Antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian medicinal plant Eremophila neglecta.

AIMS: To determine the antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian plant Eremophila neglecta. METHODS AND RESULTS: Antimicrobial activities of serrulatane compounds 8,19-dihydroxyserrulat-14-ene (1) and 8-hydroxyserrulat-14-en-19-oic acid (2) were tested against Gram-negative and Gram-positive bacteria including human and veterinary pathogens and some multidrug-resistant isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were determined by broth microdilution assay. Both compounds exhibited antibacterial activity against all Gram-positive test strains. They showed antimycobacterial activity against isolates of Mycobacterium fortuitum and Mycobacterium chelonae. Of the five Gram-negative bacteria tested, only Moraxella catarrhalis showed susceptibility to the compounds. Cytotoxic activities were tested in the Vero cell line. Compound 1 showed more activity than 2 in both antibacterial and cytotoxicity assays with cytotoxicity at concentrations similar to the MBC. CONCLUSIONS: Serrulatane compounds showed significant activity against medically important bacteria, with 1 exhibiting stronger antibacterial activity. However, they also displayed toxicity to mammalian cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Serrulatanes are of interest as novel antibacterial compounds for use in biomedical applications; this study reports data obtained with a range of bacterial strains and mammalian cells, essential for assessing the capabilities and limitations of potential applicability of these compounds.

Epithelialization of a synthetic polymer in the feline cornea: a preliminary study.

PURPOSE: This study examined the potential of a synthetic polymer to support stable epithelial growth when implanted in the feline cornea. METHODS: A perfluoropolyether-based polymer was cast into lenticules that were coated with collagen I and implanted in four feline corneas. Epithelial growth onto the lenticules was monitored clinically for 6 weeks, after which time the animals were killed, and three corneas were evaluated histologically. Immunohistochemistry was used to identify proteins associated with the formation of a basement membrane (laminin) and adhesion complexes (bullous pemphigoid antigen and collagen VII). Electron microscopy was used to examine the tissue-polymer interface for evidence of the assembly of these adhesive structures. RESULTS: Postoperative epithelial growth began on days 2 to 3, and lenticules were fully epithelialized by days 5 to 9. Lenticules were clinically well tolerated and histology showed epithelium consisting of multiple layers adherent to the lenticule's surface. Laminin, bullous pemphigoid antigen and collagen VII were identified at the tissue-polymer interface using immunohistochemistry. Ultrastructural examination showed evidence of assembly of these proteins into a recognizable basement membrane and hemidesmosomal plaques. CONCLUSIONS: A perfluoropolyether-based polymer coated with collagen I was implanted in the feline cornea and supported epithelial growth that showed signs of persistent adhesion, both clinically and histologically. This polymer shows potential for ophthalmic applications that require sustained epithelialization.

Mechanism of initial attachment of corneal epithelial cells to polymeric surfaces.

The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: 'tissue culture' polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)-80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85-95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture neophytes can employe endogenous mechanisms for the initial attachment to TCPS, whereas cells established in culture are dependent on exogenous vitronectin in order to attach.

Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels.

A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups ('charge') on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.

Irreversible adsorption of human serum albumin to hydrogel contact lenses: a study using electron spin resonance spectroscopy.

Human serum albumin (HSA) was specifically spin labelled with 4-maleimido-tempo (MSL) at its cysteine 34 residue (HSA-MSL). The irreversible adsorption of HSA-MSL to hydrogel contact lenses (etafilcon A, tefilcon and vifilcon A) was investigated using electron spin resonance (ESR) spectroscopy. Changes in ESR spectral characteristics of adsorbed HSA-MSL as compared to HSA-MSL in solution displayed an additional immobilisation of the spin label due to the adsorption. This immobilisation of MSL corresponds to a large conformational alteration of the HSA-MSL near the modified Cys 34 residue. For both etafilcon A and tefilcon, the rate of irreversible adsorption was relatively slow compared with that of vifilcon A where the maximum state of immobilisation and hence conformational change occurred within the first hour of adsorption. Furthermore, tefilcon produced markedly different ESR spectra where a strong conformational change to a less mobile protein was apparent. This supported a model where the direct irreversible adsorption of HSA from solution dominated on tefilcon as opposed to conversion of the adsorbed protein from the reversible to the irreversible state on both etafilcon A and vifilcon A. HSA-MSL adsorption onto hydrophobic poly(methylmethacrylate) (PMMA) and hydrophilic poly(N-ter-butylacrylamide) (PTBAM) latex beads was also investigated. The spin label MSL was found to be less mobile when HSA was adsorbed onto PMMA compared with PTBAM beads. It was also found that the rate of irreversible adsorption of HSA is far higher onto PMMA surfaces than onto PTBAM surfaces.

XPS and surface-MALDI-MS characterisation of worn HEMA-based contact lenses.

XPS and MALDI-MS were used to analyse initial adsorption events in the fouling of HEMA-based contact lenses. All of the lenses tested accumulated tear film deposits within 10 min of wear. XPS indicated the presence of mainly proteinaceous deposits, with indications of some contributions by mucins or lipids on some lenses and the nature of the deposit being influenced by the lens chemistry. MALDI-MS detected the presence of surface-adsorbed species with molecular weights < 15 kDa. While lysozyme could be identified by comparison of MALDI-MS signals with known protein mass and assignments are suggested for some other signals, several other species, with MWs less than that of lysozyme, could not be identified as no ocular proteins with corresponding MWs had been reported in previous biochemical tear film analyses. These species, and others, were also detected in MALDI-MS analysis of reflex tear film, suggesting that the adsorbed unidentified species were not simply products of surface-induced dissociation of adsorbing higher-MW proteins. This short-term wear study detected rapid interface conversion and demonstrated the utility and surface sensitivity of XPS and MALDI-MS in characterising contact lens deposits at the initial stages when sub-monolayer adsorbed amounts are present on lenses.

Surface-MALDI mass spectrometry in biomaterials research.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.

The control of Staphylococcus epidermidis biofilm formation and in vivo infection rates by covalently bound furanones.

In order to overcome the continuing infection rate associated with biomaterials, the use of covalently bound furanones as an antibiofilm coating for biomaterials has been investigated. Furanones have previously been shown to inhibit growth of Gram-positive and Gram-negative bacteria. The aim of these studies were to covalently bind furanones to polymers and to test their efficacy for inhibiting biofilm formation of Staphylococcus epidermidis and in vivo infection rate. Two methods of covalent attachment of furanones were used. The first, a co-polymerisation with a styrene polymer, and second, a plasma-1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) reaction to produce furanone-coated catheters. Biofilm formation by S. epidermidis in vitro was inhibited by 89% for polystryene-furanone disks and by 78% by furanone-coated catheters (p<0.01). In an in vivo sheep model we found furanones were effective at controlling infection for up to 65 days. Furanones have potential to be used as a coating for biomaterials to control infection caused by S. epidermidis.

A thin glycoprotein coating of a synthetic lenticule does not cause nutritional deficiency of the anterior cornea.

PURPOSE: This study investigated whether a glycoprotein coating that will be used to enhance corneal epithelialization affects in situ nutritional passage through a permeable membrane. METHODS: Sixteen adult cats were equally divided into two groups. Polycarbonate membranes with pore size of 0.1 microm and total pore area (porosity) of 3.1% were used as implant materials. The membranes for Group 1 were coated with a thin layer of Collagen I, while the membranes for Group 2 were uncoated. Each membrane with 8-mm diameter was implanted into an interlamellar pocket of the cornea. The eyes were observed for approximately 35 days to monitor clinical signs of nutritional deficiency of the cornea, and then 7 membranes were removed from the eyes. The permeability of the explanted membranes to glucose, inulin and albumin was used to predict the in situ difference between the coated and uncoated groups in regard to nutritional passage through the membranes. To investigate the long-term effect of the surface coating on corneal health, two animals from Group 1 were followed for up to two years and then both eyes of each animal underwent histological examination. RESULTS: Clinically, no post-surgical complications associated with nutritional deficiency were observed in any of the eyes. Nutritional permeability tests showed no significant differences between the coated and uncoated membranes. Histologically, the long-term animals showed no abnormal morphology associated with nutritional deficiency in the cornea anterior or posterior to the membranes. CONCLUSIONS: A thin glycoprotein coating on a permeable membrane does not appear to affect the nutritional supply of the anterior cornea and therefore can be used to enhance epithelialization of synthetic corneal onlays in vivo.

Molecular-level removal of proteinaceous contamination from model surfaces and biomedical device materials by air plasma treatment.

Established methods for cleaning and sterilising biomedical devices may achieve removal of bioburden only at the macroscopic level while leaving behind molecular levels of contamination (mainly proteinaceous). This is of particular concern if the residue might contain prions. We investigated at the molecular level the removal of model and real-life proteinaceous contamination from model and practical surfaces by air plasma (ionised air) treatment. The surface-sensitive technique of X-ray photoelectron spectroscopy (XPS) was used to assess the removal of proteinaceous contamination, with the nitrogen (N1s) photoelectron signal as its marker. Model proteinaceous contamination (bovine serum albumin) adsorbed on to a model surface (silicon wafer) and the residual proteinaceous contamination resulting from incubating surgical stainless steel (a practical biomaterial) in whole human blood exhibited strong N1s signals [16.8 and 18.5 atomic percent (at.%), respectively] after thorough washing. After 5min air plasma treatment, XPS detected no nitrogen on the sample surfaces, indicating complete removal of proteinaceous contamination, down to the estimated XPS detection limit 10ng/cm(2). Applying the same plasma treatment, the 7.7at.% nitrogen observed on a clinically cleaned dental bur was reduced to a level reflective of new, as-received burs. Contact angle measurements and atomic force microscopy also indicated complete molecular-level removal of the proteinaceous contamination upon air plasma treatment. This study demonstrates the effectiveness of air plasma treatment for removing proteinaceous contamination from both model and practical surfaces and offers a method for ensuring that no molecular residual contamination such as prions is transferred upon re-use of surgical and dental instruments.

[New viewpoints on the clinical picture, diagnosis and pathophysiology of reflex sympathetic dystrophy (Sudeck's disease)]. / Neue Gesichtspunkte zur Klinik, Diagnostik und Pathophysiologie der sympathischen Reflexdystrophie (Morbus Sudeck).

Reflex sympathetic dystrophy can be elicited by various factors (e. g. trauma, herpes zoster, myocardial infarction). Independent of kind and site of a lesion, symptoms occur most often in the whole distal part of the affected extremity. There in most cases, a triad of autonomic, motor and sensory disturbances can be found clinically. For early diagnosis--beside clinical investigation--a comparative measurement of skin temperatures on both sides of finger or toe tips, respectively, is recommended. Hereby the clinical finding of a warmer or colder extremity can be proved, which supplies evidence of a disturbed skin blood flow. In case, the above mentioned triad and a disturbance of skin circulation is found, diagnosis of sympathetic reflex dystrophy can be made with great certainty. With regard to the underlying pathophysiology, symptoms can be explained at this time satisfactory only by the assumption of a vicious circle. Starting from a painful event (e.g. trauma, mark in a plaster cast, nerve lesion or myocardial infarction) a functional disturbance of the sympathetic nervous system is initiated. This results in a disturbance of the circulation in all of the affected tissues (skin, muscle, bone and joint), which finally gives rise to an abnormal excitation of afferent receptors, particularly of nociceptors. This excitation maintains the disturbance of the sympathetic nervous system at central nervous level (vicious circle). The most relevant pathomechanism in this process seems to be the occurrence of an imbalance between the activity of sympathetic vasoconstrictor neurons supplying arteries and those, supplying veins. A sympatholytic therapy, if applied in time, is able to cut off the vicious circle, which may lead to a restitutio ad integrum. Further investigations will show to what extent psychological factors are involved in developing the central nervous disturbance of the sympathetic nervous system and may also show if in addition the motor system is affected.

[Distal post-traumatic edema--symptom of a sympathetic reflex dystrophy (Sudeck's disease)?]. / Das distale posttraumatische Odem--Symptom einer sympathischen Reflexdystrophie (M. Sudeck)?

The present paper describes various mechanisms, possibly being involved in the development of the posttraumatic, distally generalized edema. New ideas point to a special importance of the sympathetic vasoconstrictor system for this clinical phenomenon, since this system could induce an enhanced venoconstriction at the exit of the capillary bed, which would result in an edema producing diminished venous return. Since the distally generalized edema is an initially and very commonly occurring symptom of reflex sympathetic dystrophy (M. Sudeck), the observation of such an edema should lead one to look for further symptoms of this disorder, especially for the typical triad of autonomic (sympathetic), motor, and sensory disturbances.

[Neurologic aspects of clinical manifestations, pathophysiology and therapy of reflex sympathetic dystrophy (causalgia, Sudeck's disease)]. / Neurologische Aspekte der Klinik, Pathophysiologie und Therapie der sympathischen Reflexdystrophie (Kausalgie, Morbus Sudeck).

The symptomatology of reflex sympathetic dystrophy (RSD), a diagnostic term which today includes causalgia and M. Sudeck, is characterized clinically by a triad of autonomic (sympathetic), motor and sensory disturbances. They develop following a noxious event--though independent of its nature and location--in a generalized distribution pattern at the distal site of the affected extremity. Pathophysiologically, a complex disturbance of the sympathetic vasoconstrictor system is involved, which mediates the dominant symptoms of RSD, namely the spontaneous pain and the swelling. This disturbance is thought to be initiated by nociceptive impulses, occurring in conjunction with the preceding noxious event, and to be maintained reflexly, in a form of a vicious circle, by means of the typical pain sensation accompanying the RSD-syndrome. From these ideas, an important part of the RSD therapy is deduced; i.e. the early interruption of the neuronal sympathetic activity by means of a sympathetic blockade. Such a blockade can interrupt the pain and at the same time also the vicious circle of RSD. Altogether, for the RSD syndrome there are relevant neurological aspects with respect to its clinical symptomatology, its pathophysiology and its therapy.

Direct detection of proteins adsorbed on synthetic materials by matrix-assisted laser desorption ionization-mass spectrometry.

The irreversible accumulation of biological material on synthetic surfaces ("biofouling") adversely affects for instance contact lenses, implantable biomedical devices, biosensors, water purification, transport and storage systems, and marine structures. It is shown here that proteins adsorbed on contact lenses can be detected directly, rapidly, and conveniently, with high sensitivity, by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers on samples retrieved from clinical usage and in vitro protein adsorption studies, at levels substantially below monolayer coverage. Identification of the detected biological molecules can be done by comparison of the detected mass peaks with known protein molecular masses or with spectra recorded of pure compounds or by separate biochemical assays. The MALDI mass spectra recorded on different contact lenses contain peaks assignable to lysozyme and a number of smaller proteins. Such sensitive characterization of the early stages of biofouling enhances the understanding of protein/materials interactions and assists in designing guided strategies toward control of biological adsorption processes.

Matrix-assisted laser desorption ionization mass spectrometry detection of proteins adsorbed in vivo onto contact lenses.

Identification of the biomolecules that form the first adsorbed monolayer, which thus effect "interface conversion", in competitive adsorption from multicomponent biological solutions can be challenging because of limitations in mass resolution and sensitivity of established techniques. In this study matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry is developed and applied as a novel surface analytical method to enable analysis of adsorbed multicomponent biomolecule layers directly on the biomaterial surfaces. We show that proteins adsorbed in vivo (on human eyes) on contact lenses can be detected rapidly and conveniently by the diagnostic highly resolved mass signals recorded by MALDI mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers at levels substantially below monolayer coverage. Identification is done by comparison with molecular masses of known proteins. Specifically, it is shown that in addition to lysozyme, other low molecular weight proteins adsorb from human tear fluid onto contact lenses; these proteins had not been detected in earlier studies using other techniques.

Covalent attachment and non-specific binding of reactive probe molecules onto surfaces.

Optimization of strategies for the covalent attachment of proteins onto polymer surfaces requires the development of analytical methods which can differentiate between proteins that are covalently attached and those that are non-covalently bound (physisorbed). We probed for the surface density of reactive amine, carbonyl, and hydrazide groups using solution phase derivatization reactions to mimic and explore protein immobilization reaction strategies. Labeling compounds investigated were fluorescein derivatives, which were quantified by adsorption spectroscopy, and fluorinated phenyl compounds which were quantified by XPS. Control experiments consisted of performing the same labeling reactions using surfaces without reactive groups, or immersing the polymer surface into the labeling solution after blocking the reactive group of the labeling compound by a covalent reaction in solution. We always found non-negligible contributions arising from physisorption of the derivatization labels. Multiple control surfaces and a novel 'crossover derivatization-XPS' method were studied with the aim of improving compensation for physisorption. Our documentation of surprisingly large physisorption components even for small molecule labels, together with the known propensity of proteins to adsorb onto polymers, suggests caution in quantitative analysis of surface groups by derivatization, and in interpreting covalent protein immobilizations onto polymeric surfaces.

Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer.

The relative effects of porosity and surface chemistry on the migration of epithelial tissue over the surface of a polymer were determined in vitro. These studies compared nonporous polymers with those having 0.1-microm diameter track-etched pores and were conducted on polycarbonate and polyester. Epithelial tissue migration over the polymer surface was stimulated by the presence of these pores. The surface chemistries of the polymers were modified by deposition of various polymer films using radio frequency gas deposition, giving a range of surfaces that varied in air:water sessile contact angle (SCA) of between 26 and 100 degrees. Tissue migration on the nonporous surfaces was affected by the surface chemistry, being generally linear as a function of the SCA and higher on hydrophilic than on hydrophobic surfaces but reduced if the hydrophilic surface had a mobile chemistry. The effects of the 0.1-microm diameter pores and the surface hydrophilicity were additive with the maximal level of epithelial tissue migration occurring on a porous, hydrophilic polymer surface.

Growth of human cells on plasma polymers: putative role of amine and amide groups.

The attachment and growth of human endothelial cells and fibroblasts was studied on polymer surfaces fabricated by the polymerization of volatile amine and amide compounds in a low pressure gas plasma, and by the treatment of various surfaces in ammonia plasmas, which served to increase the nitrogen content of the surface layers. Infrared spectra showed the presence of amide groups, including those cases where the volatile compound ('monomer') did not contain oxygen. The performance of the surfaces in cell attachment correlated with the surface hydrophilicity and the nitrogen content, although for the latter a fair degree of scatter indicated that a more complex relationship applies. All these surfaces supported the attachment and growth of human cells. Generally, amide plasma polymers were best but the individual monomer and the plasma parameters also played a role. From comparisons of the various surfaces, it is suggested that the amide group is the main promoter of cell attachment in nitrogen-containing plasma surfaces.

Effects of surface topography on corneal epithelialization in vivo: a preliminary study.

PURPOSE: In this study we investigated the relationship between surface topography and initial epithelialization of synthetic lenticules in vivo. METHODS: Millipore ultrafiltration membranes of three different pore sizes were used as model lenticule materials. The nominal membrane pore sizes were 0.1 microm, 0.45 microm, and 3 microm; the surface roughness increased in the same order The membranes were coated with a thin layer of collagen I and implanted in a circular pocket of the anterior cornea of adult cats, and were clinically evaluated for the extent of epithelialization and the persistence of epithelial attachment. RESULTS/CONCLUSIONS: It was demonstrated that a smooth surface appears more attractive for initial epithelialization than a rough surface in vivo.