Resistance development in community-acquired strains of methicillin-resistant Staphylococcus aureus: an in vitro study

This study compares in vitro antimicrobial resistance development between strains of Staphylococcus aureus including newly described community-acquired methicillin-resistant strains (CA-MRSA). High-level resistance developed in all strains of S. aureus after exposure to rifampicin and gentamicin and in some strains after fusidic acid exposure, independent of methicillin resistance phenotype. Resistance did not develop after exposure to clindamycin, cotrimoxazole, ciprofloxacin, linezolid, or vancomycin. These results have important implications for therapy of CA-MRSA infections. (C) 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor

The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C2C12 myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 μM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the α-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E214k), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia. © 2002 Cancer Research UK.

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

Development of a novel in vitro system for nasal drug delivery development

There is currently, no ideal system for studying nasal drug delivery in vitro. The existing techniques such as the Ussing chamber and cell culture all have major disadvantages. Most importantly, none of the existing techniques accurately represent the interior of the nasal cavity, with its airflow and humidity; neither do they allow the investigation of solid dosage forms.The work in this thesis represents the development of an in vitro model system in which the interior characteristics of the nasal cavity are closely represented, and solid or minimal volume dosage forms can be investigated. The complete nasal chamber consists of two sections: a lower tissue, viability chamber and an upper nasal chamber. The lower tissue viability chamber has been shown, using existing tissue viability monitoring techniques, to maintain the viability of a number of epithelial tissues, including porcine and rabbit nasal tissue, and rat ileal and Payers' patch tissue. The complete chamber including the upper nasal chamber has been shown to provide tissue viability for porcine and rabbit nasal tissue above that available using the existing Ussing chamber techniques. Adaptation of the complete system, and the development of the necessary experimental protocols that allow aerosol particle-sizing, together with videography, has shown that the new factors investigated, humidity and airflow, have a measurable effect on the delivered dose from a typical nasal pump. Similarly, adaptation of the chamber to fit under a confocal microscope, and the development of the necessary protocols has shown the effect of surface and size on the penetration of microparticulate materials into nasal epithelial tissues. The system developed in this thesis has been shown to be flexible, in allowing the development of the confocal and particle-sizing systems. For future nasal drug delivery studies, the ability to measure such factors as the size of the delivered system in the nasal cavity, the depth of penetration of the formulation into the tissue are essential. Additionally, to have access to other data such as that obtained from drug transport in the same system, and to have the tissue available for histological examination represents a significant advance in the usefulness of such an in vitro technique for nasal delivery.

The development of a novel in vitro model suitable for the prediction of bioavailability

In vitro studies of drug absorption processes are undertaken to assess drug candidate or formulation suitability, mechanism investigation, and ultimately for the development of predictive models. This study included each of these approaches, with the aim of developing novel in vitro methods for inclusion in a drug absorption model. Two model analgesic drugs, ibuprofen and paracetamol, were selected. The study focused on three main areas, the interaction of the model drugs with co-administered antacids, the elucidation of the mechanisms responsible for the increased absorption rate observed in a novel paracetamol formulation and the development of novel ibuprofen tablet formulations containing alkalising excipients as dissolution promoters.Several novel dissolution methods were developed. A method to study the interaction of drug/excipient mixtures in the powder form was successfully used to select suitable dissolution enhancing exicipents. A method to study intrinsic dissolution rate using paddle apparatus was developed and used to study dissolution mechanisms. Methods to simulate stomach and intestine environments in terms of media composition and volume and drug/antacid doses were developed. Antacid addition greatly increased the dissolution of ibuprofen in the stomach model.Novel methods to measure drug permeability through rat stomach and intestine were developed, using sac methodology. The methods allowed direct comparison of the apparent permeability values obtained. Tissue stability, reproducibility and integrity was observed, with selectivity between paracellular and transcellular markers and hydrophilic and lipophilic compounds within an homologous series of beta-blockers.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

Topographical guidance of intervertebral disc cell growth in vitro:towards the development of tissue repair strategies for the anulus fibrosus

The anulus fibrosus (AF) of the intervertebral disc consists of concentric sheets of collagenous matrix that is synthesised during embryogenesis by aligned disc cells. This highly organised structure may be severely disrupted during disc degeneration and/or herniation. Cell scaffolds that incorporate topographical cues as contact guidance have been used successfully to promote the healing of injured tendons. Therefore, we have investigated the effects of topography on disc cell growth. We show that disc cells from the AF and nucleus pulposus (NP) behaved differently in monolayer culture on micro-grooved membranes of polycaprolactone (PCL). Both cell types aligned to and migrated along the membrane's micro-grooves and ridges, but AF cells were smaller (or less spread), more bipolar and better aligned to the micro-grooves than NP cells. In addition, AF cells were markedly more immunopositive for type I collagen, but less immunopositive for chondroitin-6-sulphated proteoglycans than NP cells. There was no evidence of extracellular matrix (ECM) deposition. Disc cells cultured on non-grooved PCL did not show any preferential alignment at sub-confluence and did not differ in their pattern of immunopositivity to those on grooved PCL. We conclude that substratum topography is effective in aligning disc cell growth and may be useful in tissue engineering for the AF. However, there is a need to optimise cell sources and/or environmental conditions (e.g. mechanical influences) to promote the synthesis of an aligned ECM.

Progress in the development of in vitro human-based alternatives to animal models

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