Kevin Kavanagh

Enhancement of Chemotherapeutic Drug Toxicity to Human Tumour Cells In Vitro by a Subset of Non-steroidal Anti-inflammatory Drugs (NSAIDs)

The effect on cytotoxicity of combining a range of clinically important non-steroidal anti-inflammatory drugs (NSAIDs) with a variety of chemotherapeutic drugs was examined in the human lung cancer cell lines DLKP, A549, COR L23P and COR L23R and in a human leukaemia line HL60/ADR. A specific group of NSAIDs (indomethacin, sulindac, tolmetin, acemetacin, zomepirac and mefenamic acid) all at non-toxic levels, significantly increased the cytotoxicity of the anthracyclines (doxorubicin, daunorubicin and epirubicin), as well as teniposide, VP-16 and vincristine, but not the other vinca alkaloids vinblastine and vinorelbine. A substantial number of other anticancer drugs, including methotrexate, 5-fluorouracil, cytarabine, hydroxyurea, chlorambucil, cyclophosphamide, cisplatin, carboplatin, mitoxantrone, actinomycin D, bleomycin, paclitaxel and camptothecin, were also tested, but displayed no synergy in combination with the NSAIDs. The synergistic effect was concentration dependent. The effect appears to be independent of the cyclo-oxygenase inhibitory ability of the NSAIDs, as (i) the synergistic combination could not be reversed by the addition of prostaglandins D2 or E2; (ii) sulindac sulphone, a metabolite of sulindac that does not inhibit the cyclooxygenase enzyme, was positive in the combination assay: and (iii) many NSAIDs known to be cyclo-oxygenase inhibitors, e.g. meclofenamic acid, diclofenac, naproxen, fenoprofen, phenylbutazone, flufenamic acid, flurbiprofen, ibuprofen and ketoprofen, were inactive in the combination assay. The enhancement of cytotoxicity was observed in a range of drug sensitive tumour cell lines, but did not occur in P-170-overexpressing multidrug resistant cell lines. However, in the HL60/ADR and COR L23R cell lines, in which multidrug resistance is due to overexpression of the multidrug resistance-associated protein MRP, a significant increase in cytotoxicity was observed in the presence of the active NSAIDs. Subsequent Western blot analysis of the drug sensitive parental cell lines, DLKP and A549, revealed that they also expressed MRP and reverse-transcription-polymerase chain reaction studies demonstrated that mRNA for MRP was present in both cell lines. It was found that the positive NSAIDs were among the more potent inhibitors of [3H]-LTC4 transport into inside-out plasma membrane vesicles prepared from MRP-expressing cells, of doxorubicin efflux from preloaded cells and of glutathione-S-transferase activity. The NSAIDs did not enhance cellular sensitivity to radiation. The combination of specific NSAIDs with anticancer drugs reported here may have potential clinical applications, especially in the circumvention of MRP-mediated multidrug resistance.

Mite‐related bacterial antigens stimulate inflammatory cells in rosacea

Background Patients with papulopustular rosacea have a higher density of Demodex folliculorum mites on their faces than normal subjects but the role, if any, of their mites in initiating inflammation is disputed. Selective antibiotics are effective in reducing the inflammatory changes of papulopustular rosacea, but their mode of action is unknown.

Histatins: antimicrobial peptides with therapeutic potential

Histatins are a group of antimicrobial peptides, found in the saliva of man and some higher primates, which possess antifungal properties. Histatins bind to a receptor on the fungal cell membrane and enter the cytoplasm where they target the mitochondrion. They induce the non‐lytic loss of ATP from actively respiring cells, which can induce cell death. In addition, they have been shown to disrupt the cell cycle and lead to the generation of reactive oxygen species. Their mode of action is distinct from those exhibited by the conventional azole and polyene drugs, hence histatins may have applications in controlling drug‐resistant fungal infections. The possibility of utilising histatins for the control of fungal infections of the oral cavity is being actively pursued with the antifungal properties of topical histatin preparations and histatin‐impregnated denture acrylic being evaluated. Initial clinical studies are encouraging, having demonstrated the safety and efficacy of histatin preparations in blocking the adherence of the yeast Candida albicans to denture acrylic, retarding plaque formation and reducing the severity of gingivitis. Histatins may represent a new generation of antimicrobial compounds for the treatment of oral fungal infections and have the advantage, compared with conventional antifungal agents, of being a normal component of human saliva with no apparent adverse effects on host tissues and having a mode of action distinct to azole and polyene antifungals.

Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils

ABSTRACT The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were immunoprecipitated and analyzed by matrix-assisted laser desorption ionization—time of flight analysis. The results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Aspergillus fumigatus is a pathogenic fungus capable of causing both allergic lung disease and invasive aspergillosis, a serious, life-threatening condition in neutropenic patients. Aspergilli express an array of mycotoxins and enzymes which may facilitate fungal colonisation of host tissue. In this study we investigated the possibility of using the insect, Galleria mellonella, for in vivo pathogenicity testing of Aspergillus species. Four clinical isolates of Aspergillus fumigatus and a single strain of Aspergillus niger were characterised for catalase and elastase activity and for the production of gliotoxin. Gliotoxin is an immunosuppressive agent previously implicated in assisting tissue penetration. Results illustrated a strain dependent difference in elastase activity but no significant difference in catalase activity. Gliotoxin production was detected in vitro and in vivo by Reversed Phase-High Performance Liquid Chromatography, with highest amounts being produced by A. fumigatus ATCC 26933 (350 ng/mg hyphae). Survival probability plots (Kaplan–Meier) of experimental groups infected with Aspergillus conidia indicate that G. mellonella is more susceptible to fungal infection by A. fumigatus ATCC 26933, implicating a critical role for gliotoxin production rather than growth rate or enzymatic activity in the virulence of A. fumigatus in this model.

Self-Protection against Gliotoxin—A Component of the Gliotoxin Biosynthetic Cluster, GliT, Completely Protects Aspergillus fumigatus Against Exogenous Gliotoxin

Gliotoxin, and other related molecules, are encoded by multi-gene clusters and biosynthesized by fungi using non-ribosomal biosynthetic mechanisms. Almost universally described in terms of its toxicity towards mammalian cells, gliotoxin has come to be considered as a component of the virulence arsenal of Aspergillus fumigatus. Here we show that deletion of a single gene, gliT, in the gliotoxin biosynthetic cluster of two A. fumigatus strains, rendered the organism highly sensitive to exogenous gliotoxin and completely disrupted gliotoxin secretion. Addition of glutathione to both A. fumigatus ΔgliT strains relieved gliotoxin inhibition. Moreover, expression of gliT appears to be independently regulated compared to all other cluster components and is up-regulated by exogenous gliotoxin presence, at both the transcript and protein level. Upon gliotoxin exposure, gliT is also expressed in A. fumigatus ΔgliZ, which cannot express any other genes in the gliotoxin biosynthetic cluster, indicating that gliT is primarily responsible for protecting this strain against exogenous gliotoxin. GliT exhibits a gliotoxin reductase activity up to 9 µM gliotoxin and appears to prevent irreversible depletion of intracellular glutathione stores by reduction of the oxidized form of gliotoxin. Cross-species resistance to exogenous gliotoxin is acquired by A. nidulans and Saccharomyces cerevisiae, respectively, when transformed with gliT. We hypothesise that the primary role of gliotoxin may be as an antioxidant and that in addition to GliT functionality, gliotoxin secretion may be a component of an auto-protective mechanism, deployed by A. fumigatus to protect itself against this potent biomolecule.

Fungi : biology and applications

List of Contributors. Preface. 1. Introduction to Fungal Physiology (Graeme M. Walker and Nia A. White). 1.1 Introduction. 1.2 Morphology of yeasts and fungi. 1.3 Ultrastructure and function of fungal cells. 1.4 Fungal nutrition and cellular biosyntheses. 1.5 Fungal metabolism. 1.6 Fungal growth and reproduction. 1.7 Conclusions. 1.8 Further reading. 1.9 Revision questions. 2. Fungal Genetics (Malcolm Whiteway and Catherine Bachewich). 2.1 Introduction. 2.2 Life cycles. 2.3 Sexual analysis: Regulation of mating. 2.4 Unique characteristics of filamentous fungi that are advantageous for genetic analysis. 2.5 Genetics as a tool. 2.6 Conclusions. 2.7 Further reading. 2.8 Revision questions. 3. Fungal Genetics: A Post-Genomic Perspective (Brendan Curran and Virginia Bugeja). 3.1 Introduction. 3.2 Genomics. 3.3 Transcriptomics and proteomics. 3.4 Proteomics. 3.5 Systems biology. 3.6 Conclusions. 3.7 Further reading. 3.8 Revision questions. 4. Fungal Fermentation Systems and Products (Kevin Kavanagh). 4.1 Introduction. 4.2 Fungal fermentation systems. 4.3 Ethanol production. 4.4 Commercial fungal products. 4.5 Genetic manipulation of fungi. 4.6 Conclusion. 4.7 Further reading. 4.8 Revision questions. 5. Antibiotics, Enzymes and Chemical Commodities from Fungi (Richard A. Murphy and Karina A. Horgan). 5.1 Introduction. 5.2 Fungal metabolism. 5.3 Antibiotic production. 5.4 Pharmacologically active products. 5.5 Enzymes. 5.6 Chemical commodities. 5.7 Yeast extracts. 5.8 Enriched yeast. 5.9 Further reading. 5.10 Revision questions. 6. The Biotechnological Exploitation of Heterologous Protein Production in Fungi (Brendan Curran and Virginia Bugeja). 6.1 Fungal biotechnology. 6.2 Heterologous protein expression in fungi. 6.3 Budding stars. 6.4 Methylotrophic yeast species. 6.5 Case study - hepatitis B vaccine - a billion dollar heterologous protein from yeast. 6.6 Further biotechnological applications of expression technology. 6.7 Conclusion. 6.8 Further reading. 6.9 Revision questions. 7. Fungal Diseases of Humans (Derek Sullivan, Gary Moran and David Coleman). 7.1 Introduction. 7.2 Fungal diseases. 7.3 Superficial mycoses. 7.4 Opportunistic mycoses. 7.5 Endemic systemic mycoses. 7.6 Concluding remarks. 7.7 Further reading. 7.8 Revision questions. 8. Antifungal Agents for Use in Human Therapy (Khaled H. Abu-Elteen and Mawieh Hamad). 8.1 Introduction. 8.2 Polyene antifungal agents. 8.3 The azole antifungal agents. 8.4 Flucytosine. 8.5 Novel antifungal agents. 8.6 Miscellaneous antifungal agents. 8.7 New strategies and future prospects. 8.8 Conclusion. 8.9 Further reading. 8.10 Revision questions. 9. Fungal Pathogens of Plants (Fiona Doohan). 9.1 Fungal pathogens of plants. 9.2 Disease symptoms. 9.3 Factors influencing disease development. 9.4 The disease cycle. 9.5 Genetics of the plant-fungal pathogen interaction. 9.6 Mechanisms of fungal plant parasitism. 9.7 Mechanisms of host defence. 9.8 Disease control. 9.9 Disease detection and diagnosis. 9.10 Vascular wilt diseases. 9.11 Blights. 9.12 Rots and damping-off diseases. 9.13 Leaf and stem spots, anthracnose and scabs. 9.14 Rusts, smuts and powdery mildew diseases. 9.15 Global repercussions of fungal diseases of plants. 9.16 Conclusion. 9.17 Acknowledgements. 9.18 Further reading. 9.19 Revision questions. Answers to Revision Questions. Chapter 1. Chapter 2. Chapter 3. Chapter 4. Chapter 5. Chapter 6. Chapter 7. Chapter 8. Chapter 9. Index.

Emergence of Saccharomyces cerevisiae as a human pathogen: Implications for biotechnology

The yeast Saccharomyces cerevisiae is widely used in baking, brewing, wine making, and biotechnology and previously has had GRAS (generally regarded as safe) status. Recent evidence indicates the involvement of S. cerevisiae in a range of superficial and systemic diseases. Numerous cases of S. cerevisiae-induced vaginitis have been documented as have cases of oropharyngeal infection. Potentially fatal systemic disease due to S. cerevisiae has been recorded in bone marrow transplant patients and in those immunocompromised as a result of cancer or AIDS. A number of studies have indicated that commercially available strains of S. cerevisiae may cause disease in certain individuals. Pathogenic isolates exhibit the ability to grow at 42°C, produce proteinase, and are capable of pseudohyphal growth. In addition, a number of isolates are capable of phenotypic switching and show partial or complete resistance to commonly used antifungal agents, including fluconazole. In the light of these findings, S. cerevisiae should now be regarded as an opportunistic pathogen, albeit of relatively low virulence, and treated accordingly by those in the industrial and biotechnological sectors.

Potential role of Demodex mites and bacteria in the induction of rosacea.

Rosacea is a common dermatological condition that predominantly affects the central regions of the face. Rosacea affects up to 3 % of the worlds population and a number of subtypes are recognized. Rosacea can be treated with a variety of antibiotics (e.g. tetracycline or metronidazole) yet no role for bacteria or microbes in its aetiology has been conclusively established. The density of Demodex mites in the skin of rosacea patients is higher than in controls, suggesting a possible role for these mites in the induction of this condition. In addition, Bacillus oleronius, known to be sensitive to the antibiotics used to treat rosacea, has been isolated from a Demodex mite from a patient with papulopustular rosacea and a potential role for this bacterium in the induction of rosacea has been proposed. Staphylococcus epidermidis has been isolated predominantly from the pustules of rosacea patients but not from unaffected skin and may be transported around the face by Demodex mites. These findings raise the possibility that rosacea is fundamentally a bacterial disease resulting from the over-proliferation of Demodex mites living in skin damaged as a result of adverse weathering, age or the production of sebum with an altered fatty acid content. This review surveys the literature relating to the role of Demodex mites and their associated bacteria in the induction and persistence of rosacea and highlights possible therapeutic options.

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.