Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene-targeting

Cell-mediated immunity is important for anti-Candida host defence in mucosal tissues. In this study we used cytokine-specific gene knockout mice to investigate the requirement for T helper type 1 (Th1) and Th2 cytokines in recovery from oral candidiasis. Knockout mice used in this study included interleukin-4 (IL-4), IL-10, IL-12p40, interferon-gamma (IFN-gamma), and tumour necrosis factor (TNF). The mice were challenged either orally or systemically with Candida albicans yeasts, and levels of colonization were determined. IL-12p40 knockout mice developed chronic oropharyngeal candidiasis, but were not more susceptible to systemic challenge. On the other hand, TNF knockout mice displayed increased susceptibility to both oral and systemic challenge, but only in the acute stages of infection. TNF apparently has a protective effect in the acute stages of both oral and systemic candidiasis, whereas IL-12p40 is essential for recovery from oral but not systemic candidiasis. The role of IL-12p40, and its relation to T-cell-mediated responses remain to be determined.

Isolates of Candida albicans that differ in virulence for mice elicit strain-specific antibody-mediated protective responses

Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Patterns of infection differed significantly between both yeasts and mouse strains. Systemic infection conferred significant protection against re-challenge with the homologous, but not the heterologous yeast; however, the protective effect was more evident in the tissue-susceptible CBA/CaH mice than in the resistant BALB/c strain. In contrast, oral infection induced protection against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. CBA/CaH mice produced antibodies of both IgG1 and IgG2a subclasses, whereas BALB/c mice produced predominantly IgG1. Western blotting demonstrated considerable differences between epitopes recognised by serum antibodies from mice of both strains after immunisation with each of the three yeasts. Thus, different strains of yeast show considerable specificity in antibody responses elicited by either systemic or oral infection. (c) 2005 Elsevier SAS. All rights reserved.

Mapping the I-3 gene for resistance to Fusarium wilt in tomato: application of an I-3 marker in tomato improvement and progress towards the cloning of I-3

Fusarium wilt of tomato, caused by the fungal pathogen, Fusarium oxysporum f. sp. lycopersici (Fol), is an economically damaging disease that results in huge losses in Australia and other countries worldwide. The I-3 gene, which confers resistance to Fol race 3, has been described in wild tomato, Lycopersicon pennellii, accessions LA716 and PI414773. We are pursuing the isolation of I-3 from LA716 by map-based cloning. We have constructed a high-resolution map of the I-3 region and have identified markers closely flanking I-3 as well as markers co-segregating with I-3. In addition, construction of a physical map based on these markers has been initiated. This review describes the context of our research and our progress towards isolating the I-3 gene. It also describes some important practical outcomes of our work, including the development and use of a PCR-based marker for marker-assisted selection for I-3, and the finding that the I-3 gene from LA716 is different to that from PI1414773, which we have now designated I-7. Tomato varieties combining I-3 and I-7 have been developed and are currently being introduced into commercial production to further safeguard tomato crops against Fusarium wilt.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

Resistance to persuasive messages as a function of majority and minority source status

Three experiments examined the extent to which attitudes following majority and minority influence are resistant to counter-persuasion. In Experiment 1, participants’ attitudes were measured after being exposed to two messages which argued opposite positions (initial pro-attitudinal message and subsequent, counter-attitudinal counter-message). Attitudes following minority endorsement of the initial message were more resistant to a (second) counter-message than attitudes following majority endorsement of the initial message. Experiment 2 replicated this finding when the message direction was reversed (counter-attitudinal initial message and pro-attitudinal counter-message) and showed that the level of message elaboration mediated the amount of attitude resistance. Experiment 3 included conditions where participants received only the counter-message and showed that minority-source participants had resisted the second message (counter-message) rather than being influenced by it. These results show that minority influence induces systematic processing of its arguments which leads to attitudes which are resistant to counter-persuasion.

Pupil resistance to their schooling experience

The thesis explores the nature of pupil resistance; it investigates what constitutes it and how it can be explained. Various ethnic and national group, male and female working-class resistance if analysed in two secondary schools in Birmingham (England) and one school in Sydney (Australia). It focuses on the pupils’ experience of school. ‘Compressed ethnographies’ (Walford and Miller, 1991) were conducted in each school to examine pupil resistance. The research found that structural societal state factors, regional, community and formal, informal and physical characteristics of each school, together with the teachers and pupils characteristics and background all influence resistance. The class, gender, ethnic and national identity of each pupil shapes resistance. In all three schools that were involved with the research, girls were more likely to exhibit overt, collective forms of resistance, whereas lads were more likely to operate alone. Islander pupils in Sydney and African-Caribbean kids in Birmingham were more likely to display engaged forms of resistance. Girls tended to show more engaged forms compared to their male counterparts across all ethnic and national cultures. Resistance is complex and dynamic, the definition alters depending upon context. Dimensions of resistance are developed; including overt, covert; individual, collective; intentional, unintentional; engaged and detached forms. Resistance operates within a structure and agency framework, the pupils can shape their own schooling experience mediated within the structures of their school, community and society. Some pupils manage their resources and the structures better than others; how the pupil manages and operates within the structures influences their resistance response. Resistance is contradictory and can reinforce the status quo. To fully understand resistance, it must be contextualised.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His7 and Ala8, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu9-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu9 of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue (Lys9)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys9)GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Phytoestrogens modulate breast cancer resistance protein expression and function at the blood-cerebrospinal fluid barrier

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter expressed at the blood cerebrospinal fluid barrier (BCSFB), and influences distribution of drugs into the central nervous systems (CNS). Current inhibitors have failed clinically due to neurotoxicity. Novel approaches are needed to identify new modulators to enhance CNS delivery. This study examines 18 compounds (mainly phytoestrogens) as modulators of the expression/function of BCRP in an in vitro rat choroid plexus BCSFB model. METHODS: Modulators were initially subject to cytotoxicity (MTT) assessment to determine optimal non-toxic concentrations. Reverse-transcriptase PCR and confocal microscopy were used to identify the presence of BCRP in Z310 cells. Thereafter modulation of the intracellular accumulation of the fluorescent BCRP probe substrate Hoechst 33342 (H33342), changes in protein expression of BCRP (western blotting) and the functional activity of BCRP (membrane insert model) were assessed under modulator exposure. RESULTS: A 24 hour cytotoxicity assay (0.001 µM-1000 µM) demonstrated the majority of modulators possessed a cellular viability IC50 > 148 µM. Intracellular accumulation of H33342 was significantly increased in the presence of the known BCRP inhibitor Ko143 and, following a 24 hour pre-incubation, all modulators demonstrated statistically significant increases in H33342 accumulation (P < 0.001), when compared to control and Ko143. After a 24 hour pre-incubation with modulators alone, a 0.16-2.5-fold change in BCRP expression was observed for test compounds. The functional consequences of this were confirmed in a permeable insert model of the BCSFB which demonstrated that 17-β-estradiol, naringin and silymarin (down-regulators) and baicalin (up-regulator) can modulate BCRP-mediated transport function at the BCSFB. CONCLUSION: We have successfully confirmed the gene and protein expression of BCRP in Z310 cells and demonstrated the potential for phytoestrogen modulators to influence the functionality of BCRP at the BCSFB and thereby potentially allowing manipulation of CNS drug disposition.

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Resistance to colistin : what is the fate for this antibiotic in pig production?

Colistin, a cationic polypeptide antibiotic, has reappeared in human medicine as a last-line treatment option for multidrug-resistant Gram-negative bacteria (MDR-GNB). Colistin is widely used in veterinary medicine for the treatment of gastrointestinal infections caused by Enterobacteriaceae. GNB resistant to colistin owing to chromosomal mutations have already been reported both in human and veterinary medicine, however several recent studies have just identified a plasmid-mediated mcr-1 gene encoding for colistin resistance in Escherichia coli colistin resistance. The discovery of a non-chromosomal mechanism of colistin resistance in E. coli has led to strong reactions in the scientific community and to concern among physicians and veterinarians. Colistin use in food animals and particularly in pig production has been singled out as responsible for the emergence of colistin resistance. The present review will focus mainly on the possible link between colistin use in pigs and the spread of colistin resistance in Enterobacteriaceae. First we demonstrate a possible link between Enterobacteriaceae resistance emergence and oral colistin pharmacokinetics/pharmacodynamics and its administration modalities in pigs. We then discuss the potential impact of colistin use in pigs on public health with respect to resistance. We believe that colistin use in pig production should be re-evaluated and its dosing and usage optimised. Moreover, the search for competitive alternatives to using colistin with swine is of paramount importance to preserve the effectiveness of this antibiotic for the treatment of MDR-GNB infections in human medicine.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

JNK regulates Fas receptor mediated apoptosis in prostate cancer cell lines

Prostate Cancer is a disease that primarily affects elderly men. The incidence of prostate cancer has been progressively increasing in the western world over the last two decades. Life expectancy and diet are believed to be the main factors contributing to this increase in prevalence. Prostate cancer is a slowly progressing disorder and patients often live for over 10 years after initially being diagnosed with prostate cancer. However, patients with hormone refractory prostate cancer have a poor prognosis and generally do not survive for longer than 2 or 3 years. Hormone refractory prostate cancer is responsible for over 200,000 deaths each year and current chemotherapeutic regimens are only useful as palliative agents. The long-term survival rate is poor and chemotherapy does not significantly increase this. Cell lines derived from hormone refractory tumours usually display elevated resistance to many cytotoxic drugs. The Fas receptor is a membrane bound protein capable of binding to a ligand called Fas ligand. Engagement of Fas receptor with Fas ligand results in clustering of Fas receptor on the plasma membrane of cells. A number of proteins responsible for initiating apoptosis are recruited to the plasma membrane and are activated in response to elevated local concentrations. This series of events initiates a proteolysis cascade and that culminates in the degradation of structural and enzymatic processes and the repackaging of cellular constituents within membrane bound vesicles that can be endocytosed and recycled by surrounding phagocytic cells. The Fas receptor is believed to be a key mechanism by which immune cells can destroy damaged cells. Consequently, resistance to Fas receptor mediated apoptosis often correlates with tumour progression. It has been reported that prostate cancer cell lines display elevated resistance to Fas receptor mediated apoptosis and this correlates with the stage of tumour from which the cell lines were isolated. JNK, a stress-activated protein kinase, has been implicated both with increased survival and increased apoptosis in prostate cancer. Elevated endogenous JNK activity has been demonstrated to correlate with prostate cancer progression. It has been shown that endogenous JNK activity increases the expression of anti-apoptotic proteins and can increase the resistance of prostate cancer cell lines to chemotherapy. In addition, elevated endogenous JNK activity is required for improved proliferation and transformation of a number of epithelial tumours. However, prolonged JNK activation in response to cytotoxic stimuli can increase the sensitivity of cells to apoptosis. Prolonged JNK activity appears to induce the expression of a separate set of genes responsible for promoting apoptosis. Our group has recently shown that activation of JNK by chemotherapeutic drugs can sensitise DU 145 prostate carcinoma cells to Fas receptor mediated apoptosis. In order toidentify novel targets for treating hormone refractory prostate cancer we have investigated the role of JNK in Fas receptor mediated apoptosis. We have demonstrated that prolonged JNK activation is defective in DU 145 cells in response to Fas receptor activation alone. Co-administering anisomycin, a JNK agonist, greatly enhances the ability of DU 145 cells to undergo apoptosis by increasing the rate of Caspase 8 cleavage. We also investigated the role of endogenous JNK activity in Fas receptor mediated.

Resistance to colistin : what is the fate for this antibiotic in pig production?

Colistin, a cationic polypeptide antibiotic, has reappeared in human medicine as a last-line treatment option for multidrug-resistant Gram-negative bacteria (MDR-GNB). Colistin is widely used in veterinary medicine for the treatment of gastrointestinal infections caused by Enterobacteriaceae. GNB resistant to colistin owing to chromosomal mutations have already been reported both in human and veterinary medicine, however several recent studies have just identified a plasmid-mediated mcr-1 gene encoding for colistin resistance in Escherichia coli colistin resistance. The discovery of a non-chromosomal mechanism of colistin resistance in E. coli has led to strong reactions in the scientific community and to concern among physicians and veterinarians. Colistin use in food animals and particularly in pig production has been singled out as responsible for the emergence of colistin resistance. The present review will focus mainly on the possible link between colistin use in pigs and the spread of colistin resistance in Enterobacteriaceae. First we demonstrate a possible link between Enterobacteriaceae resistance emergence and oral colistin pharmacokinetics/pharmacodynamics and its administration modalities in pigs. We then discuss the potential impact of colistin use in pigs on public health with respect to resistance. We believe that colistin use in pig production should be re-evaluated and its dosing and usage optimised. Moreover, the search for competitive alternatives to using colistin with swine is of paramount importance to preserve the effectiveness of this antibiotic for the treatment of MDR-GNB infections in human medicine.

Synergy between histone deacetylase inhibitors and DNA- damaging agents is mediated by histone deacetylase 2 in colorectal cancer

Previous studies have associated the overexpression of histone deacetylase 2 (HDAC2) and the presence of TP53 mutations with the progression to advanced stage drug resistant colorectal cancer (CRC). However, the mechanistic link between HDAC2 expression and the TP53 mutational status has remained unexplored. Here, we investigated the function of HDAC2 in drug resistance by assessing the synergistic effects of DNA-targeted chemotherapeutic agents and HDAC inhibitors (HDACis) on two TP53-mutated colorectal adenocarcinoma CRC cell lines (SW480 and HT-29) and on the TP53-wild type carcinoma cell line (HCT116 p53+/+) and its TP53 deficient sub-line (HCT116 p53-/-). We showed that in the untreated SW480 and HT-29 cells the steady-state level of HDAC2 was low compared to a TP53-wild type carcinoma cell line (HCT116 p53+/+). Increased expression of HDAC2 correlated with drug resistance, and depletion by shRNA sensitised the multi-drug resistance cell line HT-29 to CRC chemotherapeutic drugs such as 5-fluorouracil (5-FU) and oxaliplatin (Oxa). Combined treatment with the HDACi suberoylanilide hydroxamic acid plus 5-FU or Oxa reduced the level of HDAC2 expression, modified chromatin structure and induced mitotic cell death in HT-29 cells. Non-invasive bioluminescence imaging revealed significant reductions in xenograft tumour growth with HDAC2 expression level reduced to <50% in treated animals. Elevated levels of histone acetylation on residues H3K9, H4K12 and H4K16 were also found to be associated with resistance to VPA/Dox or SAHA/Dox treatment. Our results suggest that HDAC2 expression rather than the p53 mutation status influences the outcome of combined treatment with a HDACi and DNA-damaging agents in CRC.

FLIP: a targetable mediator of resistance to radiation in non-small cell lung cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non-small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor, FLIP, is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were down-regulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the anti-tumor activity of IR in vivo. Therefore, FLIP down-regulation induced by HDAC inhibitors is a potential clinical strategy to radio-sensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR.