Modelling of microstructural evolution in multi-layered overlay coatings.

Functionally graded, multi-layered coatings are designed to provide corrosion protection over a range of operating conditions typically found in industrial gas turbines. A model incorporating diffusion, equilibrium thermodynamics and oxidation has been developed to simulate the microstructural evolution within a multi-layered coating system. The phase and concentration profiles predicted by the model have been compared with an experimental multi-layered system containing an Al-rich outer layer, a Cr-enriched middle layer and an MCrAlY-type inner layer deposited on a superalloy substrate. The concentration distribution and many microstructural features observed experimentally can be predicted by the model. The model is expected to be useful for assessing the microstructural evolution of multilayer coated systems which can be potentially used on industrial gas turbine aerofoils.

Helium ion microscopy based wall thickness and surface roughness analysis of polymer foams obtained from high internal phase emulsion.

Due to their wide range of applications, porous polymers obtained from high internal phase emulsions have been widely studied using scanning electron microscopy. However, due to their lack of electrical conductivity, quantitative information of wall thicknesses and surface roughness, which are of particular interest to tissue engineering, has not been obtained. Here, Helium Ion Microscopy is used to examine uncoated polymer foams and some very strong but unexpected contrast is observed, the origin of which is established here. Based on this analysis, a method for the measurement of wall thickness variations and wall roughness measurements has been developed, based on the modeling of Helium ion transmission. The results presented here indicate that within the walls of the void structure there exist small features with height variations of ~30 nm and wall thickness variations from ~100 nm to larger 340 nm in regions surrounding interconnecting windows within the structure. The suggested imaging method is applicable to other porous carbon based structures with wall thicknesses in the range of 40-340 nm.

Differential interactions of Streptococcus gordonii and Staphylococcus aureus with cultured osteoblasts.

The impedance of normal osteoblast function by microorganisms is at least in part responsible for the failure of dental or orthopedic implants. Staphylococcus aureus is a major pathogen of bone, and exhibits high levels of adhesion and invasion of osteoblasts. In this article we show that the commensal oral bacterium Streptococcus gordonii also adheres to and is internalized by osteoblasts. Entry of S. gordonii cells had typical features of phagocytosis, similar to S. aureus, with membrane protrusions characterizing initial uptake, and closure of the osteoblast membrane leading to engulfment. The sensitivities of S. gordonii internalization to inhibitors cytochalasin D, colchicine and monensin indicated uptake through endocytosis, with requirement for actin accumulation. Internalization levels of S. gordonii were enhanced by expression of S. aureus fibronectin-binding protein A (FnBPA) on the S. gordonii cell surface. Lysosomal-associated membrane protein-1 phagosomal membrane marker accumulated with intracellular S. aureus and S. gordonii FnBPA, indicating trafficking of bacteria into the late endosomal/lysosomal compartment. Streptococcus gordonii cells did not survive intracellularly for more than 12 h, unless expressing FnBPA, whereas S. aureus showed extended survival times (>48 h). Both S. aureus and S. gordonii DL-1 elicited a rapid interleukin-8 response by osteoblasts, whereas S. gordonii FnBPA was slower. Only S. aureus elicited an interleukin-6 response. Hence, S. gordonii invades osteoblasts by a mechanism similar to that exhibited by S. aureus, and elicits a proinflammatory response that may promote bone resorption.

SulA-induced filamentation in Salmonella enterica serovar Typhimurium: effects on SPI-1 expression and epithelial infection.

AIMS: Salmonella enterica serovar Typhimurium is capable of adopting a filamentous phenotype in response to damage. How this adaptive response affects bacterial virulence is unclear. We have examined the hypothesis that filamentation affects the ability of Salmonella to infect host cells. METHODS AND RESULTS: Expression of the cell division inhibitor SulA in Salm. Typhimurium SL1344 from an arabinose-inducible plasmid resulted in filamentation. We examined expression of the type 3 secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) using SL1344 expressing a chromosomal PprgH-gfp reporter. Single cell analysis of SulA-induced SL1344 PprgH-gfp revealed a relationship between increasing cell length and decreasing propensity for prgH expression, but there was no evidence of a significant change in prgH expression evident at the whole population level. Filamentous Salm. Typhimurium were capable of initiating membrane ruffling on MDCK epithelial cells, but only nonfilamentous bacteria (< 6 µm) invade. CONCLUSIONS: Induction of SulA expression in Salmonella inhibits septation. Increasing filament length is associated with down-regulation of SPI-1 gene expression, but a significant proportion of filaments retain the ability to produce SPI-1 T3SS and induce membrane ruffles on epithelia. Despite an active SPI-1 T3SS, filamentous Salmonella are unable to invade epithelial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings that filamentous Salmonella can express an invasive phenotype but fail to invade cells suggest that their presence in food does not constitute an immediate risk of infection until septation occurs. The described SulA expression model provides a convenient model for studying the impact of filamentation in the absence of additional stresses.

Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins.

INTRODUCTION: Hereditary spherocytosis (HS) and hereditary pyropoikilocytosis (HPP, severe form of hereditary elliptocytosis) are unrelated red cell disorders caused by defects in distinct regions of the red cell cytoskeleton. The high predictive value of the eosin-5-maleimide (EMA)-binding test for the diagnosis of HS is because of its interaction with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47, which are reduced on HS red cells. Our study was undertaken to determine why EMA-labelled HPP red cells were previously found to give much lower fluorescence readings than HS. METHODS: Flow cytometry was used to determine the relative amounts of monoclonal antibodies bound to red cells from normal adults, HS and HPP groups. Confocal microscopy was used to visualise the overall staining pattern of the red cells with selected antibodies. RESULTS: In flow cytometry, HPP red cells gave lower antibody binding to the four EMA-reactive membrane proteins than HS red cells and bound less antibody to glycophorins A and C, and CD59. Confocal images of Rh protein and band 3 immunostaining revealed a greater number of HPP red cells having partial or no fluorescence than in HS and normal controls. CONCLUSION: Lesser amounts of EMA-reactive membrane proteins were detected in HPP than HS red cells, thus confirming their lower fluorescence readings in the EMA-binding test. The concomitant reduction in glycophorins A and C, and CD59 in HPP could have caused cellular contraction, resulting in poikilocytosis.

Energy selective scanning electron microscopy to reduce the effect of contamination layers on scanning electron microscope dopant mapping.

We demonstrate that energy selective scanning electron microscopy can lead to substantial dopant contrast and resolution improvements (compared to standard SEM) when the energy selection is carried out based on Monte Carlo modelled secondary electron spectra in combination with detector transfer modelling.

The role of helium ion microscopy in the characterisation of complex three-dimensional nanostructures.

This work addresses two major issues relating to Helium Ion Microscopy (HeIM). First we show that HeIM is capable of solving the interpretation difficulties that arise when complex three-dimensional structures are imaged using traditional high lateral resolution techniques which are transmission based, such as scanning transmission electron microscopy (STEM). Secondly we use a nano-composite coating consisting of amorphous carbon embedded in chromium rich matrix to estimate the mean escape depth for amorphous carbon for secondary electrons generated by helium ion impact as a measure of HeIM depth resolution.

Norepinephrine increases the pathogenic potential of Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni can cause a spectrum of diseases in humans, ranging from enteritis and diarrhoea to severe inflammation, profuse bloody diarrhoea and chronic relapsing infection. Norepinephrine (NE) levels in the intestine increase under conditions of stress and trauma, and are thought to result in spill over of NE into the intestinal lumen. NE is known to stimulate the growth of a range of bacterial species, and to increase the pathogenicity of Escherichia coli. AIM: To determine the effects of NE on the pathogenic potential of C jejuni in a model system. METHODS: C jejuni was grown in iron-replete and iron-limited media in the presence and absence of 100 microM NE. Several virulence-associated characteristics, including motility and cell invasion, were measured. RESULTS: When C jejuni was grown in iron-limited media in the presence of NE, growth rate, motility and invasion of cultured epithelial cells were increased compared with cultures grown in the absence of NE. Bacteria exposed to NE during growth also caused greater subsequent disruption of cultured epithelial cell monolayers, inducing widespread breakdown of tight junctions. CONCLUSION: Exposure to NE causes an increase in the virulence-associated properties of Campylobacter. Stress and concomitant infection could therefore be contributory factors to the variable presentation of this disease.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture.

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only non-O157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to HEp-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli O157:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed.

Disruption of epithelial barrier function by H2O2: distinct responses of Caco-2 and Madin-Darby canine kidney (MDCK) strains.

Modulation of epithelial function by oxidants such as hydrogen peroxide may contribute to a variety of disease processes. The effects of hydrogen peroxide (H2O2) on epithelial barrier function and tight junction protein distribution were compared in three distinct types of polarised epithelial cell, each of which was found to respond differently to H2O2. Of the cell-lines examined, Madin-Darby canine kidney (MDCK) stain II was the most susceptible to H2O2 and Caco-2 the least H2O2 induced a decrease in transepithelial electrical resistance in all three epithelial cell-lines which was accompanied by redistribution of the tight junction protein occludin in Caco-2 and MDCK II but not MDCK I, cell layers. The effects of H2O2 on epithelia displayed marked asymmetry, each cell-line being affected more by basal than apical application of H2O2. Genistein partially prevented the effects of H2O2 on Caco-2 cells suggesting a role for protein tyrosine phosphorylation in H2O2-induced epithelial barrier dysfunction. However, genistein was without effect on the responses of MDCK cells to H2O2. Taken together these data indicate that H2O2 has distinct effects onthe tight junctions of epithelial cells from different origins and suggest that enhanced tyrosine phosphorylation is a contributory factor inthe enhanced permeability of some, but possibly not all, epithelial cell-lines.

Butyrate downregulates alpha2beta1 integrin: a possible role in the induction of apoptosis in colorectal cancer cell lines.

BACKGROUND: Integrins mediate cell matrix adhesion and regulate cell growth and survival. In colonic epithelial cells, alpha(2)beta(1) integrin controls glandular differentiation and proliferation. Butyrate stimulates differentiation and induces apoptosis in vitro. AIMS: We investigated whether butyrate induction of apoptosis was associated with perturbation of integrin mediated cell matrix adhesion. METHODS: Three colonic cancer cell lines (SW1222, SW620, LS174T) were studied. Adhesion to extracellular matrix proteins, expression of alpha(2)beta(1) integrin, and apoptosis were studied in adherent cells after treatment with 4 mM butyrate. RESULTS: Butyrate decreased the attachment to type I collagen in SW620 cells and type I and IV collagen in LS174T cells. The decreased cell attachment was associated with downregulation of alpha(2)beta(1) integrin and increased apoptosis in adherent cells. No changes in alpha(2)beta(1) expression or matrix adhesion were seen in SW1222 cells, which were also found to be less sensitive to butyrate induction of apoptosis. Downregulation of alpha(2)beta(1) integrin preceded the detection of apoptosis. CONCLUSION: Apoptosis induced by butyrate is associated with downregulation of expression and functional activity of alpha(2)beta(1) integrin. Perturbation of cell matrix adhesion may be a novel mechanism by which butyrate induces apoptosis in colorectal cancer cells.

Analysis of pathogen-host cell interactions in purpura fulminans: expression of capsule, type IV pili, and PorA by Neisseria meningitidis in vivo.

The pattern of meningococcal surface structure expression in different microenvironments following bloodstream invasion in vivo is not known. We used immunohistochemistry to determine the expression of capsule, type IV pili, and PorA by meningococci residing in the skin lesions of children with purpura fulminans. All the skin biopsy samples showed evidence of thrombosis and, frequently, a perivascular inflammatory cell infiltrate consisting of neutrophils (elastase positive) and monocytes/macrophages (CD68 positive). Modified Gram staining revealed 20 to over 100 gram-negative diplococci in each 4-microm-thick section, usually grouped into microcolonies. Immunoperoxidase staining demonstrated that the invading meningococci expressed PorA, capsule, and type IV pilin. Expression of these antigens was not restricted to any particular environment and was found in association with meningococci located in leukocytes, small blood vessels, and the dermal interstitium. Confocal laser scanning microscopy demonstrated coexpression of pilin and capsule by numerous microcolonies. However, there was some discordance in capsule and pilin expression within the microcolonies, suggesting phase variation. The strategy employed in this study will be helpful in investigating invasive bacterial diseases where antigenic and phase variation has a significant impact on virulence and on vaccine design.

Exploiting M cells for drug and vaccine delivery.

The specialised antigen sampling M cells represent an efficient portal for mucosal drug and vaccine delivery. Delivery may be achieved using synthetic particulate delivery vehicles including poly(DL-lactide-co-glycolide) microparticles and liposomes. M cell interaction of these delivery vehicles is highly variable, and is determined by the physical properties of both particles and M cells. Delivery may be enhanced by coating with reagents including appropriate lectins, microbial adhesins and immunoglobulins which selectively bind to M cell surfaces. Live attenuated microorganisms are also suitable as vaccines and mucosal vectors and many, including Salmonella typhimurium, innately target to M cells. After cell surface adhesion, delivery vehicles are rapidly transported across the M cell cytoplasm to underlying lymphoid cells and may subsequently disseminate via the lymphatics. Further definition of M cell development and function should permit exploitation of their high transcytotic capacity for safe and reliable mucosal delivery.

Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells.

Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1-encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin-Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F-actin in tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin-stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild-type and sipA mutant strains of S. typhimurium. Time-lapse phase-contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild-type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild-type bacteria or failed to invade at all. Furthermore, although wild-type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild-type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.

The role of M cells in Salmonella infection.

Intestinal M cells, the specialised antigen-sampling cells of the mucosal immune system, are exploited by Salmonella and other pathogens as a route of invasion. Salmonella entry into M cells and colonisation of Peyer's patches involve mechanisms critical for infection of cultured cells as well as factors not accurately modelled in vitro.

Localization of dysfunctional tight junctions in Salmonella enterica serovar typhimurium-infected epithelial layers.

Infection of polarized MDCK epithelial layers by Salmonella enterica serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for Salmonella-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.

Lectin-mediated mucosal delivery of drugs and microparticles.

Absorption of drugs and vaccines at mucosal surfaces may be enhanced by conjugation to appropriate bioadhesins which bind to mucosal epithelia. Bioadhesins might also permit cell- and site-selective targeting. One approach is to exploit surface carbohydrates on mucosal epithelial cells for lectin-mediated delivery. We review work supporting the use of lectins as mucosal bioadhesins in the gastrointestinal and respiratory tracts, the oral cavity and the eye. The gastrointestinal tract is particularly favoured for mucosal delivery. Many studies have demonstrated that the antigen sampling intestinal M cells offer a portal for absorption of colloidal delivery vehicles. Evidence is presented that M cell targeting may be achieved using M cell-specific lectins, microbial adhesins or immunoglobulins. While many hurdles must be overcome before mucosal bioadhesins can guarantee consistent, safe, effective mucosal delivery, this is an exciting area of research that has important implications for future drug and vaccine formulation.

Studying M cells and their role in infection.

In addition to sampling antigens, M cells are a common route for pathogen invasion. Recent studies have partly defined the mechanisms by which pathogens interact with and exploit M cells as a gateway into the host. New research tools are facilitating studies on M cell infection, differentiation and function.

Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia.

Polarized monolayers of strain II Madin-Darby canine kidney cells (MDCK II) were treated with vanadate/H2O2, known inhibitors of protein tyrosine phosphatase activity. Vanadate/H2O2 treatment resulted in a rapid increase in paracellular permeability as revealed by decreased transepithelial resistance and increased permeability to inulin. These alterations in epithelial barrier function coincided with increased phosphotyrosine immunofluorescence in the vicinity of intercellular junctions and with redistribution of F-actin, the adherens junction protein E-cadherin and the tight junction protein ZO-1. The effects of vanadate/H2O2 on intercellular junction permeability and protein distribution were completely blocked by the specific protein tyrosine kinase (PTK) inhibitor tyrphostin 25 and partially inhibited by the alternative PTK inhibitor genistein. The relative potency of these two inhibitors in blocking the effects of vanadate/H2O2 on intercellular junctions correlated with their abilities to inhibit tyrosine phosphorylation. The potent ser/thr protein kinase inhibitor staurosporine had only a small influence on the vanadate/H2O2-induced increase in paracellular permeability and did not affect the observed redistribution of intercellular junction proteins or phosphotyrosine immunofluorescence. The relative potencies of these distinct protein kinase inhibitors in reversing the effects of vanadate/H2O2 indicate that these effects are directly related to tyrosine phosphorylation. In conclusion, our data provide evidence that enhanced tyrosine phosphorylation of intercellular junction proteins in MDCK epithelia increases paracellular permeability and can also induce prominent reorganization of the junctional complex.

The rat mucosal mast cell chymase, RMCP-II, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and occludin.

Mucosal mast cells undergo hyperplasia in a variety of inflammatory bowel diseases including nematode infection in man and animals. The intra-epithelial localization of these cells make their soluble mediators prime candidates for modulators of epithelial function. In particular previous in vivo and ex vivo studies have established a link between the release of the highly soluble mast cell granule chymases and increased mucosal permeability. The hypothesis that the rat mast cell protease, RMCP-II, directly increases permeability to macromolecules via the paracellular route is tested in this study. Monolayers of epithelial cells (Madin-Darby canine kidney cell line) were exposed to varying concentrations of RMCP-II in vitro, in the absence of other cell types or mediators, and the effect on permeability and tight junction associated proteins was investigated. Basolateral, but not apical, exposure of polarized MDCK monolayers on porous supports to RMCP-II led to concentration- (> 100 microg/ml) and time-dependent increases in electrical conductance and permeability to mannitol (MW182) and inulin (MW5000), which was accompanied by decreases in the immunostaining of the tight junction-associated proteins occludin and ZO-1. Furthermore, prolonged exposure to RMCP-II (> 12 hours) resulted in the formation of identifiable gaps separating adjacent epithelial cells, in the absence of evidence of cytotoxicity. Inhibition of RMCP-II with Soya bean trypsin inhibitor completely abrogated the response, demonstrating that proteolysis was required. These data provide direct evidence that the rat mast cell chymase RMCP-II can, in the absence of other inflammatory mediators, increase epithelial permeability via an effect on the paracellular route.