CD59 Protects Primary Human Cerebrovascular Smooth Muscle Cells from Cytolytic Membrane Attack Complex.

Cerebral amyloid angiopathy is characterized by a weakening of the small- and medium-sized cerebral arteries, as their smooth muscle cells are progressively replaced with acellular amyloid ß, increasing vessel fragility and vulnerability to microhemorrhage. In this context, an aberrant overactivation of the complement system would further aggravate this process. The surface protein CD59 protects most cells from complement-induced cytotoxicity, but expression levels can fluctuate due to disease and varying cell types. The degree to which CD59 protects human cerebral vascular smooth muscle (HCSM) cells from complement-induced cytotoxicity has not yet been determined. To address this shortcoming, we selectively blocked the activity of HCSM-expressed CD59 with an antibody, and challenged the cells with complement, then measured cellular viability. Unblocked HCSM cells proved resistant to all tested concentrations of complement, and this resistance decreased progressively with increasing concentrations of anti-CD59 antibody. Complete CD59 blockage, however, did not result in a total loss of cellular viability, suggesting that additional factors may have some protective functions. Taken together, this implies that CD59 plays a predominant role in HCSM cellular protection against complement-induced cytotoxicity. The overexpression of CD59 could be an effective means of protecting these cells from excessive complement system activity, with consequent reductions in the incidence of microhemorrhage. The precise extent to which cellular repair mechanisms and other complement repair proteins contribute to this resistance has yet to be fully elucidated.

CD59 Protects Primary Human Cerebrovascular Smooth Muscle Cells from Cytolytic Membrane Attack Complex.

Cerebral amyloid angiopathy is characterized by a weakening of the small and medium sized cerebral arteries, as their smooth muscle cells are progressively replaced with acellular amyloid ß, increasing vessel fragility and vulnerability to microhemorrhage. In this context, an aberrant overactivation of the complement system would further aggravate this process. The surface protein CD59 protects most cells from complement-induced cytotoxicity, but expression levels can fluctuate due to disease and vary between cell types. The degree to which CD59 protects human cerebral vascular smooth muscle (HCSM) cells from complement-induced cytotoxicity has not yet been determined. To address this shortcoming, we selectively blocked the activity of HCSM-expressed CD59 with an antibody and challenged the cells with complement, then measured cellular viability. Unblocked HCSM cells proved resistant to all tested concentrations of complement, and this resistance decreased progressively with increasing concentrations of anti-CD59 antibody. Complete CD59 blockage, however, did not result in total loss of cellular viability, suggesting that additional factors may have some protective functions. Taken together, this implies that CD59 plays a predominant role in HCSM cellular protection against complement-induced cytotoxicity. Over-expression of CD59 could be an effective means of protecting these cells from excessive complement system activity, with consequent reduction in the incidence of microhemorrhage. The precise extent to which cellular repair mechanisms and other complement repair proteins contribute to this resistance has yet to be fully elucidated.

Depyrogenation using Plasmas: A Novel Approach for Endotoxin Deactivation Using a Dielectric Barrier Discharge at Atmospheric Pressure.

Developing a low-cost depyrogenation process is vital in extending medical applicability of polymers that can be used in medicine. We present an overview of the plasma-based depyrogenation literature and address the need to develop a non-thermal plasma-based depyrogenation process for delicate materials such as chitosan. We present a low-cost plasma apparatus to treat chitosan powder in hermetically sealed bags. We decouple the experiments into two; depyrogenation experiments for dried standard endotoxin on glass slides, and chitosan modifications analysis through FTIR spectroscopy. We demonstrate depyrogenation efficacy with up to a 4-log reduction in endotoxin levels and discuss minor changes observed in plasma-treated chitosan.

Evaluation of a Chitosan Hemostat in a Porcine Laparoscopic Partial Nephrectomy Model: A Pilot Study.

Background and Objective: The ideal hemostatic agent for laparoscopic partial nephrectomy (LPN) would provide complete hemostasis and sealing of the collecting system at a low cost. Chitosan (CS) is an established topical hemostatic agent, but standard sterilization techniques affect its functional and biologic properties, thereby preventing parenteral uses. This study sought to characterize the safety and efficacy of an implanted CS hemostat sterilized with either a standard technique, electron beam (e-beam) irradiation, or a novel technique, nonthermal nitrogen plasma, in a porcine LPN model. Methods: Laparoscopic partial nephrectomies were performed on six farm pigs and hemostasis achieved using only a CS hemostatic agent (Clo-Sur P.A.D.) that was e-beam (n = 3) or plasma sterilized (PS) (n = 3). Number of pads needed to achieve hemostasis, estimated blood loss, operative time, mass of kidney resection, and warm ischemia time were measured. Animals were monitored for 14 weeks and at harvest, retrograde ureteropyelography and histologic analysis were performed. Results: Complete hemostasis and collection system sealing were achieved in both groups. There was a trend toward less pads required for hemostasis (p = 0.056) and reduced blood loss (p = 0.096) with PS pads, although this did not achieve statistical significance. No complications were observed for 14 weeks and gross examination showed the implanted CS was encapsulated in a fibrous capsule. Histologic analysis revealed a healed nephrectomy site with residual CS and associated chronic inflammation, reactive fibrosis, and foreign body giant cell formation. Importantly, the adjacent renal tissue was intact and viable with no residual parenchymal inflammation or cytologic damage. Conclusion: CS pads alone provided safe and effective hemostasis in a porcine LPN model. PS may enhance hemostatic efficacy and resorption compared with e-beam.

Markers of oxidative damage to lipids, nucleic acids and proteins and antioxidant enzymes activities in Alzheimer's disease brain: A meta-analysis in human pathological specimens.

Oxidative stress and decreased cellular responsiveness to oxidative stress are thought to influence brain aging and Alzheimer's disease, but the specific patterns of oxidative damage and the underlying mechanism leading to this damage are not definitively known. The objective of this study was to define the pattern of changes in oxidative-stress related markers by brain region in human Alzheimer's disease and mild cognitive impairment brain tissue. Observational case-control studies were identified from systematic queries of PubMed, ISI Web of Science and Scopus databases and studies were evaluated with appropriate quality measures. The data was used to construct a region-by-region meta-analysis of malondialdehyde, 4-hydroxynonenal, protein carbonylation, 8-hydroxyguanine levels and superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase activities. We also evaluated ascorbic acid, tocopherol, uric acid and glutathione levels. The analysis was complicated in several cases by publication bias and/or outlier data. We found that malondialdehyde levels were slightly increased in the temporal and occipital lobes and hippocampus, but this analysis was significantly impacted by publication bias. 4-hydroxynonenal levels were unchanged in every brain region. There was no change in 8-hydroxyguanine level in any brain region and protein carbonylation levels were unchanged except for a slight increase in the occipital lobe. Superoxide dismutase, glutathione peroxidase and reductase and catalase activities were not decreased in any brain region. There was limited data reporting non-enzymatic antioxidant levels in Alzheimer's disease brain, although glutathione and tocopherol levels appear to be unchanged. Minimal quantitative data is available from brain tissue from patients with mild cognitive impairment. While there is modest evidence supporting minor regional changes in markers of oxidative damage, this analysis fails to identify a consistent pattern of pro-oxidative changes and accumulation of oxidative damage in bulk tissue analysis in the setting of Alzheimer's disease, as has been widely reported.

Stability and bioactivity of chitosan as a transfection agent in primary human cell cultures: A case for chitosan-only controls.

Chitosan polymers (Cs), from which microparticles (CsM) may be precipitated to deliver various intracellular payloads, are generally considered biologically inert. We examined the impact of cell culture conditions on CsM size and the effect of chitosan on CD59 expression in primary human smooth muscle cells. We found that particle concentration and incubation time in biological buffers augmented particle size. Between pH 7.0 and pH 7.5, CsM size increased abruptly. We utilized CsM containing a plasmid with a gene for CD59 (pCsM) to transfect cells. Both CD59 mRNA and the number of CD59-positive cells were increased after pCsM treatment. Unexpectedly, CsM also augmented the number of CD59-positive cells. Cs alone enhanced CD59 expression more potently than either pCSM or CsM. This observation strongly suggests that chitosan is in fact bioactive and that chitosan-only controls should be included to avoid misattributing the activity of the delivery agent with that of the payload.

Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity.

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Formulation and characterization of a plasma sterilized, pharmaceutical grade chitosan powder.

Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was micronized using cryo-ball and cryo-jet milling and subsequently sterilized with nitrogen plasma. Micronized chitosan was characterized by laser diffraction, scanning electron microscopy (SEM), conductometric titration, viscometry, loss on drying, FTIR, and limulus amebocyte lysate (LAL) assays. Cryo-jet milling produced mean particle size of 16.05µm, 44% smaller than cryo-ball milling. Cryomilled chitosan demonstrated increased hygroscopicity, but reduced molecular weight and degree of deacetylation (DD). SEM imaging showed highly irregular shapes. FTIR showed changes consistent with reduced DD and an unexplained shift at 1100cm(-1). Plasma treated chitosan was sterile with <2.5EU/g after low-pressure plasma and <1.3EU/g after atmospheric pressure plasma treatment. Plasma treatment decreased the reduced viscosity of chitosan flake and powder, with a greater effect on powder. In conclusion, pharmaceutical grade, sterile chitosan powder was produced with cryo-jet milling and plasma sterilization.

Effect of Plasma Sterilization on the Hemostatic Efficacy of a Chitosan Hemostatic Agent in a Rat Model.

INTRODUCTION: The United States military has had success with chitosan (CS)-based hemostatic agents to control trauma-induced hemorrhages. Despite the positive reviews, additional physical forms of CS may enhance its hemostatic efficacy. Additionally, standard sterilization techniques may negatively affect the hemostatic efficacy of CS. We studied the effects of a CS-based hemostatic pad, the Clo-Sur P.A.D.™ (Scion Cardio-Vascular, Inc.), on severe femoral vessel bleeding in a rat model. The effects of different sterilization techniques on the bioadhesivity, surface atomic concentrations, and hemostatic efficacy of the P.A.D. were also evaluated. METHODS: Hemostatic efficacy, bioadhesivity, and surface atomic concentrations of the P.A.D. were evaluated in its unsterilized form, after sterilization with standard e-beam treatment, and after sterilization with one of three types of non-thermal nitrogen plasma: nitrogen gas, air, or nitrous oxide plasma. After standardized puncture of the femoral artery or transection of the femoral vessels, rats were treated with either a CS P.A.D. or gauze pad. RESULTS: The Clo-Sur P.A.D., regardless of sterilization technique, stopped arterial and mixed arterial/venous bleeding in all cases in <90 s with the time to hemostasis (TTH) significantly less for all P.A.D. treatment groups (P < 0.001; n = 4-5/group) compared to gauze-treated controls (n = 3). E-beam sterilized P.A.D.s consistently showed non-significant trends toward increased TTH and worse hemostasis scores compared to unsterilized and plasma sterilized P.A.D.s. Treating e-beam sterilized P.A.D.s with N2 plasma reverted the hemostatic efficacy to levels equivalent to native, unsterilized PADs. CONCLUSION: A CS-based hemostatic pad successfully controlled severe bleeding in a rat model with combined e-beam and plasma sterilized P.A.D.s showing the most promising results. Further studies are warranted.

A chi-square goodness-of-fit test for autoregressive logistic regression models with applications to patient screening.

We propose a chi-square goodness-of-fit test for autoregressive logistic regression models. General guidelines for a two-dimensional binning strategy are provided, which make use of two types of maximum likelihood parameter estimates. For smaller sample sizes, a bootstrap p-value procedure is discussed. Simulation studies indicate that the test procedure satisfactorily approximates the correct size and has good power for detecting model misspecification. In particular, the test is very good at detecting the need for an additional lag. An application to a dataset relating to screening patients for late-onset Alzheimer's disease is provided.

From development to dysfunction: microglia and the complement cascade in CNS homeostasis.

Of the many mysteries that surround the brain, few surpass the awe-inspiring complexity of its development. The intricate wiring of the brain at both the system and molecular level is both spatially and temporally regulated in perfect synchrony. How such a delicate, yet elegant, system arises from an embryo's most basic cells remains at the forefront of neuroscientific research. At the cellular level, the competitive dance between synapses struggling to gain dominance seems to be refereed by both neurons themselves and microglia, the innate immune cells of the nervous system. Additionally, the unexpected complement cascade, a major effecter arm of the innate immune system, is almost certainly involved in synaptic remodeling by tagging destined neurons and synapses for destruction. As suddenly as they appear, the mechanisms of neurogenesis recede entering into adulthood. However, with age and insult, these mechanisms boisterously return, resulting in neurodegeneration. This review describes some of the mechanisms involved in synaptogenesis and wiring of the brain from the point of view of the innate immune system and then covers how similar molecular processes return with age and disease, specifically in the context of Alzheimer's disease.

A shift in microglial ß-amyloid binding in Alzheimer's disease is associated with cerebral amyloid angiopathy.

Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) are two common pathologies associated with ß-amyloid (Aß) accumulation and inflammation in the brain; neither is well understood. The objective of this study was to evaluate human post-mortem brains from AD subjects with purely parenchymal pathology, and those with concomitant CAA (and age-matched controls) for differential expression of microglia-associated Aß ligands thought to mediate Aß clearance and the association of these receptors with complement activation. Homogenates of brain parenchyma and enriched microvessel fractions from occipital cortex were probed for levels of C3b, membrane attack complex (MAC), CD11b and α-2-macroglobulin and immunoprecipitation was used to immunoprecipitate (IP) CD11b complexed with C3b and Aß. Both C3b and MAC were significantly increased in CAA compared to AD-only and controls and IP showed significantly increased CD11b/C3b complexes with Aß in AD/CAA subjects. Confocal microscopy was used to visualize these interactions. MAC was remarkably associated with CAA-affected blood vessels compared to AD-only and control vessels. These findings are consistent with an Aß clearance mechanism via microglial CD11b that delivers Aß and C3b to blood vessels in AD/CAA, which leads to Aß deposition and propagation of complement to the cytolytic MAC, possibly leading to vascular fragility.

Targeted proteomics for quantification of histone acetylation in Alzheimer's disease.

The epigenetic remodeling of chromatin histone proteins by acetylation has been the subject of recent investigations searching for biomarkers indicative of late onset cognitive loss. Histone acetylations affect the regulation of gene transcription, and the loss of learning induced deacetylation at specific histone sites may represent biomarkers for memory loss and Alzheimer's disease (AD). Selected-reaction-monitoring (SRM) has recently been advanced to quantitate peptides and proteins in complex biological systems. In this paper, we provide evidence that SRM-based targeted proteomics can reliably quantify specific histone acetylations in both AD and control brain by identifying the patterns of H3 K18/K23 acetylations Results of targeted proteomics assays have been validated by Western blot (WB) analysis. As compared with LC-MS/MS-TMT (tandem-mass-tagging) and WB methods, the targeted proteomics method has shown higher throughput, and therefore promised to be more suitable for clinical applications. With this methodology, we find that histone acetylation is significantly lower in AD temporal lobe than found in aged controls. Targeted proteomics warrants increased application for studying epigenetics of neurodegenerative diseases.

Deferiprone reduces amyloid-ß and tau phosphorylation levels but not reactive oxygen species generation in hippocampus of rabbits fed a cholesterol-enriched diet.

Accumulation of amyloid-ß (Aß) peptide and the hyperphosphorylation of tau protein are major hallmarks of Alzheimer's disease (AD). The causes of AD are not well known but a number of environmental and dietary factors are suggested to increase the risk of developing AD. Additionally, altered metabolism of iron may have a role in the pathogenesis of AD. We have previously demonstrated that cholesterol-enriched diet causes AD-like pathology with iron deposition in rabbit brain. However, the extent to which chelation of iron protects against this pathology has not been determined. In this study, we administered the iron chelator deferiprone in drinking water to rabbits fed with a 2% cholesterol diet for 12 weeks. We found that deferiprone (both at 10 and 50 mg/kg/day) significantly decreased levels of Aß40 and Aß42 as well as BACE1, the enzyme that initiates cleavage of amyloid-ß protein precursor to yield Aß. Deferiprone also reduced the cholesterol diet-induced increase in phosphorylation of tau but failed to reduce reactive oxygen species generation. While deferiprone treatment was not associated with any change in brain iron levels, it was associated with a significant reduction in plasma iron and cholesterol levels. These results demonstrate that deferiprone confers important protection against hypercholesterolemia-induced AD pathology but the mechanism(s) may involve reduction in plasma iron and cholesterol levels rather than chelation of brain iron. We propose that adding an antioxidant therapy to deferiprone may be necessary to fully protect against cholesterol-enriched diet-induced AD-like pathology.

Assessing candidate serum biomarkers for Alzheimer's disease: a longitudinal study.

Because of the growing impact of late onset cognitive loss, considerable effort has been directed toward the development of improved diagnostic techniques for Alzheimer's disease (AD) that may pave the way for earlier (and more effective) therapeutic efforts. Serum-based biomarkers are the least expensive and invasive modality for screening and routine monitoring. We systematically reviewed the literature to assemble a list of serum biomarkers relevant to AD. In parallel, we conducted a proteomic LC-MS/MS analysis of serum collected from neurologically normal subjects and subjects with mild cognitive impairment (MCI) and early AD (n = 6 in all). Complement C3 and alpha-2-macroglobulin were identified from both the literature review and our proteomic screen for further validation. For these two candidates, ELISA was performed on serum collected from a small independent cohort of subjects for longitudinal analysis. Serum was serially collected from neurologically normal subjects (n = 5) and subjects with MCI who were subsequently followed for a period of two years (n = 5) and regrouped into stable MCI and progressive MCI or AD (n = 6). The ability of each marker to predict which subjects with MCI would progress to dementia and which would remain cognitively stable was assessed. Patients with probable cerebral amyloid angiopathy were also identified (n = 3). This preliminary analysis tested the most-promising serum protein biomarkers for AD and we concluded that none are yet ready for use in the clinical diagnosis and management of dementia. However, a more thorough assessment in longitudinal studies with higher statistical power is warranted.

Altered serum iron and copper homeostasis predicts cognitive decline in mild cognitive impairment.

Alzheimer's disease (AD) brain is marked by severe neuronal death which has been partly attributed to increased oxidative stress. The pathophysiology accounting for this free radical injury is not well-delineated at this point, but one hypothesis is that a derangement in transition metal metabolism contributes to the process. We tested the hypothesis that peripheral derangement of transition metal metabolism is present early in the dementing process. We analyzed non-heme iron and copper levels in serum from subjects with normal cognition, mild cognitive impairment, and early stage senile dementia and followed these subjects over 5 years. An increase in the ratio of serum copper to non-heme iron levels predicted which subjects with mild cognitive impairment would progress to dementia versus those that would remain cognitively stable. This increase did not correlate with changes in expression of iron regulatory protein 2 or selected downstream targets in peripheral lymphocytes. A cDNA-based microarray (IronChip) containing genes relevant to iron and copper metabolism was used to assess transition metal metabolism in circulating lymphocytes from cognitively normal and demented subjects. No gene was identified as being dysregulated more than 2-fold, and verification using quantitative RT-PCR demonstrated no significant changes in expression for ALAS2, FOS, and CTR1. The increased ratio of serum copper to serum iron prior to dementia has potential as a biomarker for cognitive decline and mirrors other changes in serum previously reported by others, but iron and copper metabolism pathways appear to be broadly unaffected in peripheral blood in AD.

Increased ceramide in brains with Alzheimer's and other neurodegenerative diseases.

Ceramide has been suggested to participate in the neuronal cell death that leads to Alzheimer's disease (AD), but its role is not yet well-understood. We compared the levels of six ceramide subspecies, which differ in the length of their fatty acid moieties, in brains from patients who suffered from AD, other neuropathological disorders, or both. We found elevated levels of Cer16, Cer18, Cer20, and Cer24 in brains from patients with any of the tested neural defects. Moreover, ceramide levels were highest in patients with more than one neuropathologic abnormality. Interestingly, the range of values was higher among brains with neural defects than in controls, suggesting that the regulation of ceramide synthesis is normally under tight control, and that this tight control may be lost during neurodegeneration. These changes, however, did not alter the ratio between the tested ceramide species. To explore the mechanisms underlying this dysregulation, we evaluated the expression of four genes connected to ceramide metabolism: ASMase, NSMase 2, GALC, and UGCG. The patterns of gene expression were complex, but overall, ASMase, NSMase 2, and GALC were upregulated in specimens from patients with neuropathologic abnormalities in comparison with age-matched controls. Such findings suggest these genes as attractive candidates both for diagnostic purposes and for intervening in neurodegenerative processes.

Proteomic analysis of eccrine sweat: implications for the discovery of schizophrenia biomarker proteins.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.

In vivo iron quantification in collagenase-induced microbleeds in rat brain.

Brain microbleeds (BMB) are associated with chronic and acute cerebrovascular disease. Because BMB present in the brain is a source of potentially cytotoxic iron proportional to the volume of extravasated blood, BMB iron content is a potentially valuable biomarker both to assess tissue risk and small cerebral vessel health. We recently reported methods to quantify focal iron sources using phase images that were tested in phantoms and BMB in postmortem tissue. In this study, we applied our methods to small hemorrhagic lesions induced in the in vivo rat brain using bacterial collagenase. As expected by theory, measurements of geometric features in phase images correlated with lesion iron content measured by graphite furnace atomic absorption spectrometry. Iron content estimation following BMB in an in vivo rodent model could shed light on the role and temporal evolution of iron-mediated tissue damage and efficacy of potential treatments in cerebrovascular diseases associated with BMB.