Evaluation of Copper Chelation Therapy in a Transgenic Rat Model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of the amyloid ß (Aß) protein in blood vessels and leads to hemorrhages, strokes, and dementia in elderly individuals. Recent reports have shown elevated copper levels colocalized with vascular amyloid in human CAA and Alzheimer's disease patients, which have been suggested to contribute to cytotoxicity through the formation of reactive oxygen species. Here, we treated a transgenic rat model of CAA (rTg-DI) with the copper-specific chelator, tetrathiomolybdate (TTM), via intraperitoneal (IP) administration for 6 months to determine if it could lower copper content in vascular amyloid deposits and modify CAA pathology. Results showed that TTM treatment led to elevated Aß load in the hippocampus of the rTg-DI rats and increased microbleeds in the wild type (WT) animals. X-ray fluorescence microscopy was performed to image the distribution of copper and revealed a surprising increase in copper colocalized with Aß aggregates in TTM-treated rTg-DI rats. Unexpectedly, we also found an increase in the copper content in unaffected vessels of both rTg-DI and WT animals. These results show that IP administration of TTM was ineffective in removing copper from vascular Aß aggregates in vivo and increased the development of disease pathology in CAA.

Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame.

Cryomicroscopy is commonly hampered by frost accumulation, reducing the visual clarity of the specimen. Pulling a vacuum or purging with nitrogen gas can greatly reduce the sample chamber's humidity, but at cryogenic temperatures, even minute concentrations of water vapor can still result in frost deposition. Here, a hygroscopic ice frame was created around the specimen to suppress frost growth during cryomicroscopy. Specifically, fluorescently tagged rat brain vessels were frozen on a silicon nitride window with an ice frame, and the luminescence of the fluorescent tag was improved by a factor of 6 compared to a similar specimen in only a nitrogen purge environment. These findings suggest that the simple implementation of a hygroscopic ice frame surrounding the specimen can substantially improve the visual clarity for cryomicroscopy, beyond that of a vacuum or nitrogen purge system.

Plasma-initiated graft polymerization of carbon nanoparticles as nano-based drug delivery systems.

Plasma-initiated free radical polymerization was used to engineer carbon nanoparticles (CNPs) with tailored chemical and physical properties. Following surface modification, CNPs were loaded with a highly effective anti-infection agent called metal-free Russian propolis ethanol extract (MFRPEE), thus, creating nano-based drug delivery systems (NBDDSs). The loading of MFRPEE onto grafted CNPs occurred naturally through both electrostatic interactions and hydrogen bonding. When constructed under optimal experimental conditions, the NBDDSs were stable under physiologic conditions, and demonstrated enhanced anti-biofilm activity when compared with free MFRPEE. Mechanistic studies revealed that the enhanced anti-infectious activity of the NBDDSs was attributed to the modified surface chemistry of grafted CNPs. More specifically, the overall positive surface charge on grafted CNPs, which stems from quaternary ammonium polymer brushes covalently bound to the CNPs, provides NBDDSs with the ability to specifically target negatively charged components of biofilms. When studying the release profile of MFRPEE from the modified CNPs, acidic components produced by a biofilm triggered the release of MFRPEE bound to the NBDDS. Once in its free form, the anti-infectious properties of MFRPEE became activated and damaged the extracellular polymeric matrix (EPM) of the biofilm. Once the architecture of the biofilm became compromised, the EPM was no longer capable of protecting the bacteria encapsulated within the biofilm from the anti-infectious agent. Consequently, exposure of bacteria to MFRPEE led to bacterial cell death and biofilm inactivation. The results obtained from this study begin to examine the potential application of NBDDSs for the treatment of healthcare-associated infections (HCAIs).

Copper accumulation and the effect of chelation treatment on cerebral amyloid angiopathy compared to parenchymal amyloid plaques.

Accumulation of fibrillar amyloid ß-protein (Aß) in parenchymal plaques and in blood vessels of the brain, the latter condition known as cerebral amyloid angiopathy (CAA), are hallmark pathologies of Alzheimer's disease (AD) and related disorders. Cerebral amyloid deposits have been reported to accumulate various metals, most notably copper and zinc. Here we show that, in human AD, copper is preferentially accumulated in amyloid-containing brain blood vessels compared to parenchymal amyloid plaques. In light of this observation, we evaluated the effects of reducing copper levels in Tg2576 mice, a transgenic model of AD amyloid pathologies. The copper chelator, tetrathiomolybdate (TTM), was administered to twelve month old Tg2576 mice for a period of five months. Copper chelation treatment significantly reduced both CAA and parenchymal plaque load in Tg2576 mice. Further, copper chelation reduced parenchymal plaque copper content but had no effect on CAA copper levels in this model. These findings indicate that copper is associated with both CAA deposits and parenchymal amyloid plaques in humans, but less in Tg2576 mice. TTM only reduces copper levels in plaques in Tg2576 mice. Reducing copper levels in the brain may beneficially lower amyloid pathologies associated with AD.

Plasma-initiated graft polymerization as an immobilization platform for metal free Russian propolis ethanol extracts designed specifically for biomaterials.

The antibacterial and anti-biofilm activities of propolis have been intensively reported. However, the application of this folk remedy as a means to prevent biomedical implant contamination has yet to be completely evaluated. In response to the significant resistant and infectious attributes of biofilms, biomaterials engineered to possess specific chemical and physical properties were immobilized with metal free Russian propolis ethanol extracts (MFRPEE), a known antibacterial agent. The results obtained from this study begin to examine the application of MFRPEE as a novel alternative method for the prevention of medical and biomedical implant infections. When constructed under specific experimental conditions, immobilized biomaterials showed excellent stability when subjected to simulated body fluid and fetal bovine serum. The ability of immobilized biomaterials to specifically target pathogens (both Gram-positive and Gram-negative biofilm forming bacteria), while promoting tissue cell growth, renders these biomaterials as potential candidates for clinical applications.

Anti-infection silver nanoparticle immobilized biomaterials facilitated by argon plasma grafting technology.

Many research groups have attained slow, persistent, continuous release of silver ions through careful experimental design using existing methods. Such methods effectively kill planktonic bacteria and therefore prevent surface adhesion of pathogens. However, the resultant modified coatings cannot provide long-term antibacterial efficacy due to sustained anti-microbial release. In this study, the anti-infection activity of AgNP immobilized biomaterials was evaluated, facilitated by argon plasma grafting technology and activated by bacterial colonization. The modified materials generated in this study showed excellent specificity and were active against both Gram-positive and Gram-negative biofilm forming bacteria, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli. The anti-infection biomaterials developed in this study demonstrate several attractive advantages in comparison to traditional anti-bacterial surfaces loaded with antibiotics or other types of antibacterial agents and include (1) broad spectrum of activity against antibiotic resistant bacteria, (2) the unlikelihood of bacterial resistance, (3) specificity, (4) biocompatibility, and (5) stability.

Are Russian propolis ethanol extracts the future for the prevention of medical and biomedical implant contaminations?

BACKGROUND: Most studies reveal that the mechanism of action of propolis against bacteria is functional rather than structural and is attributed to a synergism between the compounds in the extracts. HYPOTHESIS/PURPOSE: Propolis is said to inhibit bacterial adherence, division, inhibition of water-insoluble glucan formation, and protein synthesis. However, it has been shown that the mechanism of action of Russian propolis ethanol extracts is structural rather than functional and may be attributed to the metals found in propolis. If the metals found in propolis are removed, cell lysis still occurs and these modified extracts may be used in the prevention of medical and biomedical implant contaminations. STUDY DESIGN: The antibacterial activity of metal-free Russian propolis ethanol extracts (MFRPEE) on two biofilm forming bacteria: penicillin-resistant Staphylococcus aureus and Escherichia coli was evaluated using MTT and a Live/Dead staining technique. Toxicity studies were conducted on mouse osteoblast (MC-3T3) cells using the same viability assays. METHODS: In the MTT assay, biofilms were incubated with MTT at 37°C for 30min. After washing, the purple formazan formed inside the bacterial cells was dissolved by SDS and then measured using a microplate reader by setting the detecting and reference wavelengths at 570nm and 630nm, respectively. Live and dead distributions of cells were studied by confocal laser scanning microscopy. RESULTS: Complete biofilm inactivation was observed when biofilms were treated for 40h with 2µg/ml of MFRPEE. Results indicate that the metals present in propolis possess antibacterial activity, but do not have an essential role in the antibacterial mechanism of action. Additionally, the same concentration of metals found in propolis samples, were toxic to tissue cells. Comparable to samples with metals, metal free samples caused damage to the cell membrane structures of both bacterial species, resulting in cell lysis. CONCLUSION: Results suggest that the structural mechanism of action of Russian propolis ethanol extracts stem predominate from the organic compounds. Further studies revealed drastically reduced toxicity to mammalian cells when metals were removed from Russian propolis ethanol extracts, suggesting a potential for medical and biomedical applications.