impact of the simulation using 2nd year students enrolled in genetics. Using a laboratory quiz, we found significant improvements in student performance when students used the simulation compared to those that did not. We suggest this interactive approach is a simple and effective way to enhance student learning of key aspects of laboratory pipettes. The simulation is freely available.
Publisher: MDPI AG
Date: 08-04-2022
DOI: 10.3390/IJMS23084139
Abstract: The accurate segregation of sister chromatids is complex, and errors that arise throughout this process can drive chromosomal instability and tumorigenesis. We recently showed that methylglyoxal (MGO), a glycolytic by-product, can cause chromosome missegregation events in lymphocytes. However, the underlying mechanisms of this were not explored. Therefore, in this study, we utilised shotgun proteomics to identify MGO-modified proteins, and label-free quantitation to measure changes in protein abundance following exposure to MGO. We identified numerous mitotic proteins that were modified by MGO, including those involved in the separation and cohesion of sister chromatids. Furthermore, the protein abundance of Securin, an inhibitor of sister chromatid separation, was increased following treatment with MGO. Cytological examination of chromosome spreads showed MGO prevented sister chromatid separation, which was associated with the formation of complex nuclear anomalies. Therefore, results from this study suggest MGO may drive chromosomal instability by preventing sister chromatid separation.
Publisher: The American Association of Immunologists
Date: 05-2019
DOI: 10.4049/JIMMUNOL.202.SUPP.61.4
Abstract: Hemolytic disease of the newborn (HDN, Erythroblastalis fetalis) is a potentially fatal condition. It is initiated due to Rhesus (Rh) antigens on fetal red blood cells (RBCs) being seen as foreign by the mothers Rh− immune system. In the first pregnancy, exposure of the maternal immune system to fetal Rh+ RBCs leads to immune activation, IgM production and memory cell formation. A subsequent pregnancy with another Rh+ fetus, activates memory cells, leading to class switching and IgG production, which is placentally transferred to the fetus. Through multiple effector functions, the IgG leads to hemolysis, causing anemia and in severe cases even death of the fetus. The role of the immune response is key to this disease, hence it is taught to undergraduate immunology students at the University of South Australia. Topics include the primary and secondary immune responses, placental transfer of IgG, deletion of self reactive cells and use of mAbs as therapeutic agents are addressed. Experience has shown that students find this topic challenging. In an attempt to enhance understanding, I developed an interactive simulation to teach these principles which was trialed in 2018. The simulation gave students the opportunity to blood group expecting parents and then discuss possible HDN issues and the treatment option. As with other simulations I have generated, (e.g. flow cytometry and mAbs), I suggest this approach is a new approach to teach complex immunological concepts. This approach can enhance student understanding as well as provide a new means of information transferal. The HDN simulation is freely available to all teaching staff who may wish to use it as part of their teaching approach.
Publisher: American Society of Hematology
Date: 30-06-2022
Abstract: Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.
Location: Australia
No related grants have been discovered for Maurizio Costabile.