Spelling suggestions: "subject:"RM therapeutic. pharmacology""
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Enhancing the delivery of therapeutic cargo to cancer cells using cell penetrating peptidesEissa, Noura Gamal January 2015 (has links)
Cell penetrating peptides (CPPs) have received considerable attention as cellular delivery vectors for small-molecule and macromolecular therapeutics. Hundreds of CPP sequences have now been described including bioportides that serve as single entity modifiers of cell physiology. Translation of promising CPP pre-clinical studies have however been disappointing as few identified delivery-systems have progressed to clinical trials. This project aimed to extend on studies using CPPs for delivering peptides influencing NF-B signalling and to search for novel membrane active peptides that could be classed as CPPs or bioportides. In-house methods measuring IB-α degradation and luciferase expression assays were developed to study the capacity of penetratin to influence NF-B activity via delivery of the NBD peptide (Pen-NBD: DRQIKIWFQNRRMKWKKTALDWSWLQTE). Results from these studies highlighted how two methods investigating the same signalling pathway could give very different results. Pen-NBD did not reduce TNF-α-induced IB-α degradation but induced reduction in TNF-α-induced luciferase activity. This effect was also observed in cells treated with penetratin alone or DMSO- diluent. EJP-18 (LFMRRRHIVRKRTLRRLL) represents a novel peptide from the EGFR juxtamembrane region and was found to be membrane active and internalised into cells. This thesis further characterised this peptide including two other sequences from this region of EGFR (EJP-21- ii LFMRRRHIVRKRTLRRLLQER and E-64562- RRRHIVRKRTLRRLLQER). In studied cancer cell lines, EJP-18 was toxic in an EGFR-dependant manner and aside from being a potential bioportide, also effectively delivered protein (BSA) and siRNA into cells. EJP-18/BSA was delivered to the lysosome and it remains to be determined whether it could be utilised to deliver siRNA to the cytosol. Cytotoxicity and protein delivery studies with EJP-21 and E-64562 highlighted the critical importance of terminal hydrophobicity in the EJP-18 sequence; this may extend to other studies involving CPPs. This thesis provides new information on in vitro activity of CPPs and CPP-chimeras. Additionally, new peptides were discovered that could potentially be classed as bioportides and CPPs.
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Activation of CD8+ T-Cell with abacavir and HLA-B*57:01 binding peptides and elimination of T-Cell responses through chemical modificationAlhaidari, Mohammad January 2015 (has links)
Adverse drug reactions are responsible for 6.5% of all hospital admissions. They also affect 10 to 20% of hospitalized patients. Drug-induced hypersensitivity reactions constitute an important major subtype of type B adverse drug reactions. Low molecular weight (i.e. penicillins, sulfamethoxazole and abacavir) and high molecular weight (biologics) drugs are both associated with the inadvertent activation of the adaptive immune system, which manifests as hypersensitivity in certain individuals. Pharmacogenomic research revealed that several forms of immunological drug reactions are strongly associated with specific human leukocyte antigens (HLA) alleles. The underlying mechanisms of HLA associated immunological drug reactions are still under exploration, however abacavir hypersensitivity represent a prototype model that exclusively occurs in individuals carrying the risk allele HLA- B*57:01. One hundred and ten CD8+ T-cell clones out of 1304 clones generated from 3 HLA-B*57:01 healthy volunteers displayed proliferative responses and/or secreted interferon gamma in the presence of abacavir which have been used for further mechanistic studies to evaluate mechanisms of drug antigen presentation. Approximately half of the clones were activated with abacavir in the absence of antigen presenting cells. Of the remaining clones, interestingly about half were activated with abacavir pulsed APC. The APC pulse negative clones are likely activated via a PI mechanism involving the direct binding of abacavir to surface HLA-B*57:01. In contrast, the pulse positive clones seem to be activated via the altered peptide repertoire mechanism involving abacavir binding to endogenous HLA-B*57:01. Some clones were also activated with APC from certain allogeneic donors expressing different HLA-B alleles. APC-dependent abacavir-specific clones were activated by the drug in the presence of APC from at least 3 allogenic different donors. Sixteen 6-amino substituted abacavir analogues were synthesized. Computational docking studies were completed to predict capacity for analogue binding within HLA-B*57:01. Abacavir-specific CD8+ clones were generated to study the association between HLA-B*57:01 analogue binding and T-cell activation. Antiviral activity and the direct inhibitory effect of analogues on proliferation were assessed. MHC class I-restricted CD8+ clones proliferated and secreted interferon gamma following abacavir binding to surface and endogenous HLA-B*57:01. Several analogues retained antiviral activity and showed no overt inhibitory effect on T-cell proliferation, but displayed highly divergent antigen-specific T-cell responses. Abacavir and N-propyl abacavir were equally potent at activating clones, while the closely-related analogues N-isopropyl and N-methyl isopropyl abacavir were devoid of T-cell activity. Docking abacavir analogues to HLA-B*57:01 revealed a quantitative relationship between drug-protein binding and the T-cell response. These studies demonstrate that the unwanted T-cell activity of abacavir can be eliminated whilst maintaining the favourable antiviral profile. A relatively large panel of abacavir-dependent HLA-B*57:01 binding self peptides were utilised to understand the mechanism of T-cell activation. Our data provides experimental evidence that a 9mer peptide (Peptide 15; NTVELRVKI) is responsible for the polyclonal CD8+ T-cell activation seen in abacavir hypersensitive patients. Furthermore, the discovery that most discovered unique altered self peptides are unable to induce CD8+ T-cell activation indicated that the immune response is highly peptide specific. Collectively the data generated enhances our understanding of how a single drug abacavir interacts with immunological receptors to initiate an unwanted and often pathogenic immune response. Even for this most highly HLA-restricted immunological drug reaction the nature of the drug receptor interaction is much more complex than initially anticipated. Thus, further studies are needed to determine how the results presented herein relate to other forms of immunological drug reaction.
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The role of Orai inhibition in acute pancreatitisWen, Li January 2015 (has links)
Background and aims: Prolonged elevation of cytosolic Ca2+ concentration is the key trigger of pancreatic damage and acute pancreatitis (AP). Ca2+ release-activated Ca2+ modulator Orai1 channel is the most abundant store-operated Ca2+ entry (SOCE) channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce AP. The studies in this thesis investigated the roles of Orai1channel in pancreatic acinar cell injury and the development of AP in mice. Methods: Freshly isolated mouse and human acinar cells were hyper-stimulated or incubated with human bile acid orthapsigargin to induce Ca2+ entry. Effects of GSK-7975A or CM_128 on Ca2+ entry and necrotic cell death pathway activation were analysed by confocal and video microscopy. Experimental acute pancreatitis was induced in C57BL/6J mice by 1) ductal injections of taurolithocholic acid 3-sulphate (TLCS-AP); 2) intraperitoneal (IP) administration of caerulein (CER-AP) or 3) a combined IP administration of ethanol and palmitoleic acid (FAEE-AP). Two Orai1 inhibitors were administrated at different time points and the disease severity was assessed by various local and systemic parameters, including pancreatic trypsin and myeloperoxidase (MPO) activity, serum interleukin (IL)-6 and lung MPO activityas well as blinded histopathology. Results: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of SOCE via Orai1 channels in a concentration dependent manner, in mouse and human pancreatic acinar cells (inhibition > 90% the levels observed in control cells). GSK-7975A and CM_128 each inhibited all local and systemic features of AP in all three representative models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given early versus late after induction of AP. Conclusion: Cytosolic Ca2+ overload mediated via Orai1 plays a pivotal role in the pathogenesis of AP. Inhibition of Orai1 channel is a useful therapeutic approach for human acute pancreatitis.
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In vitro evaluation of cytotoxicity caused by carbamazepine and its metabolites in association to carbamazepine-induced hypersensitivity reactionsMarlot, Philippe January 2014 (has links)
Carbamazepine (CBZ), an anticonvulsant and mood-stabilising drug, is known to cause delayed type hypersensitivity reactions. These reactions occur only in a minority of patients treated with the drug, but often result in severe clinical outcomes. Although an association between CBZ-induced hypersensitivity reactions and HLA alleles has been demonstrated, the underlying mechanism(s) of toxicity are poorly understood. Cell death caused by CBZ and one of its metabolites, 9-acridinecarboxaldehyde (9-AC) was investigated. CBZ did not show cytotoxic effects in concentrations ranging from sub-therapeutic to supra-therapeutic. By contrast, 9-AC caused apoptosis in the lymphoblastoid cell line (50µM and 24 hours of exposure) and primary PBMCs (50 µM and 2 hours of exposure). PBMCs from 20 CBZ-naïve individuals showed significant inter-individual variability in the susceptibility to the cytotoxic effect of 9-AC. To further investigate the observed inter-individual variability, 331 immortalised lymphoblast cell lines of unrelated individuals from 4 populations were exposed to CBZ, CBZ-10,11 epoxide or 9-AC and cell viability was measured after 24 hour exposure. Considerable inter-individual variability in the cytotoxic response was observed for all three compounds. The genome wide association study (GWAS) revealed two genetic polymorphisms in dual oxidase 1 (DUOX1) and RP11-354|13.2 that were linked to cell toxicity at low concentrations of all three compounds. A SNP in DUOX1 was investigated further because of its biological plausibility. Genotyping of 153 patients did not show an association between this SNP and CBZ-induced hypersensitivity in Caucasians. Due to the higher than normal frequency of the DUOX1 variant in non-Caucasian patients (20%), the involvement of DUOX1 in the predisposition to CBZ-induced hypersensitivity reactions in non-Caucasians could not be excluded. To elucidate how T cell activation occurs in CBZ-induced hypersensitivity reactions, the protein binding capability of CBZ and two of its metabolites, CBZ-10,11 epoxide (CBZE) and 9-AC, to human serum albumin and glutathione S-transferase π was assessed. Only CBZE was found to bind covalently to these proteins. For 9-AC, no covalent products were observed but an indication of reversible binding was detected. Finally, newly developed genotyping methods for HLA-A*31:01 were investigated in comparison to sequence based typing. The methods were based on SSP-PCR. One of the methods showed exact accordance with the current gold standard but PCR failed to amplify the gene of interest and the control gene in considerable amount of samples (13.1%), while the second SSP-PCR typing method showed less reliability. In conclusion, the mechanisms of CBZ hypersensitivity has been investigated using a number of approaches designed to elucidate bioactivation of the drug, and how cytotoxicity links with genetic factors. The genomic approach may have the potential to identify novel biomarkers, but needs further studies with larger sample size.
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Self-assembling antitumour prodrugs for localised drug deliveryCitossi, Francesca January 2016 (has links)
Localised cancer therapy is a developing strategy used to overcome the systemic toxicity associated with intravenous systemic chemotherapy, which still represents the primary route of administration for the majority of current anticancer agents. Low molecular weight gelators (LMWGs) have recently gained increasing popularity as drug delivery platforms for localised cancer therapy: they are small molecules, which self-assemble into a 3D network via non-covalent interactions. Due to their inherent biocompatibility, LMWGs represent a viable alternative to the extensively explored polymer based drug delivery systems. One such drug delivery approach, based on LMWGs, involves the synthesis of gelator-based prodrugs of chemotherapeutic agents; however, to date, there remains a limited number of anticancer prodrugs that have gelation properties. Therefore, the present work aims to find new chemotherapeutic agents that display gelation properties by modification of the parent drug with known self-assembling groups. Two anticancer agents have been evaluated: the clinical antimetabolite methotrexate (MTX) and the experimental benzothiazole derivative 5F 203, both characterised by significant anticancer activity but systemic toxicity. Therefore, formulation of MTX and 5F 203 as LMWGs-based prodrugs for localised cancer therapy was considered a useful strategy to overcome the systemic toxicity of these antitumour drugs. Different synthetic approaches were explored to formulate self-assembling MTX prodrugs. The most successful one involved the synthesis of MTX derivatives bearing alkyl chains and aromatic groups at the α or both α and γ carboxylic acid terminals of MTX. Unfortunately, preliminary gelation tests performed on MTX acyl derivatives via a solvent-switch method, revealed lack of self-assembling properties. Therefore, the MTX conjugates developed were not suitable for localised drug delivery applications. Due to the absence of self-assembling properties of MTX conjugates, the potential gelation behaviour of novel derivatives of another anticancer candidate, the benzothiazole agent 5F 203, was considered, and two amide prodrugs series of 5F 203 were investigated. Amongst the compounds tested for gelation, 5F 203 succinic acid conjugate (68a) revealed formation of a hydrogel at physiological pH. Rheological measurements confirmed its LMWG nature, showing formation of a cross-linked gel network. In vitro growth inhibitory assays against breast (MCF-7) and ovarian (IGROV-1 and OVCAR-4) carcinoma cell lines showed overall activity of 5F 203 amide prodrugs in inhibiting cell proliferation. Release studies from the gel matrix of 68a revealed a release of the derivative and the active drug over 3 days, thus confirming its potential application as a depot formulation for localised delivery of 5F 203. In order to improve the rate of conversion of prodrugs into the parent amine 5F 203, compared to the previous amide series developed, acyloxyalkoxycarbonyl derivatives of 5F 203 were synthesised. Gelation tests displayed self-assembling behaviour for derivatives 76a-76c and rheological studies confirmed the LMWG nature of the new entities. The acyloxyalkoxycarbonyl prodrugs revealed in vitro potencies similar to those displayed by 5F 203, when tested against MCF-7 and IGROV-1 cell lines. The isobutyl carbamate prodrug (76a) proved to be the most potent of the series, showing hydrolysis into the active drug 5F 203, when incubated in either PBS buffer, rat or human plasma; release from the gel matrix also showed release of 5F 203 in PBS within 72 h. The outcomes from this work have therefore provided a basis for future optimisation and development of LMWG derivatives of 5F 203, as depot formulations for localised delivery of this anticancer agent.
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Investigation of the adaptive immune response in multiple sclerosisRathbone, Emma January 2018 (has links)
In multiple sclerosis (MS), clonally-expanded brain-resident B cells may sustain chronic disease, however their relative contributions versus recently recruited B cells is unclear. Furthermore, pro-inflammatory CD20+ T cells may also be involved in MS pathogenesis. This study aimed to characterise the cerebrospinal fluid (CSF) B cell response in MS and investigate the features of CD20+ T cells. CSF B cells and antibody-secreting cells (ASC) displayed an activated phenotype and were identified in MS CSF at a higher frequency than controls. In contrast to the periphery, CSF ASC almost exclusively expressed IgG and were strongly lgK-biased, whereas memory B cells displayed similar immunoglobulin expression profiles in both compartments. MS CSF antibodies were frequently reactive towards EBNA-1, which preferentially induced an lgK-biased antibody response. Finally, CD20+ T cells displayed a highly activated effector phenotype and were present in the CSF, although their frequencies were no different between MS and OND groups. These findings suggest that most CSF B cells result from non-specific recruitment, whereas ASC are involved in a persistent lgK-biased antigen-driven immune response, which may primarily be directed towards EBNA-1. Despite their highly activated phenotype, a role for CD20+ T cells in MS pathogenesis, if any, remains to be determined.
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A critical evaluation of the interplay between relationship quality and pricing mechanisms in physicians' prescription decision makingKhan, Sohail M. January 2018 (has links)
Pharmaceutical managers and sales representatives (PSRs) rely greatly on relationship marketing activities in order to develop relationship quality (RQ) with physicians. The aim is to influence physicians' prescription behaviour to engender positive sales outcomes based on their RQ. There is little known about how PSR-physician RQ develops in the context of Pakistan, or how far RQ helps a PSR to achieve objective outcomes for a firm (i.e. positive sales), when it interacts or interplays with product price, particularly in specific economic conditions such as those of patients in Pakistan - who pay directly for their medicines. Drawing on available literature on RQ, this research firstly aims to discover what determinants are required and why they help to achieve RQ between a PSR and physician. Secondly, by integrating the literature on physician's prescription decision making, it seeks to identify how far product price affects RQ’s objective (or sales) outcomes under contingent patient economic conditions. Based on critical realism's guiding philosophical principles, this qualitative research adopted an embedded case study strategy; and using a semi-structured interview method 22 participants (i.e. pharmaceutical sales managers and physicians) from urban and rural sales areas of Multan in Pakistan were purposively interviewed in two sequential phases. Findings revealed that along with determinants identified through RQ literature, i.e. firm's image product quality, PSR's visit frequency, product knowledge, ethical selling behaviour and relationship investments, some additional determinants such as PSR's appearance, communication, and flexible responses to varied situations were also required by a PSR to achieve RQ with physicians in the context of Pakistan. These determinants were essential in order to fulfil physicians' technical, as well as social needs. When a PSR fulfils these physician needs, both physician's self-interest and emotional sentiments for a PSR are engendered, which serve as mechanisms that further foster the physician's reciprocity mechanism for a PSR. The presence of a reciprocity mechanism for a PSR thus influences the physician's prescription decision making in terms of a PSR's product. However, findings also indicated that higher product price serves as a barrier, which mitigates the effect of reciprocity mechanism because of the presence of more prevalent mechanisms, i.e. patient's non-affordability due to overall poor economic conditions. This engenders further mechanisms such as physician's emotional sentiments for patients, physician's self-interest in patient's wellbeing, their retention and physician's own practice viability. Therefore, the price of a product did not affect subjective or behavioural outcomes, such as physicians providing more time and priority to the PSR, when they had RQ. However, PSR-physician RQ worked more effectively for PSR in terms of achieving his objective (sales) outcomes, when product price and quality was competitive. Thus, the findings of this research suggest that RQ determinants are contingent to the research context and the context of the research should be taken into account. Furthermore, RQ's objective outcomes cannot be seen in isolation or by just predicting that RQ between the partners will ultimately lead to objective outcomes. For its comprehensive understanding and implementation, it is crucial to investigate other prevailing factors, such as: broader economic conditions and the presence of various relationships in the value chain that either support, or restrain, RQ between partners. Because, RQ works more effectively in terms of achieving its objective (or positive sales) outcomes in the context where overall economic conditions and product price related mechanisms support salespeople-customer's RQ related reciprocity mechanism.
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Analysis of potential driver genes in oral squamous cell carcinomaDavidson, Matthew Alexander January 2018 (has links)
The 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) has remained at ~50% for over 50 years. HNSCC is categorised by multiple anatomical sites, but oral (oral SCC) and oropharyngeal squamous cell carcinoma (OPSCC) account for approximately 90% of all cases. At the time of writing, only one targeted agent, cetuximab (a monoclonal antibody targeting the epithelial growth factor receptor), has been approved for the treatment of recurrent/metastatic HNSCC. However, despite the high expression of EGFR in oral SCC tumour samples, the clinical benefit of cetuximab has been modest thus far. Using a phenotypic screening approach, I sought to identify putative therapeutic targets. A whole genome siRNA screen carried out using an aggressive patient-derived cell line (‘Liv7k’) in normoxic and hypoxic conditions provided the foundation for this project. In addition, a drug-repurposing screen tested the efficacy of 1,351 compounds, approved for cancer and non-cancer indications. A number of approaches were used to identify potential targets, including a whole genome siRNA screen in normoxic and hypoxic conditions, a drug-repurposing screen, and a data multiplexing approach combining the two screens with pathway analysis and datasets from The Cancer Genome Atlas and the International Cancer Genome Consortium. Genomic characterisation of oral cancer cell lines confirmed the importance of a previously identified frequently amplified region of chromosome three, which contains a number of driver genes in HNSCC. In addition, a differential susceptibility of oral SCC cells in hypoxia formed the basis of a line of inquiry centred on triglyceride and ether lipid metabolism. Finally, compound screening identified a dependence of oral SCCs on cysteinyl leukotriene signalling, which is involved in inflammatory conditions such as asthma.
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Characterisation of grape and grape pomace polyphenolics : their absorption and metabolism and potential effects on hypertension in a SHR rat modelKy, Isabelle January 2013 (has links)
This study investigated the beneficial potential effects of grape pomaces obtained after winemaking of different Mediterranean grape varieties from crude materials to their in vivo effectiveness. Grapes and their respective grape pomaces from six different V. vinifera L. cultivar were studied namely Grenache (from two different locations [GRE1 and GRE2]), Syrah (from two different locations [SYR1 and SYR2]), Carignan (CAR), Mourvèdre (MOU), Counoise (COU) and Alicante (ALI) grape varieties from the Rhône Valley. The comparison of several wine industry by-products with their respective grapes provided evidence that pomace remaining at the end of the winemaking process can be very rich sources of antioxidants. The quantitative and qualitative distribution of polyphenols by HPLC-PDA-Fluo-MS in grape pomaces showed significant differences through varieties and vintages varying from 15% to 70% of polyphenols extracted. Seeds from Grenache (GRE1), Syrah (SYR1) and skins from Syrah (SYR1), Carignan and Alicante were of particular interest because of their higher polyphenol contents in terms of flavan-3-ols (monomers, dimers and trimers) up to 8.7 mg/g DW and anthocyanins (glycosides, acetylated and coumaroylated derivatives up to 17.40, 1.57 and 2.38 mg/g DW, respectively). The investigation of aqueous and hydro-alcoholic 70% extracts of seeds from Carignan and Syrah (SYR1) and skins from Carignan and Alicante was carried out as they contained high levels of total phenols and antioxidant activity. Several extracts, were tested in order to evaluate their in vivo biological effects on hypertension using a spontaneously hypertensive rat (SHR) model. A series of different grape pomace extracts were tested in association with verapamil. All in vivo experiments demonstrated that some grape pomace extracts administrated with or without co-ingestion with verapamil possessed an anti-hypertensive activity. This was evident with GRE1 (EA70) seed pomace extract, SYR1 (EA70) seed pomace extract, ALI (EA70) skin pomace extract administrated alone and with GRE1 (EA70) seed pomace extract, SYR1 (EAQ) seed pomace extract, ALI (EA70) skin pomace extract and SYR2 (EAQ) skin pomace extract administrated in association with verapamil. Grape pomace extracts with or without co-ingestion with verapamil were absorb as phase II metabolites mainly including glucuronide, O-methyl glucuronide, sulfate, and O-methyl sulfate derivatives of (epi)catechin which arise from the metabolism of monomeric flavan-3-ols. The detection by HPLC-PDA-Fluo-MSn and GC-MS of microbial-derived metabolites of flavan-3-ols, hydroxyphenyl-γ-valerolactones in their glucuronide and sulfate forms confirmed the absorption of metabolites derived from both monomeric and polymeric flavan-3-ols from grape pomace extracts and subsequent post-absorption conjugation. Numerous metabolites derived from further microbial degradation of hydroxyvalerolactones were also detected. The urinary excretion of these metabolites accounted for a larger proportion of the total polyphenol ingested than phase II metabolites of monomeric flavan-3-ols, indicating the important role of intestinal bacteria in the metabolism of polymerized procyanidins. All these metabolites may have exerted biological effects during the period in which they circulated in the bloodstream. This study constitutes the first step of assessing grape pomace as an enhancer of the verapamil, an anti-hypertensive drug. Substantial levels of polyphenols, especially flavan-3-ols, procyanidins and anthocyanins, remain in pomace after the winemaking process in quantities sufficient to exert anti-hypertensive effects. In addition, according to the extract used and its composition, it is feasible to modulate anti-hypertensive effects by amplifying or decreasing polyphenols and/or verapamil absorption.
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Evaluating Candida albicans biofilm formation and novel antifungal treatmentSherry, Leighann January 2014 (has links)
Candida biofilms have become an increasingly important clinical problem. The widespread use of antibiotics, frequent use of indwelling medical devices, and a trend towards increased patient immuno-suppression has resulted in a creation of opportunity for clinically important yeasts to form biofilms. Whilst there is growing evidence of the importance of Candida biofilms in clinical medicine, not all clinical isolates are able to form biofilms. There is therefore a fundamental gap in understanding exactly what drives biofilm formation and its clinical implications. These structures have become increasingly recognised as a significant clinical problem. One of the major reasons behind this is the impact that these have upon treatment, as antifungal therapy often fails and surgical intervention is required. This places a large financial burden on health care providers. Therefore, the discovery of alternative antifungal agents to be used in the treatment of fungal biofilms is in great demand for the management of these infections. A panel of Candida albicans bloodstream isolates were assessed for their biofilm forming ability by using the crystal violet assay and measuring cellular surface hydrophobicity. Scanning electron microscopy was used to visualise differences in the clinical biofilms. The impact of amphotericin B (AMB) treatment was determined next by broth microdilution method to assess differences in susceptibility profiles of the clinical isolates. The virulence of these clinical isolates was evaluated in vivo using a Galleria mellonella model and transcriptional analysis used to assess the expression of various genes associated with C. albicans biofilm formation within clinical isolates. Extracellular DNA (eDNA) in clinical biofilms was quantified using a microplate fluorescence assay and chitinase activity measured using a biochemical assay. Moreover, the potential of a novel antimicrobial agent Carbohydrate-derived fulvic acid (CHD-FA) was assessed against a panel of fungal and bacterial species. The mechanism of action of CHD-FA was determined using membrane assays include ATP release, and propidium iodide fluorescence, with various inhibitors used to determine whether CHD-FA activity is affected by known resistance mechanisms. Finally, the immunomodulatory properties of CHD-FA were investigated using ELISA and PCR arrays. The results from this study have shown C. albicans biofilm formation is differential within clinical isolates, where those with high biofilm formation (HBF) predominately consisted of hyphal cells, were more virulent in vivo and had decreased susceptibility to AMB, when compared to those with low biofilm formation (LBF). Furthermore, transcriptional analysis identified a number of genes that positively correlated with C. albicans biofilm formation. The novel agent carbohydrate-derived fulvic acid (CHD-FA) was shown to not only be highly active against C. albicans biofilms, but also against a range or orally relevant bacteria through non-specific membrane activity. Furthermore, CHD-FA was shown to down-regulate a number of pro-inflammatory mediators in an oral epithelial cell line. In conclusion, this study has characterised C. albicans clinical isolates based on their biological characteristics, where clear difference in virulence and antifungal treatment have been shown. It may be possible to develop a panel of genetic markers that could be used as a diagnostic tool for detecting biofilm formation in clinical isolates. CHD-FA is a microbiocidal compound that may serve as a potential novel antiseptic agent for the treatment of oral candidiasis and other candidal biofilm infections, whereby the immunomodulatory properties of CHD-FA could be exploited for controlling inflammation in a number of diseases.
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