Eliciting Exploratory Patient Preference Data: A Case Study in a Rare Disease.

INTRODUCTION: Qualitative and quantitative methods provide different and complementary insights into patients' preferences for treatment. OBJECTIVE: The aim of this study was to use a novel, mixed-methods approach employing qualitative and quantitative approaches to generate preliminary insights into patient preferences for the treatment of a rare disease-generalized myasthenia gravis (gMG). METHODS: We conducted a mixed-methods study to collect exploratory qualitative and quantitative patient preference information and generate informative results within a condensed timeline (about 4 months). Recruitment was facilitated by an international health research firm. Study participants first reviewed a brief document describing six treatment attributes (to facilitate more efficient review of the material during the focus groups) and were then provided a link to complete an online quantitative survey with a single risk threshold task. They then participated in online focus groups, during which they discussed qualitative questions about their experience with gMG treatment and completed up to three quantitative threshold tasks, the first of which repeated the threshold task from the online survey. RESULTS: The study elicited both quantitative data on 18 participants' risk tolerance and qualitative data on their treatment experience, additional treatment attributes of importance, the reasoning behind their preferences, and the trade-offs they were willing to make. Most participants (n = 15) chose the same hypothetical treatment in the first threshold task in the online survey and the focus groups. Focus group discussions provided insights into participants' choices in the threshold tasks, confirmed that all the attributes were relevant, and helped clarify what was important about the attributes. CONCLUSIONS: Patient preference information can be collected using a variety of approaches, both qualitative and quantitative, tailored to fit the research needs of a study. The novel mixed-methods approach employed in this study efficiently captured patient preference data that were informative for exploratory research, internal decision making, and future research.

Safety of intravenous lacosamide in hospitalized children and neonates.

OBJECTIVE: Seizures are common in critically ill children and neonates, and these patients would benefit from intravenous (IV) antiseizure medications with few adverse effects. We aimed to assess the safety profile of IV lacosamide (LCM) among children and neonates. METHODS: This retrospective multicenter cohort study examined the safety of IV LCM use in 686 children and 28 neonates who received care between January 2009 and February 2020. RESULTS: Adverse events (AEs) were attributed to LCM in only 1.5% (10 of 686) of children, including rash (n = 3, .4%), somnolence (n = 2, .3%), and bradycardia, prolonged QT interval, pancreatitis, vomiting, and nystagmus (n = 1, .1% each). There were no AEs attributed to LCM in the neonates. Across all 714 pediatric patients, treatment-emergent AEs occurring in >1% of patients included rash, bradycardia, somnolence, tachycardia, vomiting, feeling agitated, cardiac arrest, tachyarrhythmia, low blood pressure, hypertension, decreased appetite, diarrhea, delirium, and gait disturbance. There were no reports of PR interval prolongation or severe cutaneous adverse reactions. When comparing children who received a recommended versus a higher than recommended initial dose of IV LCM, there was a twofold increase in the risk of rash in the higher dose cohort (adjusted incidence rate ratio = 2.11, 95% confidence interval = 1.02-4.38). SIGNIFICANCE: This large observational study provides novel evidence demonstrating the tolerability of IV LCM in children and neonates.

Efficacy and safety of PICOPREP tailored dosing compared with PICOPREP day-before dosing for colon cleansing: a multi-centric randomised study.

Background and study aims The success of any colonoscopy procedure depends upon the quality of bowel preparation. We evaluated the efficacy and safety of a new tailored dosing (TD) regimen compared with the approved PICOPREP day-before dosing regimen (DBD) in the European Union. Patient and methods Patients (≥ 18 years) undergoing colonoscopy were randomised (2:1) to TD (Dose 1, 10 - 18 hours; Dose 2, 4 - 6 hours before colonoscopy) or DBD (Dose 1 before 8:00AM on the day before colonoscopy; Dose 2, 6 - 8 hours after Dose 1). The primary endpoint of overall colon cleansing efficacy was based on total Ottawa Scale (OS) scores (0 - 14, excellent-worst). The key secondary endpoint was a binary endpoint based on the ascending colon OS (success 0 or 1, failure [≥ 2]). Convenience and satisfaction were evaluated similar to the primary and key secondary endpoints. Safety and tolerability were also evaluated. Results Use of the PICOPREP TD regimen resulted in a statistically significant reduction in the mean total Ottawa Scale score compared to the PICOPREP DBD regimen (-3.93, 95 % confidence intervals [CI]: - 4.99, - 2.97; P < 0.0001) in the intent-to-treat analysis set. The PICOPREP TD regimen also resulted in a statistically significant increase in the odds of achieving an ascending colon OS score ≤ 1, compared to the PICOPREP DBD regimen (estimated odds ratio 9.18, 95 % CI: 4.36, 19.32; P < 0.0001). There was no statistically significant difference in the overall rate of treatment-emergent adverse events (12 % (TD) and 5.7 % (DBD), respectively, P = 0.2988). The convenience and satisfaction were comparable in the two groups. Conclusion The TD regimen was superior to the DBD regimen for overall and ascending colon cleansing efficacy. ClinicalTrials.gov Identifier: NCT02239692.

Metallic gold slows disease progression, reduces cell death and induces astrogliosis while simultaneously increasing stem cell responses in an EAE rat model of multiple sclerosis.

Multiple sclerosis (MS) is the most common neurodegenerative disease in the Western world affecting younger, otherwise healthy individuals. Today no curative treatment exists. Patients suffer from recurring attacks caused by demyelination and underlying neuroinflammation, ultimately leading to loss of neurons. Recent research shows that bio-liberation of gold ions from metallic gold implants can ameliorate inflammation, reduce apoptosis and promote proliferation of neuronal stem cells (NSCs) in a mouse model of focal brain injury. Based on these findings, the present study investigates whether metallic gold implants affect the clinical signs of disease progression and the pathological findings in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Gold particles 20-45 µm suspended in hyaluronic acid were bilaterally injected into the lateral ventricles (LV) of young Lewis rats prior to EAE induction. Comparing gold-treated animals to untreated and vehicle-treated ones, a statistically significant slowing of disease progression in terms of reduced weight loss was seen. Despite massive inflammatory infiltration, terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed reduced apoptotic cell death in disease foci in the brain stem of gold-treated animals, alongside an up-regulation of glial fibrillary acidic protein-positive reactive astrocytes near the LV and in the brain stem. Cell counting of frizzled-9 and nestin-stained cells showed statistically significant up-regulation of NSCs migrating from the subventricular zone. Additionally, the neuroprotective proteins Metallothionein-1 and -2 were up-regulated in the corpus callosum. In conclusion, this study is the first to show that the presence of small gold implants affect disease progression in a rat model of MS, increasing the neurogenic response and reducing the loss of cells in disease foci. Gold implants might thus improve clinical outcome for MS patients and further research into the long-term effects of such localized gold treatment is warranted.

Uptake of silver from metallic silver surfaces induces cell death and a pro-inflammatory response in cultured J774 macrophages.

In clinical medicine metallic silver is used as anti-bacterial coating on various catheters, bandages and prostheses. By means of dissolucytosis, i.e. extracellular macrophage-mediated bio-liberation of metal ions, silver ions are continuously liberated from silver surfaces starting within minutes of exposure. The present study investigates how bio-liberation and subsequent cellular uptake of silver ions affects cell viability and cell signalling within the first 3-24 hours of exposure when J774 macrophages are grown directly on a silver surface. Autometallography (AMG) was applied to demonstrate cytoplasmatic silver uptake and localisation after 1, 3, 12 and 24 hours of exposure to metallic silver. From 12 hours onwards the cells were completely filled with silver enhanced silver-sulphur nanocrystals (AMG-silver grains). At the ultrastructural level, the silver accumulations were located to lysosome-like structures. An immunoassay cell death kit found silver-induced apoptosis after 12 and 24 hours of exposure. Necrosis was seen at the same times. Judged by mRNA analysis silver exposure statistically significantly induces TNF-α and m-CSF gene expression, especially at 3 hours. Furthermore, anti-inflammatory IL-10 transcription is reduced by silver uptake and 24 hours of silver exposure induces massive iNOS-2 gene expression. At the same time silver exposure increases the gene expression of metallothionein (MT-I/MT-II), a cystein-rich protein known for its role in detoxifying heavy metals. Our data suggest that silver ions liberated from metallic silver surfaces accumulate in lysosomes, reduce macrophage viability by apoptosis and necrosis and induce a pro-inflammatory response.

Metallic silver fragments cause massive tissue loss in the mouse brain.

Silver is a metal with well-known antibacterial effects. This makes silver an attractive coating material for medical devices for use inside the body, e.g. orthopaedic prostheses and catheters used in neurosurgery as it has been found to reduce the high risk of infections. Lately, the use of nano-silver particles in the industry, e.g. woven into fabrics and furniture has increased, and thus the exposure to silver particles in daily life increases. To study the effect of metallic silver particles on nervous tissue, we injected micron-sized silver particles into the mouse brain by stereotactic procedures. After 7, 14 days and 9 months, the silver-exposed animals had considerable brain damage seen as cavity formation and inflammation adjacent to the injected metallic silver particles. The tissue loss involved both cortical and hippocampal structures and resulted in enlargement of the lateral ventricles. Autometallographic silver enhancement showed silver uptake in lysosomes of glia cells and neurons in the ipsilateral cortex and hippocampus alongside a minor uptake on the contralateral side. Silver was also detected in ependymal cells and the choroid plexus. After 9 months, spreading of silver to the kidneys was seen. Cell counts of immunostained sections showed that metallic silver induced a statistically significant inflammatory response, i.e. increased microgliosis (7 days: p < 0.0001; 14 days: p < 0.01; 9 months: p < 0.0001) and TNF-α expression (7 and 14 days: p < 0.0001; 9 months: p = 0.91). Significant astrogliosis (7, 14 days and 9 months: p < 0.0001) and increased metallothionein (MT I + II) expression (7 and 14 days: p < 0.0001; 9 months: p < 0.001) were also seen in silver-exposed brain tissue. We conclude that metallic silver implants release silver ions causing neuroinflammation and a progressive tissue loss in the brain.

Apical localization of zinc transporter ZnT4 in human airway epithelial cells and its loss in a murine model of allergic airway inflammation.

The apical cytoplasm of airway epithelium (AE) contains abundant labile zinc (Zn) ions that are involved in the protection of AE from oxidants and inhaled noxious substances. A major question is how dietary Zn traffics to this compartment. In rat airways, in vivo selenite autometallographic (Se-AMG)-electron microscopy revealed labile Zn-selenium nanocrystals in structures resembling secretory vesicles in the apical cytoplasm. This observation was consistent with the starry-sky Zinquin fluorescence staining of labile Zn ions confined to the same region. The vesicular Zn transporter ZnT4 was likewise prominent in both the apical and basal parts of the epithelium both in rodent and human AE, although the apical pools were more obvious. Expression of ZnT4 mRNA was unaffected by changes in the extracellular Zn concentration. However, levels increased 3-fold during growth of cells in air liquid interface cultures and decreased sharply in the presence of retinoic acid. When comparing nasal versus bronchial human AE cells, there were significant positive correlations between levels of ZnT4 from the same subject, suggesting that nasal brushings may allow monitoring of airway Zn transporter expression. Finally, there were marked losses of both basally-located ZnT4 protein and labile Zn in the bronchial epithelium of mice with allergic airway inflammation. This study is the first to describe co-localization of zinc vesicles with the specific zinc transporter ZnT4 in airway epithelium and loss of ZnT4 protein in inflamed airways. Direct evidence that ZnT4 regulates Zn levels in the epithelium still needs to be provided. We speculate that ZnT4 is an important regulator of zinc ion accumulation in secretory apical vesicles and that the loss of labile Zn and ZnT4 in airway inflammation contributes to AE vulnerability in diseases such as asthma.

Chemical blocking of zinc ions in CNS increases neuronal damage following traumatic brain injury (TBI) in mice.

BACKGROUND: Traumatic brain injury (TBI) is one of the leading causes of disability and death among young people. Although much is already known about secondary brain damage the full range of brain tissue responses to TBI remains to be elucidated. A population of neurons located in cerebral areas associated with higher cognitive functions harbours a vesicular zinc pool co-localized with glutamate. This zinc enriched pool of synaptic vesicles has been hypothesized to take part in the injurious signalling cascade that follows pathological conditions such as seizures, ischemia and traumatic brain injury. Pathological release of excess zinc ions from pre-synaptic vesicles has been suggested to mediate cell damage/death to postsynaptic neurons. METHODOLOGY/PRINCIPAL FINDINGS: In order to substantiate the influence of vesicular zinc ions on TBI, we designed a study in which damage and zinc movements were analysed in several different ways. Twenty-four hours after TBI ZnT3-KO mice (mice without vesicular zinc) were compared to littermate Wild Type (WT) mice (mice with vesicular zinc) with regard to histopathology. Furthermore, in order to evaluate a possible neuro-protective dimension of chemical blocking of vesicular zinc, we treated lesioned mice with either DEDTC or selenite. Our study revealed that chemical blocking of vesicular zinc ions, either by chelation with DEDTC or accumulation in zinc-selenium nanocrystals, worsened the effects on the aftermath of TBI in the WT mice by increasing the number of necrotic and apoptotic cells within the first 24 hours after TBI, when compared to those of chemically untreated WT mice. CONCLUSION/SIGNIFICANCE: ZnT3-KO mice revealed more damage after TBI compared to WT controls. Following treatment with DEDTC or selenium an increase in the number of both dead and apoptotic cells were seen in the controls within the first 24 hours after TBI while the degree of damage in the ZnT3-KO mice remained largely unchanged. Further analyses revealed that the damage development in the two mouse strains was almost identical after either zinc chelation or zinc complexion therapy.

Elevation of zinc transporter ZnT3 protein in the cerebellar cortex of the AbetaPP/PS1 transgenic mouse.

The presence of senile plaques containing abundant amyloid-beta (Abeta) peptide is one of the major pathological hallmarks of Alzheimer's disease (AD). Recent studies support the notion that overexpression of zinc transporters (ZnT) is involved in zinc metabolic disturbances and Abeta aggregation in AD brains. Here we present data showing an elevated expression of zinc transporter 3 (ZnT3) protein, revealed by immunoblotting assay, in the cerebellum of the amyloid-beta protein precursor (AbetaPP)/presenilin 1 (PS1) transgenic mouse. Confocal microscopic and autometallographic results showed that ZnT3 immunofluorescence and zinc ions were predominantly located in the amyloid plaques. ZnT3 protein was abundantly distributed throughout the plaques, whereas zinc ions were mainly located in the peripheral parts of rosette-shaped plaques with a lightly stained center. Collectively, our results suggest that ZnT3 protein is involved in the Abeta aggregation in the cerebellum of the AbetaPP/PS1 mouse.

Bio-released gold ions modulate expression of neuroprotective and hematopoietic factors after brain injury.

The discovery of neural stem cells (NSCs) provides new therapeutic strategies for brain injury by means of endogenous cell renewal. In the injured mouse brain, bio-liberated gold ions from gold implants mediate anti-inflammatory and antiapoptotic effects and activation of NSCs. This paper investigates the neuroprotective effects of gold following brain injury in mice. We show for the first time that endogenous NSCs express macrophage colony-stimulating factor (M-CSF) as part of their post-injury activation and that gold implants increase this response. Also, gold increases expression of neurotrophin (NT)-4, transforming growth factor-beta 3 (TGF-beta 3), leukemia inhibitory factor (LIF) and metallothionein I+II (MT-I+II) post-injury. This paper shows that gold ions modulate neurotrophic factors after injury and that hematopoietic factor M-CSF is expressed in activated NSCs.

Altered expression and distribution of zinc transporters in APP/PS1 transgenic mouse brain.

Pathological accumulation of beta-amyloid peptide (Abeta) is an early and common feature of Alzheimer's disease (AD). An increased zinc concentration can initiate the deposition of Abeta. The present study aimed to study the expression and distribution patterns of six members of the zinc transporter (ZnT) family, ZnT1, ZnT3, ZnT4, ZnT5, ZnT6, and ZnT7, in the APPswe/PS1dE9 transgenic mouse brain. Our results demonstrated a statistically significant (P<0.05) increase of ZnT1, ZnT3, ZnT4, ZnT6, and ZnT7 in both hippocampus and neo-cortex using Western blot method and an abundant distribution of zinc ions in the plaques and amyloid angiopathic vessels using immersion autometallography. Furthermore, all ZnT immunoreactions were detected in most amyloid plaques and amyloid angiopathic vessels. ZnT1 and ZnT4 were extensively expressed in all parts of the plaques. ZnT3, ZnT5, and ZnT6 were expressed most prominently in the degenerating neurites in the peripheral part of the plaques, while ZnT7 was present in the core of the plaques. The amyloid angiopathic vessels showed a strong ZnT3 immunoreactivity. These results might suggest multiple roles of ZnTs in the deposition and organization of the Abeta composition.

Biodistribution of gold nanoparticles in mouse lung following intratracheal instillation.

BACKGROUND: The fate of gold nanoparticles, 2, 40 and 100 nm, administered intratracheally to adult female mice was examined. The nanoparticles were traced by autometallography (AMG) at both ultrastructural and light microscopic levels. Also, the gold content was quantified by inductively coupled plasma mass spectrometry (ICP-MS) and neutron activation analysis (NAA). The liver is the major site of deposition of circulating gold nanoparticles. Therefore the degree of translocation was determined by the hepatic deposition of gold. Mice were instilled with 5 intratracheal doses of gold nanoparticles distributed over a period of 3 weeks and were killed 24 h after the last dose. One group of mice were given a single intratracheal dose and were killed after 1 h. RESULTS: The instilled nanoparticles were found in lung macrophages already 1 h after a single instillation. In mice instilled treated repeatedly during 3 weeks, the load was substantial. Ultrastructurally, AMG silver enhanced gold nanoparticles were found in lysosome-/endosome-like organelles of the macrophages and analysis with AMG, ICP-MS and NAA of the liver revealed an almost total lack of translocation of nanoparticles. In mice given repeated instillations of 2 nm gold nanoparticles, 1.4 per thousand (by ICP-MS) to 1.9 per thousand (by NAA) of the instilled gold was detected in the liver. With the 40 nm gold, no gold was detected in the liver (detection level 2 ng, 0.1 per thousand) except for one mouse in which 3 per thousand of the instilled gold was found in the liver. No gold was detected in any liver of mice instilled with 100 nm gold (detection level 2 ng, 0.1 per thousand) except in a single animal with 0.39 per thousand of the dose in the liver. CONCLUSION: We found that that: (1) inert gold nanoparticles, administered intratracheally are phagocytosed by lung macrophages; (2) only a tiny fraction of the gold particles is translocated into systemic circulation. (3) The translocation rate was greatest with the 2 nm gold particles.

SLC30A3 responds to glucose- and zinc variations in beta-cells and is critical for insulin production and in vivo glucose-metabolism during beta-cell stress.

BACKGROUND: Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. beta-cells depend on zinc for both insulin crystallization and regulation of cell mass. METHODOLOGY/PRINCIPAL FINDINGS: This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in beta-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a beta-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced beta-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals. CONCLUSION/SIGNIFICANCE: Zinc transporting proteins in beta-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

Cell death in the injured brain: roles of metallothioneins.

In traumatic brain injury (TBI), the primary, irreversible damage associated with the moment of impact consists of cells dying from necrosis. This contributes to fuelling a chronic central nervous system (CNS) inflammation with increased formation of proinflammatory cytokines, enzymes and reactive oxygen species (ROS). ROS promote oxidative stress, which leads to neurodegeneration and ultimately results in programmed cell death (secondary injury). Since this delayed, secondary tissue loss occurs days to months following the primary injury it provides a therapeutic window where potential neuroprotective treatment could alleviate ongoing neurodegeneration, cell death and neurological impairment following TBI. Various neuroprotective drug candidates have been described, tested and proven effective in pre-clinical studies, including glutamate receptor antagonists, calcium-channel blockers, and caspase inhibitors. However, most of the scientific efforts have failed in translating the experimental results into clinical trials. Despite intensive research, effective neuroprotective therapies are lacking in the clinic, and TBI continues to be a major cause of morbidity and mortality. This paper provides an overview of the TBI pathophysiology leading to cell death and neurological impairment. We also discuss endogenously expressed neuroprotectants and drug candidates, which at this stage may still hold the potential for treating brain injured patients.

The role of metallothionein in oncogenesis and cancer prognosis.

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+II's role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.

Metallic gold reduces TNFalpha expression, oxidative DNA damage and pro-apoptotic signals after experimental brain injury.

Brain injury represents a major health problem and may result in chronic inflammation and neurodegeneration. Due to antiinflammatory effects of gold, we have investigated the cerebral effects of metallic gold particles following a focal brain injury (freeze-lesion) in mice. Gold particles 20-45 microm in size or the vehicle (placebo) were implanted in the cortical tissue followed by a cortical freeze-lesioning. At 1-2 weeks post-injury, brains were analyzed by using immunohistochemistry and markers of inflammation, oxidative stress and apoptosis. This study shows that gold treatment significantly reduces the cerebral levels of tumor necrosis factor alpha (TNFalpha), oxidative DNA damage (as judged by 8-oxoguanine levels), and pro-apoptotic markers (cleaved caspase-3, cytochrome c leakage), when compared to those of controls. The data presented here points toward gold particles as a tool to modulate the cerebral response to injury.

Metallic gold treatment reduces proliferation of inflammatory cells, increases expression of VEGF and FGF, and stimulates cell proliferation in the subventricular zone following experimental traumatic brain injury.

UNLABELLED: Traumatic brain injury represents a leading cause of morbidity in young individuals and there is an imperative need for neuroprotective treatments limiting the neurologic impairment following such injury. It has recently been demonstrated that bio-liberated gold ions liberated from small metallic gold implants reduce inflammation and neuronal apoptosis, while generating an increased neuronal stem cell response following focal brain damage. In this study mice were subjected to a unilateral traumatic cryo-lesion with concomitant injection of 25-45 microm gold particles near the lesion. Placebo-treated mice subjected to cryo-lesion served as controls. The effects of gold-treatment were investigated by examining gold-induced growth factor expression (VEGF and FGF) in the first two weeks after the insult, and the extent of the neurostimulatory effect of gold was explored by comparing cell proliferation in the subventricular zone as judged by immunohistochemical staining for CDC47. Vimentin staining revealed a decrease in activated microglia and a transient astrogliosis in response to the gold liberation. Moreover, gold ions significantly increase the expression of VEGF and FGF following trauma and a significant increase in cell proliferation in both the ipsilateral and the contralateral subventricular zone was found in response to gold-treatment. IN CONCLUSION: we confirmed the previously demonstrated anti-inflammatory effect of bio-liberated gold ions, and further show that metallic gold increases growth factor expression and adult neurogenesis.

Protracted elimination of gold nanoparticles from mouse liver.

The present study aims at revealing the fate of 40-nm gold nanoparticles after intravenous injections. The gold nanoparticles were traced histochemically with light and transmission electron microscopy using autometallographic (AMG) staining, and the gold content in the liver was determined with inductively coupled plasma mass spectrometry (ICP-MS). Gold nanoparticles were identified in almost all Kupffer cells one day after the injection, but the fraction of gold-loaded cells gradually decreased to about one fifth after 6 months. Transmission electron microscopic analysis showed that the gold nanoparticles had accumulated inside the vesicular lysosome/endosome-like structures of the macrophages. At day 1, about 4.5 per thousand of the area of the liver sections was AMG-stained, after 1 month it had decreased to 0.7 per thousand, and thereafter no further significant reduction was recorded. Because ICP-MS only showed a 9% fall in the gold content over the observed 6 months, the AMG finding of a significant reduction in the stained area of the liver sections and number of macrophages loaded with gold nanoparticles reveals that over time an increasing part of the total amount of gold nanoparticles in the liver is contained in fewer macrophages accumulated in growing clusters.

Hydroxyapatite coatings did not increase TGF-beta and BMP-2 secretion in murine J774A.1 macrophages, but induced a pro-inflammatory cytokine response.

Hydroxyapatite (Ca(10)(PO(2))(6)(OH)(2), HA) coatings are widely used in un-cemented total hip arthroplasties because it increases implant fixation and bone in-growth. HA adsorbs high levels of proteins (e.g., fibronectin) and adhering macrophages release higher levels of BMP-2 compared to non-coated surfaces. Inflammation, however, can block the BMP-2 production from macrophages and it has been suggested that roughened surfaces activate macrophages. Therefore, the aim of the present study was to investigate whether commercially available 3D surfaces would generate increased BMP-2 and TGF-beta production and establish how this production is affected by macrophage activation. A mouse macrophage cell line (J774A.1) was incubated with 3D coatings resembling clinically used protestic surfaces, i.e., plasmaspray TiAlV coating with or without HA and porous TiAlV coating with or without HA. BMP-2 and TGF-beta production was determined after 24 h with ELISA and real time-reverse transcriptase-PCR (real time RT-PCR). Changes in pro-inflammatory response were established with ELISA after 6 and 24 h. The increased surface roughness of HA-coated surfaces was characterized with confocal microscopy. HA coating on plasmasprayed TiAlV coating was not found to induce BMP-2 and TGF-beta secretion in the J774A.1 macrophage cell line. In contrast, LPS-activated macrophages increased their TGF-beta, but not BMP-2, secretion when exposed to the prosthetic surfaces. These results imply that the well-known bone-inductive effects of HA may not be dependent on macrophage BMP-2 and TGF-beta secretion. Surface topography and protein adhesion may play a more pivotal role.

Cultured macrophages cause dissolucytosis of metallic silver.

The present study proves that cultured macrophages can liberate silver ions from metallic silver surfaces by a process called dissolucytosis. Macrophages (J774) were grown on a silver plate for different periods of time and after fixation in glutaraldehyde, they were subjected to autometallograhy in order to amplify possible cellular silver-sulphur nanocrystals. Light and electron microscopic analysis of the cells revealed that silver ions released from the plate had been taken up by the macrophages and accumulated in lysosome-like structures. We found that the liberation of silver ions takes place extracellularly and is caused by chemical activity in a dissolution membrane, most likely secreted and organized by the macrophages. The liberation and the subsequent uptake of silver ions in the macrophages is a relatively fast process and the resulting silver-sulphur nanocrystals can be observed in macrophages that have been in contact with metallic silver for only a few minutes. Our findings indicate that the speed of dissolucytosis is highly influenced by the chemical nature of the object exposed to the dissolucytotic process which is likely to occur whenever macrophages encounter a non-phagocytosable foreign object.