Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

Microneedle-Assisted Transfersomes as a Transdermal Delivery System for Aspirin.

Transdermal drug delivery systems offer several advantages over conventional oral or hypodermic administration due to the avoidance of first-pass drug metabolism and gastrointestinal degradation as well as patients' convenience due to a minimally invasive and painless approach. A novel transdermal drug delivery system, comprising a combination of transfersomes with either solid silicon or solid polycarbonate microneedles has been developed for the transdermal delivery of aspirin. Aspirin was encapsulated inside transfersomes using a "thin-film hydration sonication" technique, yielding an encapsulation efficiency of approximately 67.5%. The fabricated transfersomes have been optimised and fully characterised in terms of average size distribution and uniformity, surface charge and stability (shelf-life). Transdermal delivery, enhanced by microneedle penetration, allows the superior permeation of transfersomes into perforated porcine skin and has been extensively characterised using optical coherence tomography (OCT) and transmission electron microscopy (TEM). In vitro permeation studies revealed that transfersomes enhanced the permeability of aspirin by more than four times in comparison to the delivery of unencapsulated "free" aspirin. The microneedle-assisted delivery of transfersomes encapsulating aspirin yielded 13-fold and 10-fold increases in permeation using silicon and polycarbonate microneedles, respectively, in comparison with delivery using only transfersomes. The cytotoxicity of different dose regimens of transfersomes encapsulating aspirin showed that encapsulated aspirin became cytotoxic at concentrations of ≥100 µg/mL. The results presented demonstrate that the transfersomes could resolve the solubility issues of low-water-soluble drugs and enable their slow and controlled release. Microneedles enhance the delivery of transfersomes into deeper skin layers, providing a very effective system for the systemic delivery of drugs. This combined drug delivery system can potentially be utilised for numerous drug treatments.

Porphyromonas gingivalis Conditioned Medium Induces Amyloidogenic Processing of the Amyloid-ß Protein Precursor upon in vitro Infection of SH-SY5Y Cells.

Background: Cleavage of the amyloid-ß protein precursor (AßPP) mediated by host secretase enzymes, releases several fragments including amyloid-ß (Aß40 and Aß42). Objective: To determine if Porphyromonas gingivalis conditioned medium cleaved AßPP to release Aß40 and Aß42. Methods: The SH-SY5Y cell line was challenged, in vitro, with P. gingivalis (Pg381) conditioned medium in the presence/absence of cytokines. The cells and their supernatants were assessed for AßPP cleavage fragments by immunoblotting and transmission electron microscopy. Results: Western blotting of the cell lysates with the anti-AßPP C-terminal antibody demonstrated variable molecular weight bands corresponding to full length and fragmented AßPP in lanes treated with the following factors: Tryptic soy broth (TSB), Pg381, IL-6, Pg381 + IL-1ß, and Pg381 + TNF-α. The low molecular weight bands corresponding to the C99 dimerized fragment were observed in the Pg381 and interlukin-6 (IL-6) treated groups and were significantly more intense in the presence of Pg381 with either IL-6 or TNF-α. Bands corresponding to the dimerized C83 fragment were observed with cells treated with TNF-α alone and with Pg381 combined with IL-1ß or IL-6 or TNF-α. The anti-Aß antibody detected statistically significant Aß40 and Aß42, levels when these two Aß species were pooled across test samples and compared to the untreated group. Electron microscopic examination of the supernatants demonstrated insoluble Aß40 and Aß42. Conclusion: These observations strongly imply that AßPP is an infection responsive protein cleaved via the amyloidogenic pathway on exposure to conditioned medium and in the presence of pro-inflammatory mediators.

Antimicrobial, Polarizing Light, and Paired Helical Filament Properties of Fragmented Tau Peptides of Selected Putative Gingipains.

BACKGROUND: Tau is an established substrate for gingipains secreted by Porphyromonas gingivalis. Hyperphosphorylation of tau and neurofibrillary tangle (NFT) formation is a defining lesion of Alzheimer's disease (AD) where NFT distribution is related to Braak stage and disease severity. OBJECTIVE: To assess gingipains'-fragmented tau peptides for their antimicrobial properties and for the likelihood of paired helical/straight filament (PHF/SF) formation with implications for the NFT lesion. METHODS: Seven non-phosphorylated (A-G) and three phosphorylated (A-C) tau peptides, were tested for antimicrobial properties against P. gingivalis. Polarizing light properties were determined using Congo Red staining. Secondary and tertiary structures of peptides B-F were determined using transmission electron microscopy (TEM) and circular dichroism (CD) was undertaken for the soluble peptides A in phosphorylated and non-phosphorylated states. RESULTS: Phosphorylated tau peptide A displayed a significant effect against planktonic P. gingivalis. The CD results demonstrated that both peptides A, in phosphorylated and non-phosphorylated states, in aqueous solution, adopted mainly ß-type structures. Non-phosphorylated peptides B-F and phosphorylated peptides B-C were insoluble and fibrillar under the TEM. The secondary and tertiary structures of the non-phosphorylated peptide B demonstrated fewer helical twists, whereas peptide C displayed significantly more helical twists along the whole fiber(s) length following its phosphorylation. CONCLUSION: Phosphorylated peptide A reduced P. gingivalis viability. CD spectroscopy demonstrated the phosphorylated and the non-phosphorylated peptide A predominantly formed from ß-sheet structures in aqueous solution with potential antimicrobial activity. Phosphorylation of tau peptides physically changed their tertiary structure into PHFs with potential for self-aggregation and binding to the NFT lesion.

Ex vivo Detection of Amyloid-ß in Naturally Formed Oral Biofilm.

Background: Oral infection has been implicated in the possible etiology of Alzheimer's disease. Objective: To detect amyloid-ß (Aß) within microbial biofilms. Methods: Freshly extracted teeth (N = 87) with periodontal disease were separated into Group A (N = 11), with primary root canal infection and Group B (N = 21) with failed endodontic treatment identified by the presence of, gutta percha root filling. Biofilm characteristics were observed by scanning electron microscopy (SEM). Demineralized paraffin wax embedded tooth sections and mineralized calculus biofilm were immunostained with the anti-Aß antibody. The gutta perchas were processed either for on-section acrylic resin tissue immunocolloidal gold silver staining (IGSS) using the anti-Aß antibody or in Araldite resin for ultrastructure. Results: SEM demonstrated calculus and gutta percha in situ harboring a polymicrobial biofilm featuring extracellular polymeric substance (EPS) and water channels. Immunohistochemistry on rehydrated paraffin wax tooth sections from Group A, demonstrated Aß staining on external (calculus and plaque) and all intracanal infected regions. In Group B, the gutta percha biofilm IGSS gave an inconclusive result for Aß. Transmission electron microscopy of selected teeth with infected intra-canals (Group A) and 20% of gutta percha biofilm (Group B) EPS contained electron dense fibrils of variable sizes, some of which were typical of human Aß fibrils. Conclusion: This study detected both soluble and insoluble Aß fibrils within the EPS of periodontal and endodontic natural biofilm, strongly suggesting its role as an antimicrobial peptide in combatting local infection, with potential risk for cross-seeding into the brain for AD development.

The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach.

The bacterial nitroreductase NfnB has been the focus of a great deal of research for its use in directed enzyme prodrug therapy in combination with the nitroreductase prodrug CB1954 with this combination of enzyme and prodrug even entering clinical trials. Despite some promising results, there are major limitations to this research, such as the fact that the lowest reported Km for this enzyme far exceeds the maximum dosage of CB1954. Due to these limitations, new enzymes are now being investigated for their potential use in directed enzyme prodrug therapy. One such enzyme that has proved promising is the YfkO nitroreductase from Bacillus Licheniformis. Upon investigation, the YfkO nitroreductase was shown to have a much lower Km (below the maximum dosage) than that of NfnB as well as the fact that when reacting with the prodrug it produces a much more favourable ratio of enzymatic products than NfnB, forming more of the desired 4-hydroxylamine derivative of CB1954.

The use of metal-catalyzed oxidation to suppress background staining caused by marker amplification reagents and the effects of this oxidation on the stability of antibody-antigen complexes in immunohistochemistry.

Marker amplification is a powerful technique for visualizing immunohistochemically deposited markers that otherwise would be invisible. Amplification usually is achieved with physical developers, which are solutions that contain a source of silver(I) plus a reducing agent. When the marker is present in extremely small quantities, prolonged incubation in the developer is required and unwanted background staining in the form of type III argyrophilia becomes problematic. Suppression of type III argyrophilia can be achieved by metal-catalyzed oxidation using the copper/H2O2 system, which normally is applied immediately prior to amplification. Because there is no reason, in principle, why metal-catalyzed oxidation should not be employed at earlier stages in the immunohistochemical staining procedure, we investigated whether earlier oxidation might confer any advantages over the traditional methodology. Immunocolloidal gold combined with two light insensitive physical developers was chosen as the model system, because visualization by light microscopy requires extended periods in the developers. Moreover, the system does not suffer from problems concerning endogenous enzyme- or non-enzyme-catalyzed marker deposition. Applying metal-catalyzed oxidation at each stage of the immunohistochemical procedure revealed that the technique could be employed successfully prior to staining, but not following the primary or secondary antibodies. In the latter cases, specific immunolocalization was lost entirely and only generalized nonspecific staining was seen. A limited investigation into the mechanism of metal-catalyzed oxidation of aldehyde fixed tissue sections suggested that it involved the formation of aldehyde groups. We suggest that the application of metal-catalyzed oxidation prior to immunohistochemical staining would have the advantages of both suppressing type III argyrophilia and inhibiting unwanted endogenous peroxidase activity. We also suggest that metal-catalyzed oxidation might reduce the affinity of tissue for other transition metals, such as copper, whose potential for improving marker amplification techniques has been demonstrated previously in dot-blot model systems.

Massive Accumulation of Myofibroblasts in the Critical Isthmus Is Associated With Ventricular Tachycardia Inducibility in Post-Infarct Swine Heart.

OBJECTIVES: In this study the authors determined the extent of cellular infiltration and dispersion, and regional vascularization in electrophysiologically (EP) defined zones in post-myocardial infarction (MI) swine ventricle. BACKGROUND: The critical isthmus (CI) in post-MI re-entrant ventricular tachycardia (VT) is a target for catheter ablation. In vitro evidence suggests that myofibroblasts (MFB) within the scar border zone (BZ) may increase the susceptibility to slow conduction and VT, but whether this occurs in vivo remains unproven. METHODS: Six weeks after mid-left anterior descending coronary artery occlusion, EP catheter-based mapping was used to assess susceptibility to VT induction. EP data were correlated with detailed cellular profiling of ventricular zones using immunohistochemistry and spatial distribution analysis of cardiomyocytes, fibroblasts, MFB, and vascularization. RESULTS: In pigs with induced sustained monomorphic VT (mean cycle length: 353 ± 89 ms; n = 6) the area of scar that consisted of the BZ (i.e., between the normal and the low-voltage area identified by substrate mapping) was greater in VT-inducible hearts (iVT) than in noninducible hearts (non-VT) (p < 0.05). Scar in iVT hearts was characterized by MFB accumulation in the CI (>100 times that in normal myocardium and >5 times higher than that in the BZ in non-VT hearts) and by a 1.7-fold increase in blood vessel density within the dense scar region extending towards the CI. Sites of local abnormal ventricular activity potentials exhibited cellularity and vascularization that were intermediate to the CI in iVT and BZ in non-VT hearts. CONCLUSIONS: The authors reported the first cellular analysis of the VT CI following an EP-based zonal analysis of iVT and non-VT hearts in pigs post-MI. The data suggested that VT susceptibility was defined by a remarkable number of MFB in the VT CI, which appeared to bridge the few remaining dispersed clusters of cardiomyocytes. These findings define the cellular substrate for the proarrhythmic slow conduction pathway.

Nano-analyses of wear particles from metal-on-metal and non-metal-on-metal dual modular neck hip arthroplasty.

Increased failure rates due to metallic wear particle-associated adverse local tissue reactions (ALTR) is a significant clinical problem in resurfacing and total hip arthroplasty. Retrieved periprosthetic tissue of 53 cases with corrosion/conventional metallic wear particles from 285 revision operations for ALTR was selected for nano-analyses. Three major classes of hip implants associated with ALTR, metal-on-metal hip resurfacing arthroplasty (MoM HRA) and large head total hip replacement (MoM LHTHA) and non-metal-on-metal dual modular neck total hip replacement (Non-MoM DMNTHA) were included. The size, shape, distribution, element composition, and crystal structure of the metal particles were analyzed by conventional histological examination and electron microscopy with analytic tools of 2D X-ray energy dispersive spectrometry and X-ray diffraction. Distinct differences in size, shape, and element composition of the metallic particles were detected in each implant class which correlate with the histological features of severity of ALTR and variability in implant performance.

Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis.

Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ.

Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity.

Retinal ganglion cell dendritic pruning has been reported in association with a 50% reduction in Opa1 transcript and protein in retinal and neural tissue, which manifests as visual dysfunction in the heterozygous mutant mouse, B6;C3-Opa1(Q285STOP). Here we report a marked reduction in retinal ganglion cell synaptic connectivity in the absence of soma loss and explore the mechanism and relationship between mitochondrial integrity and synaptic connectivity. We observed decreased levels of postsynaptic density protein 95 in Opa1(+/-) mutant mice consistent with synaptic loss in the inner plexiform layer. Glutamatergic but not γ-aminobutyric acid-ergic synaptic sites were reduced in Opa1(+/-) mice. We observed increased synaptic vesicle number in bipolar cell terminal arbours assessed by immunohistochemistry, electron microscopy and western blot analysis. These changes occur without significant loss of mitochondrial membrane potential in retina and optic nerve. Analysis of biolistically transfected retinal ganglion cells shows the retraction of mitochondria towards the soma, and mitochondrial fragmentation, preceding dendritic loss. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina.

Defects in cell polarity underlie TSC and ADPKD-associated cystogenesis.

Clinical trials are underway for the treatment of tuberous sclerosis (TSC)-associated tumours using mTOR inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+/- and Tsc2+/- mice do not exhibit mTOR activation, suggesting that pharmacological targeting of an alternative pathway may be necessary to prevent tumour formation. Patients with TSC often develop renal cysts and those with inherited co-deletions of the autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) develop severe, early onset, polycystic kidneys. Using mouse models, we showed a genetic interaction between Tsc1 and Tsc2 with Pkd1 and confirmed an mTOR-independent pathway of renal cystogenesis. We observed that the Tsc and Pkd1 gene products helped regulate primary cilia length and, consistent with the function of this organelle in modulating cell polarity, found that many dividing pre-cystic renal tubule and hepatic bile duct cells from Tsc1, Tsc2 and Pkd1 heterozygous mice were highly misoriented. We therefore propose that defects in cell polarity underlie TSC and ADPKD-associated cystic disease and targeting of this pathway may be of key therapeutic benefit.

Can the choice of intermediate solvent or resin affect glomerular basement membrane thickness?

BACKGROUND: Establishing glomerular basement membrane (GBM) thickness is important in the diagnosis of some renal diseases. It is widely believed that GBM thickness varies according to the processing method, yet there appear to be no published data to support this. In this study we aimed to determine whether the choice of intermediate solvent and embedding resin influenced the thickness of the GBM and to assess the magnitude of any such effect. METHODS: Subcapsular renal cortical rat tissue was processed for electron microscopy using four different intermediate solvents [propylene oxide (PO), xylene (Xyl), acetone (Ac) or ethanol (Et)], and three epoxy resins [Araldite (Aral), TAAB embedding resin (TER) or TAAB low viscosity resin (TLV)]. GBM thickness was estimated by the orthogonal intercept method. RESULTS: Compared with PO-Aral embedding, GBM was significantly thinner in Xyl-Aral, Ac-Aral and Xyl-TER, and significantly thicker in PO-TER-, Ac-TER-, Et-TER- and PO-TLV-embedded tissue (P < 0.05 in all cases). No significant difference was seen with Xyl-TLV- and Ac-TLV-embedded tissue. Et-Aral and Et-TLV embedding resulted in poor quality blocks. CONCLUSIONS: The results suggest that the choice of both intermediate solvent and resin influences shrinkage of the GBM during TEM processing.

Immunolocalization of caveolin-1 in rat and human mesothelium.

Flask-shaped vesicles have been described as caveolae in mesothelial cells in a number of animal species based on morphological criteria only. Using an antibody against caveolin-1, said to be a biochemical marker of caveolae, immunoelectron microscopy suggests that many but not all such vesicles in mesothelial cells are caveolae. Mesothelial cells from different anatomical sites showed obvious variations in both the population density and distribution of these flask-shaped vesicles and in their density of immunostaining. Lung and pericardial sac had the highest staining density. In some sites (e.g., lung, bladder, colon) caveolae were equally distributed between apical and basolateral surfaces, whereas in others (e.g., spleen, liver), they were predominantly apical. Additional immunopositive sites in the peritoneal membrane were identified, including the epineurium of peripheral nerves and the endothelium of lymphatic vessels. We further suggest that variations in the number of mesothelial cell caveolae and the density of their immunolabeling may have implications for our understanding of certain diseases such as malignant mesothelioma, especially in view of the recent hypothesis that it may be caused by SV40, a virus that appears to enter cells via caveolae.

Can artifact mimic the pathology of the peritoneal mesothelium?

OBJECTIVE: A peritoneal biopsy registry was established to examine morphological and functional changes to the peritoneum during peritoneal dialysis (PD). During the early stages of this study, it became clear that surgical trauma to the peritoneum at the time of biopsy could cause a variety of changes to the surface. We examined the effects of surgical trauma in a rat biopsy model. DESIGN: Rat peritoneum was subjected to a variety of traumas that might occur at biopsy and compared with peritoneal biopsies that had been collected, using the suture method described here, from PD patients. Changes in the quality of non-PD biopsies taken before and after the development of the suture technique were evaluated. RESULTS: In the rat model, external massaging of the peritoneum induced moderate loss of microvilli. Brief light touching caused distortion of the mesothelial surface. Pressing resulted in mesothelial denudation and thin strands of presumed cellular remains. Rubbing caused complete loss of mesothelial cells and their basement membrane. Air drying caused progressive loss of microvilli and eventual cellular distortion. Comparison with peritoneal biopsies from PD patients revealed similarities with certain types of trauma, namely, air drying and pressing. Collection of peritoneal biopsies using the suture method significantly improved specimen quality compared with specimens taken before its introduction (p < 0.025%). CONCLUSION: These results illustrate the sensitivity of the mesothelium to mechanical trauma, the possibility of confusing trauma with genuine pathology, and, hence, the necessity of employing a trauma-free method of biopsy collection, such as the technique described here.

Morphologic changes in the peritoneal membrane of patients with renal disease.

This study examined the morphologic features of the parietal peritoneal membranes of 130 patients undergoing peritoneal dialysis (PD) and compared them with the features of the peritoneal membranes of normal individuals, uremic predialysis patients, and patients undergoing hemodialysis. The median thickness of the submesothelial compact collagenous zone was 50 microm for normal subjects, 140 microm for uremic patients, 150 microm for patients undergoing hemodialysis, and 270 microm for patients undergoing PD (P < 0.001 for all versus normal subjects). Compact zone thickness increased significantly with the duration of PD therapy [0 to 24 mo, 180 microm (n = 58); 25 to 48 mo, 240 microm (n = 24); 49 to 72 mo, 300 microm (n = 13); 73 to 96 mo, 750 microm (n = 16); >97 mo, 700 microm (n = 19)]. Vascular changes included progressive subendothelial hyalinization, with luminal narrowing or obliteration. These changes were absent in samples from normal subjects but were present in 28% of samples from uremic patients and 56% of biopsies from patients undergoing PD. In the PD group, the prevalence of vasculopathy increased significantly with therapy duration (P = 0.0001). The density of blood vessels per unit length of peritoneum was significantly higher for patients with membrane failure and was correlated with the degree of fibrosis (P = 0.01). For the first time, a comprehensive cross-sectional analysis of the morphologic changes in the peritoneal membranes of patients undergoing PD is provided. The infrequency of fibrosis in the absence of vasculopathy suggests that vasculopathy may predispose patients to the development of fibrosis. This study provides a sufficiently large cohort of samples to allow structure-function relationships to be established, as well as providing a repository of tissue for further studies.