Micropatterning of polymer brushes: grafting from dewetting polymer films for biological applications.

In this novel platform, a micropatterned polymer brush was obtained by grafting poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMA)) from a thin macroinitiator film using atom transfer radical polymerization (ATRP). A pattern of holes was formed in the macroinitiator film by taking advantage of its spontaneous dewetting above the glass transition temperature from a bottom polystyrene film, driven by unfavorable intermolecular forces. Patterning by dewetting can be achieved at length-scales from a few hundred nanometers to several tens of micrometers, by simply thermally annealing the bilayer above the glass transition temperature of the polymer. This approach is substrate-independent, as polymer films can be cast onto surfaces of different size, shape, or material. As a demonstration of its potential, proteins, and individual cells were attached on targeted bioadhesive polystyrene areas of the micropatterns within poly(PEGMA) protein-repellent brushes. We anticipate this approach will be suitable for the patterning of brushes, especially for biomedical applications such as in the study of single cells and of cell cocultures.

Printability and bio-functionality of a shear thinning methacrylated xanthan-gelatin composite bioink.

3D bioprinting is a recent technique that can create complex cell seeded scaffolds and therefore holds great promise to revolutionize the biomedical sector by combining materials and structures that more closely mimic the 3D cell environment in tissues. The most commonly used biomaterials for printing are hydrogels, however, many of the hydrogels used still present issues of printability, stability, or poor cell-material interactions. We propose that bioinks with intrinsic self-assembling and shear thinning properties, such as xanthan gum, can be methacrylated (XGMA) and combined with a bio-functional material such as gelatin methacryloyl (GelMa) to create a stable, cell-interactive bioink with improved properties for 3D bioprinting. These biomaterials have reduced viscosity under high shear and recover their viscosity rapidly after the shear is removed, retaining their shape, which translates to easier extrusion whilst maintaining accurate fidelity after printing. This was confirmed in printing studies, with measured normalized strand widths of 1.2 obtained for high gel concentrations (5+5 % XGMA-GelMA). Furthermore, the introduction of a secondary photo-cross-linking method allowed tuning of the mechanical properties of the hydrogel with stiffness between 15 and 30 kPa, as well as improving the stability of the hydrogel with retention of 75 % of its mass after 90 d. The hydrogel was shown to be biocompatible and bio-active with 97 % cell viability, and cell spreading after 7 d of culture for low gel concentrations (3+3 % XGMA-GelMA). Shear stresses were relatively low while printing (1 kPa) as a result of the shear thinning property of the material, which supported cell viability during extrusion. Finally, printed hydrogels retained high cell viability for lower gel concentrations, and showed improved cell viability for more concentrated hydrogels when compared to cells cultured in bulk hydrogels, presumably due to improved nutrient/oxygen diffusion and cell migration. In conclusion, stability and formulation of a XGMA-GelMA shear thinning composite hydrogel has been optimized to create a bio-functional bioink, with improved printability, andin vitroculture stabilityviasecondary photo-induced cross-linking, making this composite a promising bioink for 3D bioprinting.

Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

Elevation of cytosolic calcium ([Ca2+]i) has been reported to induce apoptosis in a number of cell types. However, in the neutrophil, which undergoes apoptosis constitutively during aging in vitro, activation by inflammatory mediators elevates [Ca2+]i and prolongs lifespan via inhibition of apoptosis. To examine this paradox, we investigated the effects of modulation of [Ca2+]i upon apoptosis of neutrophils in vitro. Calcium ionophores (A23187, ionomycin) retarded apoptosis in neutrophil populations after 20 h (P < 0.001). Conversely, intracellular Ca(2+)-chelation, using bis-(o-aminophenoxy)-N,N,N'N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) promoted apoptosis (P < 0.02). W-7 (an inhibitor of calmodulin) also promoted apoptosis (P < 0.05). Measurements of [Ca2+]i, using fura-2, showed (a) increased apoptosis in neutrophil populations was not associated with elevated [Ca2+]i, (b) neutrophils cultured with ionophore at concentrations inhibiting apoptosis exhibited transient (< 1 h) elevations of [Ca2+]i, to levels previously reported with receptor-mediated stimuli, and (c) BAPTA was able to prevent the elevation of [Ca2+]i and the inhibition of apoptosis produced by ionophore. Modulation of apoptosis occurred without alterations in intracellular pH. Thus, in the neutrophil, unlike lymphoid cells, elevation of [Ca2+]i exerts an inhibitory effect upon apoptosis. Furthermore, these data suggest that transient elevation of [Ca2+]i elicits signaling events leading to prolonged inhibition of apoptosis.

The neutrophil.

In 'beneficial inflammation', which is the major component of our innate immune system, it is possible to predict an 'ideal' sequence of cellular events: neutrophil migration would be rapid; time of contact with endothelial cells minimized; matrix degradation localized, with specific turn-on and turn-off of degradation mechanisms; neutrophil secretion and disintegration would be kept to a minimum during bacterial killing; and finally, rapid cessation of neutrophil migration and rapid removal of intact senescent cells would occur. Any doubts that the cellular events of the early stages of acute inflammation normally involve highly sophisticated cellular interactions, presumably designed to minimize tissue perturbation, should be dispelled by two elegant recent studies of neutrophil-endothelial interaction. Clearly, defects in the control of these processes could tip the balance towards cell injury or excessive matrix degradation and initiate amplification mechanisms leading to persistent inflammation and disease. The further identification of molecular mechanisms of these events should permit specific intervention in neutrophil-mediated disease. However, it is important to remember, firstly, that the neutrophil is just a part of the highly redundant inflammatory process and the removal of one 'strand' does not mean that the whole 'web' breaks down, and secondly, that impairment of neutrophil mechanisms may critically impair our anti-bacterial defences. Therefore, continued attempts should be made to define how cells and mediators interact in concert, to determine the fine specificity of molecular mechanisms and, in parallel, to identify 'time windows' in diseases, during which these mechanisms are more critical to the processes damaging the host than they are essential to its defences.

Parkinsonism-hyperpyrexia syndrome: the role of electroconvulsive therapy.

Herein, we present a case of a parkinsonism-hyperpyrexia syndrome (PHS) in a 58-year-old man with a 10-year history of Parkinson's disease. The patient presented with a 2-week history of fever and increasing confusion, in the context of a number of changes to his medication regimen. On presentation, he was noted to be febrile with autonomic instability, diaphoresis and marked rigidity. He was disoriented and responding to visual hallucinations. Investigations revealed an elevated creatine kinase and a provisional diagnosis of PHS was made. After the patient failed to respond during a 2-week period to supportive measures, electroconvulsive therapy (ECT) treatment was commenced. A good response to eight bilateral ECT treatments was achieved, with resolution of his confusional state and associated psychotic phenomena. We discuss the nosological and management issues associated with this case and discuss the role of ECT as a treatment modality in this condition.

Regulation of a multigenic invasion programme by the transcription factor, AP-1: re-expression of a down-regulated gene, TSC-36, inhibits invasion.

The transcription factor AP-1 (activator protein-1) is required for transformation by many oncogenes, which function upstream of it in the growth factor-ras signal transduction pathway. Previously, we proposed that one role of AP-1 in transformation is to regulate the expression of a multigenic invasion programme. As a test of this proposal we sought to identify AP-1 regulated genes based upon their differential expression in 208F rat fibroblasts transformed by FBR-v-fos (FBR), and to determine if they functioned in the invasion programme. Subtracted cDNA libraries specific for up- or down-regulated genes in FBRs compared to 208Fs were constructed and analysed. Northern analysis revealed that the cDNAs in both libraries represented differentially expressed genes. Nucleic acid sequence analysis of randomly selected cDNA clones from each library coupled with searches of nucleic acid and amino acid sequence databases determined that many of the cDNAs represented proteins that function in various aspects of the invasion process. Functional analysis of one the down-regulated genes, TSC-36/follistatin-related protein (TSC-36/Frp), which has not previously been associated with invasion, demonstrated that its expression in FBRs inhibited in vitro invasion. These results support the proposal that AP-1 in transformed cells regulates a multigenic invasion programme.

Phagocytosis of apoptotic neutrophils does not induce macrophage release of thromboxane B2.

Senescent human neutrophils undergo programmed cell death (apoptosis), leading to their recognition and phagocytosis by mature macrophages. At inflamed sites in vivo these processes may represent a neutrophil removal mechanism with the potential to limit the histotoxic capacity of these cells. Phagocytosis can provoke marked proinflammatory responses by macrophages. A macrophage proinflammatory response to the ingestion of apoptotic neutrophils would limit the efficacy of this neutrophil removal mechanism as a component of inflammatory resolution. In the present study we examined two macrophage proinflammatory responses; secretion of the granule enzyme N-acetyl-beta-D-glucosaminidase (NAG) and release of the membrane lipid-derived inflammatory mediator thromboxane A2 (TxA2, measured as TxB2). By contrast with the marked release of NAG and TxB2 elicited by phagocytosis of control particles (opsonised zymosan and immunoglobulin G-coated erythrocytes), macrophage ingestion of apoptotic neutrophils resulted in minimal release of NAG and no release of TxB2; indeed, there was a small depression of TxB2 release that was not due to a toxic effect of neutrophil uptake because macrophages ingesting apoptotic neutrophils retained marked TxB2 responses to subsequent stimulation with opsonised zymosan. Furthermore, there was significant TxB2 release in response to macrophage phagocytosis of apoptotic neutrophils that had been coated with opsonic serum, demonstrating that the lack of macrophage response was determined by the mechanism of recognition rather than the properties of the apoptotic particle itself. These observations are consistent with the hypothesis that macrophage clearance of senscent neutrophils undergoing apoptosis is an injury-limiting mechanism that favors resolution rather than persistence of the inflammatory response and are consistent with observations that the waves of apoptotic cell removal seen in embryological removal and thymic involution do not trigger an inflammatory response.

Coupling of neutrophil apoptosis to recognition by macrophages: coordinated acceleration by protein synthesis inhibitors.

Onset of apoptosis in many cell types, including the neutrophil granulocyte, leads to recognition and ingestion by macrophages, a key regulatory step in clearance of inflammatory cells from inflamed sites. These studies examined the requirement for protein synthesis in neutrophil apoptosis and in the recognition of apoptotic neutrophils by monocyte-derived macrophages. Treatment with cycloheximide or actinomycin D produced a time- and concentration-dependent acceleration of apoptosis in populations of neutrophils purified from human peripheral blood. Both compounds caused significant promotion of apoptosis after 8 h (apoptosis was 7.7 +/- 2.9%, mean +/- SEM, in control populations, 57.5 +/- 4.9% in cycloheximide-treated, and 73.4 +/- 5.5% in actinomycin D-treated populations, n = 4, P < 0.001), which was associated with loss of neutrophil functional ability (assessed by shape change on N-formyl-methionyl-leucyl-phenylalanine stimulation) and increased macrophage recognition and ingestion of neutrophil populations with accelerated apoptosis. These results support the existence of survival proteins, which act as intracellular suppressors of programmed cell death. However, protein synthesis was not required for the recognition process because macrophage recognition was increased pari passu with the morphology of apoptosis.

Transcriptional regulation of cell invasion: AP-1 regulation of a multigenic invasion programme.

The focus of this review will be on the regulation of the multigenic invasion programme by activator protein-1 (AP-1). Investigation of AP-1-regulated gene expression in transformed cells can be used to identify the genes in the multigenic invasion programme and to validate them as targets for diagnosis or therapy.

Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.

Stimulation of cultured human umbilical vein endothelial cells by cytokines such as interleukin-1 and tumour necrosis factor induces de novo synthesis and expression of the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In general, alterations in cell surface expression of these molecules are known to be related to increased gene transcription and altered levels of mRNA. The extension of these observations to the study of inflammatory processes in different human organs necessitates the development of techniques for the quantification of mRNA in small tissue samples. Here we present a method for the quantification of mRNA for E-selectin, VCAM-1 and ICAM-1 using reverse transcription and the polymerase chain reaction (RT-PCR). For each molecule of interest a mutant RNA was synthesised consisting of the wild-type sequence deleted of 15-20 bases. The mutant and wild-type RNA sequences are recognised by the same primers, and can therefore be amplified competitively in the same tube by RT-PCR. As the mutant and wild-type RNAs compete for the primers, the amount of wild-type RNA can be determined by the size of the dominant product that results after addition of known quantities of mutant RNA. Using this detection and quantification method we have examined the dose dependency and time course of mRNA accumulation following TNF-alpha stimulation of HUVEC. Similar time-courses of E-selectin, ICAM-1 and VCAM-1 mRNA accumulation were observed by competitive RT-PCR as by laser densitometry of Northern blots. Finally we were able to show that the technique could measure changes in levels of mRNA for these three molecules in human skin biopsies taken at different times during the development of a delayed hypersensitivity response to tuberculin purified protein derivative. This technique should be useful for the study of adhesion molecule mRNA in small tissue culture samples and in biopsies.

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis.

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 microg/ml, and 100% inhibition at 1000 microg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05-0.01) larvae from passing through the LMI sieves. At 500 microg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200-500 microg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.

Successful treatment of Paecilomyces lilacinus endophthalmitis after foreign body trauma to the cornea.

PURPOSE: To report the successful treatment of a patient with Paecilomyces lilacinus endophthalmitis infection after foreign body (FB) trauma to the cornea. METHODS: A 30-year-old man presented to us with a corneal abscess and iritis 2 months after removal of a metal corneal FB. Initial corneal biopsy culture was negative. Treatment with topical 5% natamycin, 0.9% fortified gentamycin, and 5% cephalothin hourly was commenced. As a result of developing signs of endophthalmitis, two more biopsies were taken, a week apart, from the vitreous and anterior chamber, successively. The last biopsy yielded positive microbiologic results of the specious Paecilomyces lilacinus. Intravitreal injection of 50 microg/0.5 mL of amphotericin was administered during the vitreal biopsy. Soon after isolating the specious Paecilomyces lilacinus, the following treatment was administered: 200 mg of itraconazole bd by mouth, 5% topical natamycin every hour, 2 mg/mL of topical fluconazole every 2 hours, three anterior chamber injections of 0.35 mL of 0.1% fluconazole and two amphotericin B injections to the anterior chamber of 50 microg/0.5 mL each. RESULTS: There appeared to be no sign of infection 6 months after initial treatment. A large, dense scar existed in the medial part of the cornea only. The pupil was secluded. The patient's visual acuity was 6/21. The eye was comfortable and all topical antifungal medication was ceased.

Isolation and characterization of the lignans, isolariciresinol and pinoresinol, in flaxseed meal.

The isolation and characterization of the lignans, isolariciresinol, pinoresinol, secoisolariciresinol, and matairesinol, potent phytoestrogens, from flaxseed meal are described. This is the first report of isolariciresinol and pinoresinol being detected in a food. The extraction method selected combined the removal of the lignan glycosides from the plant matrix with an alcoholic solvent system, followed by acid hydrolysis to release the aglycons. A reversed-phase high-performance liquid chromatography with diode array detection system was used for initial separation and detection of the lignans at 280 nm in the acid-hydrolyzed methanolic extract. Lignan trimethylsilyl ether derivatives were characterized by gas chromatography/mass spectrometry. Secoisolariciresinol is the major lignan in flaxseed; isolariciresinol, pinoresinol, and matairesinol were identified as minor lignan components.

Green tea flavan-3-ols and oligomeric proanthocyanidins inhibit the motility of infective larvae of Teladorsagia circumcincta and Trichostrongylus colubriformis in vitro.

The effects of a hot water infusion and an aqueous acetone extract of green tea (Camellia sinensis) on the motility of infective larvae of the sheep nematodes Teladorsagia circumcincta and Trichostrongylus colubriformis were investigated under in vitro conditions. The infusion and extract dose-dependently inactivated the infective larvae as assessed by the larval migration inhibition (LMI) assay. To determine the components responsible for the inhibitory activity, the hot water infusion and aqueous acetone extract of green tea were fractionated on Sephadex LH-20 and the green tea extract fractions (GTE-I-VIII) characterised by mass spectrometry. The larvae were exposed to increasing concentrations of these GTE fractions. Fractions containing epigallocatechin gallate (EGCG) and proanthocyanidin oligomers were most effective. GTE fractions were more effective against T. circumcincta than T. colubriformis larvae as assessed by the LMI assay.

Vehicle head restraint positioning knowledge and behaviours in a sample of Irish drivers.

A correctly positioned vehicle head restraint (HR) can reduce whiplash injury risk in collisions, however, HRs are often sub-optimally positioned. The primary aim of this study was to investigate vehicle HR position and driver knowledge of correct HR positioning in an Irish population. Secondary aims were to investigate the associations with driver age, gender and vehicle age. Data collection involved HR measurement and a driver questionnaire (n = 110). Just 27% of drivers had optimal HR positioning, while 30% had poor or marginal positioning. Newer vehicles (<5 years old) had better positioned HR in the horizontal plane (p = 0.036), than older vehicles. Younger drivers (<30 years) were more likely to have poorer positioning of HR (p = 0.002), than the 30 years or over group. Females were more likely to have better vertical positioning of their HR (p = 0.003) than males. Driver knowledge of correct position was variable, and not associated with actual HR position, with 65% knowing the correct vertical positioning standard but only 27% identifying the correct horizontal position. Many drivers have inadequately positioned HR, which needs to be addressed by improved vehicle design and public education.

In vivo anthelmintic activity of Dorycnium rectum and grape seed extract against Ostertagia (Teladorsagia) circumcincta and Trichostrongylus colubriformis in sheep.

AIM: To assess the in vivo anthelmintic activity of condensed tannins (CT) in the forage species Dorycnium rectum and Medicago sativa, and in an extract from grape (Vitus vinifera) seeds (GSE), against two species of parasite, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus colubriformis, at different stages of their life cycle, in sheep that were parasite-naïve or previously exposed to nematodes. METHODS: In Trial 1, a factorial treatment structure was used to compare faecal nematode egg counts (FEC) and worm burdens in 40 weaned Romney lambs fed either the CT-containing forage D. rectum (12% dry matter; DM) or M. sativa (lucerne; 0.2% DM). Twenty naïve and 20 previously-exposed lambs were drenched free of parasites then reinfected with known species and numbers of parasites, and housed in pens indoors on a diet of lucerne pellets and chaffed hay. Groups of lambs (n=5 lambs per group) were fed one of the forages over one of two time periods within the parasite's life cycle. Six to nine days after the last feeding of fresh forages, faecal samples were collected for FEC, and all lambs were slaughtered and worm counts conducted. In Trial 2, 12 Suffolk x Romney lambs were surgically implanted with an abomasal cannula and then housed indoors in metabolism crates. After infection with parasites, six lambs were infused continuously over a 14-day period with a commercially available CT GSE (96% DM, made up to 34 g/L in water); the remaining lambs were infused with water. During infusion, samples were collected for egg hatch and larval development assays. After infusion, samples were collected for FEC, and all lambs were slaughtered and worm counts conducted. RESULTS: In Trial 1, there was a significant (p<0.001) difference in burdens of O. circumcincta between naïve lambs and those previously exposed to parasites, but no other differences were recorded. In Trial 2, lambs infused with GSE had significantly (p<0.05) fewer T. colubriformis at slaughter and significantly (p<0.001) fewer eggs hatched in the egg hatch assay (EHA) than for lambs infused with water. Overall, the differences attributable to GSE were small in magnitude, being an 11% drop in egg hatch, and an 18% drop in numbers of adult T. colubriformis after 14 days of continuous infusion. No other differences were recorded. CONCLUSION: The results indicate that the in vivo anthelmintic activity of these CT sources is, at best, modest and is unlikely to be of any practical value. Further, these data emphasise that in vitro activity is an unreliable indicator of in vivo efficacy for CT-containing forages and extracts.

Connexins and the vacuolar proteolipid-like 16-kDa protein are not directly associated with each other but may be components of similar or the same gap junctional complexes.

Gap junction preparations made from mouse liver plasma membranes by alkali extraction contain variable proportions of connexins (Cx32 and Cx26) and the 16-kDa protein which is closely related or may be identical to the 16-kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and the mediatophore complex. The absence of a stoichiometric relationship suggests that connexins and the 16-kDa protein are not subunits of the same channel complex, but analysis of alkali preparations by isopycnic centrifugation shows both types of protein are in membrane structures of the same buoyant density. Electron microscopic analysis of alkali preparations shows a homogeneous population of gap junctions of uniform morphology and width, suggesting the proteins are in the same or similar structures. The structures containing connexins and the 16-kDa protein can be separated by treatment of the plasma membranes with Triton X-100. After such treatment, the connexins remain associated with dense cellular or extracellular material and the gap junctional structures, after further extraction with N-lauroyl sarcosine and urea, contain only the 16-kDa protein. These detergent-extracted gap junctions are thinner (14.1 nm) than those in alkali preparations (18.4 nm).

The degranulation response in differentiated HL-60 cells.

HL-60 cells differentiate into mature granulocytes in response to treatment with a variety of chemical agents. Such HL-60 cell derived granulocytes display many of the properties associated with their peripheral blood counterpart. In this study we have investigated the development of the degranulation response in dimethylsulfoxide (DMSO) or retinoic acid differentiated HL-60 cells over a six day period. The release of a number of enzymes in response to stimulation by a variety of agents was examined. Soluble aggregated IgG (SAIgG) stimulated the release primarily of elastase from HL-60 derived granulocytes with little or no release of other granule enzymes, in particular myeloperoxidase. This contrasted to what was seen when peripheral blood granulocytes were used. The lack of myeloperoxidase release was not due to the parallel release of enzyme inhibitors or failure of the stimulus to bind to the cells. Neither was it due to variations in the kinetics of enzyme release or the presence of myeloperoxidase and elastase in discrete sub-populations of HL-60 cells. When other stimuli such as fMet-Leu-Phe, A23187, or phorbol myristate acetate (PMA) were used a relatively normal degranulation response was seen. Thus, the degranulation response in granulocytes derived from HL-60 cells appears relatively normal when a range of commonly used stimuli are used but is impaired when aggregates of IgG are used.