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.

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.

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.

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.

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.

A modified "cross-talk" between histone H2B Lys-120 ubiquitination and H3 Lys-79 methylation.

Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the cross-talk relationship between H2B ubiquitination and H3 Lys-79 methylation.

Iron quantification of microbleeds in postmortem brain.

Brain microbleeds (BMB) are associated with chronic and acute cerebrovascular disease and present a source of pathologic iron to the brain proportional to extravasated blood. Therefore, BMB iron content is potentially a valuable biomarker. We tested noninvasive phase image methods to quantify iron content and estimate true source diameter (i.e., unobscured by the blooming effect) of BMB in postmortem human tissue. Tissue slices containing BMB were imaged using a susceptibility weighted imaging protocol at 11.7T. BMB lesions were assayed for iron content using atomic absorption spectrometry. Measurements of geometric features in phase images were related to lesion iron content and source diameter using a mathematical model. BMB diameter was estimated by image feature geometry alone without explicit relation to the magnetic susceptibility. A strong linear relationship (R(2) = 0.984, P < 0.001) predicted by theory was observed in the experimental data, presenting a tentative standardization curve where BMB iron content in similar tissues could be calculated. In addition, we report BMB iron mass measurements, as well as upper bound diameter and lower bound iron concentration estimates. Our methods potentially allows the calculation of brain iron load indices based on BMB iron content and classification of BMB by size unobscured by the blooming effect.

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.

Quantification of punctate iron sources using magnetic resonance phase.

Iron-mediated tissue damage is present in cerebrovascular and neurodegenerative diseases and neurotrauma. Brain microbleeds are often present in these maladies and are assuming increasing clinical importance. Because brain microbleeds present a source of pathologic iron to the brain, the noninvasive quantification of this iron pool is potentially valuable. Past efforts to quantify brain iron have focused on content estimation within distributed brain regions. In addition, conventional approaches using "magnitude" images have met significant limitations. In this study, a technique is presented to quantify the iron content of punctate samples using phase images. Samples are modeled as magnetic dipoles and phase shifts due to local dipole field perturbations are mathematically related to sample iron content and radius using easily recognized geometric features in phase images. Phantoms containing samples of a chitosan-ferric oxyhydroxide composite (which serves as a mimic for hemosiderin) were scanned with a susceptibility-weighted imaging sequence at 11.7 T. Plots relating sample iron content and radius to phase image features were compared to theoretical predictions. The primary result is the validation of the technique by the excellent agreement between theory and the iron content plot. This research is a potential first step toward quantification of punctate brain iron sources such as brain microbleeds.

Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study.

Neuroimaging with iron-sensitive MR sequences [gradient echo T2* and susceptibility-weighted imaging (SWI)] identifies small signal voids that are suspected brain microbleeds. Though the clinical significance of these lesions remains uncertain, their distribution and prevalence correlates with cerebral amyloid angiopathy (CAA), hypertension, smoking, and cognitive deficits. Investigation of the pathologies that produce signal voids is necessary to properly interpret these imaging findings. We conducted a systematic correlation of SWI-identified hypointensities to tissue pathology in postmortem brains with Alzheimer's disease (AD) and varying degrees of CAA. Autopsied brains from eight AD patients, six of which showed advanced CAA, were imaged at 3T; foci corresponding to hypointensities were identified and studied histologically. A variety of lesions was detected; the most common lesions were acute microhemorrhage, hemosiderin residua of old hemorrhages, and small lacunes ringed by hemosiderin. In lesions where the bleeding vessel could be identified, ß-amyloid immunohistochemistry confirmed the presence of ß-amyloid in the vessel wall. Significant cellular apoptosis was noted in the perifocal region of recent bleeds along with heme oxygenase 1 activity and late complement activation. Acutely extravasated blood and hemosiderin were noted to migrate through enlarged Virchow­Robin spaces propagating an inflammatory reaction along the local microvasculature; a mechanism that may contribute to the formation of lacunar infarcts. Correlation of imaging findings to tissue pathology in our cases indicates that a variety of CAA-related pathologies produce MR-identified signal voids and further supports the use of SWI as a biomarker for this disease.

Imaging cerebral microbleeds using susceptibility weighted imaging: one step toward detecting vascular dementia.

PURPOSE: To monitor changes in the number of cerebral microbleeds (CMBs) in a longitudinal study of healthy controls (HC) and mild-cognitively impaired (MCI) patients using susceptibility weighted imaging (SWI). MATERIALS AND METHODS: SWI was used to image 28 HC and 75 MCI patients annually at 1.5 Tesla over a 4-year period. Magnitude and phase data were used to visualize CMBs for the first and last scans of 103 subjects. RESULTS: Preliminary analysis revealed that none of the 28 HC had more than three CMBs. In the 75 MCI patients, five subjects had more than three CMBs in both first and last scans, while one subject had more than three bleeds only in the last scan. In five of these six MCI patients, the number of CMBs increased over time and all six went on to develop progressive cognitive impairment (PCI). Of the 130 total CMBs seen in the last scans of the six MCI cases, most were less than 4 mm in diameter. CONCLUSION: SWI can reveal small CMBs on the order of 1 mm in diameter and this technique can be used to follow their development longitudinally. Monitoring CMBs may be a means by which to evaluate patients for the presence of microvascular disease that leads to PCI.

The effect of formalin fixation on the levels of brain transition metals in archived samples.

Reports that iron, zinc and copper homeostasis are in aberrant homeostasis are common for various neurodegenerative diseases, particularly for Huntington's disease, Parkinson's disease, and Alzheimer's disease. Manipulating the levels of these elements in the brain through the application of chelators has been and continues to be tested therapeutically in clinical trials with mixed results. Much of the data indicating that these metals are abnormally concentrated in Alzheimer's disease and Parkinson's disease brain tissue was generated through the analysis of post-mortem human tissue which was archived in formalin. In this study, we evaluated the effect of formalin fixation of brain on the levels of three important transition metals (iron, copper, and zinc) by atomic absorption spectroscopy. Paired brain specimens were obtained at autopsy for each case; one was conserved by formalin archival (averaging four years), the other was rapidly frozen. Both white and grey matter samples were analyzed and the concentrations of iron and zinc were found to decrease with fixation. Iron was reduced by 40% (P < 0.01), and zinc by 77% (P < 0.0001); copper concentrations increased by 37% (P < 0.05) by the paired T-test. The increase in copper is likely due to contamination from trace copper in the formalin. These results indicate that transition metal data obtained from fixed tissue may be heavily distorted and care should be taken in interpreting this data.

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.

Immunophenotypes in the circulation of patients with mild cognitive impairment.

Alterations in the peripheral immune system are associated with dementia and the neuropathology of Alzheimer's disease, but have yet to be studied early in the disease process. To test the hypothesis that the balance of immune cell phenotypes is disrupted in the early progression of memory deterioration, patients with mild cognitive impairment (MCI) and healthy elderly controls were examined for the distribution of subpopulations of leukocytes (lymphocytes, granulocytes, and monocytes) and lymphocyte subtypes (helper/inducer and suppressor/cytotoxic T lymphocytes and B lymphocytes) in blood. MCI subjects had a significantly higher percentage of total lymphocytes and a lower percentage of granulocytes compared to elderly controls. Furthermore, the expression of cell surface amyloid precursor protein (APP) and intracellular amyloid-beta peptide (Abeta) in lymphocytes and monocytes were determined. We found lymphocyte APP expression to be significantly increased in MCI subjects compared to controls. Our data indicate that changes in immunological parameters may be detected early in MCI, and an alteration of the immune response may precede clinical AD.

Longitudinal metabolic and cognitive changes in mild cognitive impairment patients.

Advancements in clinical therapies have identified the need for biomarkers of early Alzheimer disease that distinguish the earliest stages of pathology and target those patients who are likely to gain the most benefit. The aim of this study was to characterize the longitudinal metabolic changes measured by 1H magnetic resonance spectroscopy in correlation to neuropsychologic indices of episodic memory, attention and mental processing speed, language facility, and executive function in subjects with mild cognitive impairment (MCI). Quantitative 1H magnetic resonance spectroscopy of the posterior cingulate gyrus was performed and repeated at 11.56+/-4.3 months. N-acetyl aspartate (NAA), total choline (Cho), total creatine (Cr), myo-inositol (mI), and glutamate/glutamine (Glx) metabolite levels were measured, corrected for cerebrospinal fluid dilution, and ratios calculated in MCI and cognitively normal subjects. In the first study, MCI subjects showed lower NAA levels, NAA/Cho, and NAA/mI ratios and increased Cho/Cr and mI/Cr compared with controls. In the follow-up study, 36% of the MCI subjects [atypical MCI (atMCI)] showed interval increases in NAA, Cr, and Glx levels compared with 64% of MCI subjects (typical MCI) who showed an interval decrease in NAA, Cr, and Glx. Both MCI subgroups had higher Clinical Dementia Rating scores and lower scores on episodic memory, phonemic, and semantic word fluency tasks, compared with controls. The annualized rate of change in metabolic and cognitive status did not differ between normal aging and MCI subjects. atMCI subjects showed significant negative correlations between metabolite levels and executive function task scores, with NAA/mI showing a significant positive correlation with phonemic and semantic word fluency. There were no significant correlations between metabolite levels and cognitive performance in tMCI subjects; however, NAA/mI and mI/Cr were negatively correlated with executive function tasks. These results indicate 2 distinct evolving metabolite profiles that correlate with changes in executive function and can be used to differentiate MCI from normal aging.

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.

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.

Increased production of inflammatory cytokines in mild cognitive impairment.

Recent studies indicate that chronic inflammation plays a pathogenic role in both the central nervous system (CNS) and periphery in Alzheimer's disease (AD). We have screened for cytokines differentially produced by peripheral blood mononuclear cells (PBMCs) isolated from subjects with mild cognitive impairment (MCI) and mild AD subjects who had progressed from MCI using a commercially available cytokine array. Following determination of expressed cytokines, we quantified levels of the proinflammatory cytokines TNF-alpha, IL-6, and IL-8, and the anti-inflammatory cytokine IL-10 using flow cytometry. We have found a significant increase in the levels of IL-6, IL-8, and IL-10 produced by PBMCs stimulated for 24 h with phytohemagglutinin (PHA) in MCI subjects compared to healthy elderly controls. However, in PBMCs stimulated for 48 h with lipopolysaccharide (LPS), lower TNF-alpha/IL-10, IL-6/IL-10, and IL-8/IL-10 ratios were seen in MCI subjects. There were no differences in plasma levels of IL-8 between aged controls, MCI, and mild AD, and the levels of circulating IL-6 and IL-10 were below detection limits. Our data indicate that changes in cytokine production by PBMCs may be detected early in MCI, and an alteration of the immune response may precede clinical AD.