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Scatter plots of Luminex fluorescent signal intensities for Aβ42 and pTau in MFG, hippocampus and neostriatum. We performed correlation analyses of Aβ42 and pTau signals across all analyzed brain regions and confirmed the following statistically significant positive correlations. (a) Aβ42 levels in MFG vs. Aβ42 levels in hippocampus. (b) Aβ42 levels in MFG vs. Aβ42 levels in neostriatum. (c) Aβ42 levels in hippocampus vs. Aβ42 levels in neostriatum. (d) Aβ42 levels in neostriatum vs. pTau levels in MFG. (e) pTau levels in MFG vs. pTau levels in hippocampus. Statistically significant R 2 values are shown in each scatter plot.
Source publication
The vast majority of archived research and clinical pathological specimens are stored in the form of formalin fixed, paraffin-embedded (FFPE) tissues, but, unlike fresh frozen tissue samples, highly quantitative measures in FFPE tissues are limited to immunohistochemical and immunofluorescence thresholding image analysis studies, cell counting, and...
Contexts in source publication
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... performed correlation analyses of Aβ42 and pTau levels in all analyzed brain regions. Aβ42 levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). ...
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... performed correlation analyses of Aβ42 and pTau levels in all analyzed brain regions. Aβ42 levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). ...
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... performed correlation analyses of Aβ42 and pTau levels in all analyzed brain regions. Aβ42 levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). ...
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... levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). pTau levels in the MFG were correlated positively with pTau levels in hippocampus (Figure 4f). ...
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... levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). pTau levels in the MFG were correlated positively with pTau levels in hippocampus (Figure 4f). Although pTau levels in the neostriatum correlated significantly with Aβ42 and pTau levels in some other brain regions, these correlations cannot be interpreted because pTau levels in the neostriatum were essentially as low as PBS blanks. ...
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... performed correlation analyses of Aβ42 and pTau levels in all analyzed brain regions. Aβ42 levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). ...
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... performed correlation analyses of Aβ42 and pTau levels in all analyzed brain regions. Aβ42 levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). ...
Context 8
... performed correlation analyses of Aβ42 and pTau levels in all analyzed brain regions. Aβ42 levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). ...
Context 9
... levels in the MFG were correlated positively with Aβ42 levels in hippocampus (Figure 4a) and neostriatum (Figure 4b), and Aβ42 levels in the hippocampus were correlated positively with Aβ42 levels in the neostriatum (Figure 4c). Aβ42 levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). pTau levels in the MFG were correlated positively with pTau levels in hippocampus (Figure 4f). ...
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... levels in the neostriatum were correlated positively with pTau levels in the MFG (Figure 4d). pTau levels in the MFG were correlated positively with pTau levels in hippocampus (Figure 4f). Although pTau levels in the neostriatum correlated significantly with Aβ42 and pTau levels in some other brain regions, these correlations cannot be interpreted because pTau levels in the neostriatum were essentially as low as PBS blanks. ...
Citations
... The protocol as in [30] was followed for total protein extraction from frozen mouse cortex. Briefly, 150 μl of chilled RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 50 mM Tris hydrochloride, 0.5 mM magnesium sulfate; all from Sigma-Aldrich; St. Louis, MO) with Complete Mini protease inhibitor (Millipore Sigma-Aldrich; St. Louis, MO) was immediately added to the tube containing the sample once it was thawed and sitting on ice. ...
Alzheimer's Disease (AD) is characterized by the accumulation of extracellular amyloid-β (Aβ) as well as CNS and systemic inflammation. Microglia, the myeloid cells resident in the CNS, use microRNAs to rapidly respond to inflammatory signals. MicroRNAs (miRNAs) modulate inflammatory responses in microglia, and miRNA profiles are altered in Alzheimer's disease (AD) patients. Expression of the pro-inflammatory miRNA, miR-155, is increased in the AD brain. However, the role of miR-155 in AD pathogenesis is not well-understood. We hypothesized that miR-155 participates in AD pathophysiology by regulating microglia internalization and degradation of Aβ. We used CX3CR1CreER/+ to drive-inducible, microglia-specific deletion of floxed miR-155 alleles in two AD mouse models. Microglia-specific inducible deletion of miR-155 in microglia increased anti-inflammatory gene expression while reducing insoluble Aβ1-42 and plaque area. Yet, microglia-specific miR-155 deletion led to early-onset hyperexcitability, recurring spontaneous seizures, and seizure-related mortality. The mechanism behind hyperexcitability involved microglia-mediated synaptic pruning as miR-155 deletion altered microglia internalization of synaptic material. These data identify miR-155 as a novel modulator of microglia Aβ internalization and synaptic pruning, influencing synaptic homeostasis in the setting of AD pathology.
... The above studies specifically examined CSF levels of CHI3L1. De Fino et al. evaluated both CSF and serum levels using Luminex ® technology, [67] based on ELISA. They found significantly elevated levels in MS compared to controls in CSF but not serum [68]. ...
Purpose
For many patients, the multiple sclerosis (MS) diagnostic process can be lengthy, costly, and fraught with error. Recent research aims to address the unmet need for an accurate and simple diagnostic process through discovery of novel diagnostic biomarkers. This review summarizes recent studies on MS diagnostic fluid biomarkers, with a focus on blood biomarkers, and includes discussion of technical limitations and practical applicability.
Recent Findings
This line of research is in its early days. Accurate and easily obtainable biomarkers for MS have not yet been identified and validated, but several approaches to uncover them are underway.
Summary
Continue efforts to define laboratory diagnostic biomarkers are likely to play an increasingly important role in defining MS at the earliest stages, leading to better long-term clinical outcomes.
... While there is research into the correlation between Aβ42 levels and cognitive function in laboratory beagles [22], to our knowledge nobody has as of yet measured Aβ42 in companion dog brains in a quantitative manner and correlated these measurements with cognitive function. Here, we used a newly developed Luminex assay [23,24] to quantitatively measure Aβ42 in brains and cerebrospinal fluid (CSF) from privately owned companion dogs with known CCD scores and report statistically significant correlations between cognitive phenotype and Aβ42 abundance in these brains. ...
... They are not quantitative measures but rather ordered rankings, which limits their utility in studies that aim to correlate Aβ42 pathology and cognitive function. To overcome this limitation, we developed a Luminex-based assay for quantification of Aβ42 in human brain autopsy tissue [24], and we have further established parameters for this assay that allow quantification of Aβ42 in mutation-based animal models of AD and in natural models like non-human primates and dogs [23]. The data set from our current study in dogs constitutes one of the first applications of our Luminex assay to probe the quantitative relationship between brain Aβ42 and cognitive changes. ...
Alzheimer’s disease (AD) is a significant burden for human health that is increasing in prevalence as the global population ages. There is growing recognition that current preclinical models of AD are insufficient to recapitulate key aspects of the disease. Laboratory models for AD include mice, which do not naturally develop AD-like pathology during aging, and laboratory Beagle dogs, which do not share the human environment. In contrast, the companion dog shares the human environment and presents a genetically heterogeneous population of animals that might spontaneously develop age-associated AD-like pathology and cognitive dysfunction. Here, we quantitatively measured amyloid beta (Aβ42 or Abeta-42) levels in three areas of the companion dog brain (prefrontal cortex, temporal cortex, hippocampus/entorhinal cortex) and cerebrospinal fluid (CSF) using a newly developed Luminex assay. We found significant positive correlations between Aβ42 and age in all three brain regions. Brain Aβ42 abundance in all three brain regions was also correlated with Canine Cognitive Dysfunction Scale score in a multivariate analysis. This latter effect remained significant when correcting for age, except in the temporal cortex. There was no correlation between Aβ42 in CSF and cognitive scores; however, we found a significant positive correlation between Aβ42 in CSF and body weight, as well as a significant negative correlation between Aβ42 in CSF and age. Our results support the suitability of the companion dog as a model for AD and illustrate the utility of veterinary biobanking to make biospecimens available to researchers for analysis.
... Objective and quantitative measures of AD pathology are also essential for the testing of AD interventions. Recently, a Luminex-based approach was developed to quantify Aβ42 and pTau in frozen and formalin-fixed brain tissue from AD patients [4]. ...
... For the invertebrate species, we extracted proteins from complete fly heads and whole-worm pellets. The same extraction procedures that we used for human brain samples were used for all animal samples to generate RIPA-buffer (contain pTau) and guanidinehydrochloride (Gu-HCl, contain Aβ42) soluble fractions [4]. Total protein content was determined for each fraction using BCA assays (Pierce, Rockford IL). ...
... For quantification of Aβ42, we used the same Aβ42 standards, antibodies and procedures as described for analysis of human AD samples [4]. In short, we generated antibody-coupled (monoclonal antibody clone H31L21, Life Technologies, Carlsbad CA) magnetic Luminex beads for Aβ42-antigen capture and biotinylated antibodies (monoclonal antibody clone 6E10, Bio Legend, San Diego CA) for Aβ42-antigen detection. ...
A major obstacle for preclinical testing of Alzheimer’s disease (AD) therapies is the availability of translationally relevant AD models. Critical for the validation of such models is the application of the same approaches and techniques used for the neuropathological characterization of AD. Deposition of amyloid-β 42 (Aβ42) plaques and neurofibrillary tangles containing phospho-Tau (pTau) are the pathognomonic features of AD. In the neuropathologic evaluation of AD, immunohistochemistry (IHC) is the current standard method for detection of Aβ42 and pTau. Although IHC is indispensable for determining the distribution of AD pathology, it is of rather limited use for assessment of the quantity of AD pathology. We have recently developed Luminex-based assays for the quantitative assessment of Aβ42 and pTau in AD brains. These assays are based on the same antibodies that are used for the IHC-based diagnosis of AD neuropathologic change. Here we report the application and extension of such quantitative AD neuropathology assays to commonly used genetically engineered AD models and to animals that develop AD neuropathologic change as they age naturally. We believe that identifying AD models that have Aβ42 or pTau levels comparable to those observed in AD will greatly improve the ability to develop AD therapies.
Abbreviations: Alzheimer’s disease (AD); amyloid β 42 (Aβ42); phospho-Tau (pTau); immunohistochemistry (IHC)
... Coupling of 2H5, 3D8, 12A2 and 14E2 mAbs to fluorescent magnetic beads (Bio-Rad; Hercules, CA) was performed as previously described [25]. All couplings to beads were confirmed using serial dilutions of phycoerythrin (PE) labeled anti-mouse IgG detection antibodies (Jackson ImmunoResearch; West Grove, PA), and the beads were read using a LiquiChip Luminex instrument (QIAGEN; Germantown, MD). ...
Background:
The gene encoding HE4 undergoes alternative splicing to yield multiple protein isoforms. We investigated anti-HE4 mAbs which recognize epitopes on the C (2H5 and 3D8) or N (12A2 and 14E2) terminals.
Methods:
A Luminex assay was applied to determine mAb affinity. Binding of mAbs to sections from formaline fixed ovarian carcinomas was determined by immunohistology, binding to cultured ovarian carcinoma cells was tested by flow cytometry and immunocytochemistry, and HE4 secretion to patient serum or supernatants of cultured ovarian carcinoma was assayed by ELISA.
Results:
mAb 12A2 bound to formalin-fixed sections from 18 of 19 ovarian carcinomas, while 14E2, 2H5 and 3D8 bound to sections from 14, 7 and 4 patients, respectively. The mAbs bound independently of each other, i.e. a sample bound one mAb most strongly while another sample with similar affinity strongest bound another mAb. The intensity of binding to sections did not significantly correlate with the serum level of HE4 except for mAb 14E2, but there was a significant correlation between HE4 expression and its detection in supernatants of cultured ovarian carcinomas.
Conclusions:
HE4 epitopes are expressed independently of each other and their expression of HE4 by cultured ovarian carcinoma cells correlates with the release of HE4 into culture supernatants. he epitope recognized by mAb 14E2 was significantly more expressed by platinum resistant tumors.
Impact:
Expression of HE4 by most ovarian carcinomas makes it an excellent biomarker.. Further studies are needed to investigate the clinical relevance of overexpression of a particular epitope.
The discovery of novel metabolomic, proteomic, lipidomic, and transcriptomic (mRNA and miRNA) biomarkers in serum, plasma, brain, and CSF is a promising area that would permit the early and accurate diagnosis of diseases with complex pathology such as Alzheimer’s disease (AD). High-throughput analysis of “omic” alternations at the preclinical stages of AD would probably allow the application of novel therapeutic strategies including personalized dietary and pharmacological approaches in presymptomatic AD subjects. Liquid chromatography–mass spectrometry (LC–MS), liquid chromatography–time-off light–mass spectrometry (LC–TOF/MS), flow injection analysis/tandem mass spectrometry (FIA-MS/MS), 2DGE-MS/MS-based approach, nuclear magnetic resonance (NMR) spectroscopy, a two-dimensional gel electrophoresis combined with mass spectrometry (2DGE-MS/MS), electrospray ionization–mass spectrometry (ES-MS), gas chromatography–mass spectrometry (GC/MS), Luminex assay, SIMOA, and MALDI mass spectrometry are different high-throughput automated technological approaches that are extensively used with high sensitivity and specificity for proteomic and lipidomic biomarker discovery. Moreover, altered genes and miRNA expression levels between AD, MCI, and healthy subjects have been discovered using, respectively, the DNA microarrays and the real-time quantitative reverse transcription qRT-PCR technique. The aim of this chapter is firstly to provide information on the utility of these high-throughput omics platforms in AD area and secondly to unveil a number of specific serum and CSF brain biomarkers at different stages of AD, using highly recommended and cost-efficient analytical methods.
Human brain tissue has long been a critical resource for neuroanatomy and neuropathology, but with the advent of advanced imaging and molecular sequencing techniques, it has become possible to use human brain tissue to study, in great detail, the structural, molecular, and even functional underpinnings of human brain disease. In the century following the first description of Alzheimer’s disease (AD), numerous technological advances applied to human tissue have enabled novel diagnostic approaches using diverse physical and molecular biomarkers, and many drug therapies have been tested in clinical trials (Schachter and Davis, Dialogues Clin Neurosci 2:91-100, 2000). The methods for brain procurement and tissue stabilization have remained somewhat consistently focused on formalin fixation and freezing. Although these methods have enabled research protocols of multiple modalities, new, more advanced technologies demand improved methodologies for the procurement, characterization, stabilization, and preparation of both normal and diseased human brain tissues. Here, we describe our current protocols for the procurement and characterization of fixed brain tissue, to enable systematic and precisely targeted diagnoses, and describe the novel, quantitative molecular, and neuroanatomical studies that broadly expand the use of formalin-fixed, paraffin-embedded (FFPE) tissue that will further our understanding of the mechanisms underlying human neuropathologies.
Background:
Evidence links fine particulate matter (PM2.5) to Alzheimer's disease (AD), but no community-based prospective cohort studies in older adults have evaluated the association between long-term exposure to PM2.5 and markers of AD neuropathology at autopsy.
Objective:
Using a well-established autopsy cohort and new spatiotemporal predictions of air pollution, we evaluated associations of 10-year PM2.5 exposure prior to death with Braak stage, Consortium to Establish a Registry for AD (CERAD) score, and combined AD neuropathologic change (ABC score).
Methods:
We used autopsy specimens (N = 832) from the Adult Changes in Thought (ACT) study, with enrollment ongoing since 1994. We assigned long-term exposure at residential address based on two-week average concentrations from a newly developed spatiotemporal model. To account for potential selection bias, we conducted inverse probability weighting. Adjusting for covariates with tiered models, we performed ordinal regression for Braak and CERAD and logistic regression for dichotomized ABC score.
Results:
10-year average PM2.5 from death across the autopsy cohort was 8.2 (1.9) μg/m3. Average age at death was 89 (±7) years. Each 1μg/m3 increase in 10-year average PM2.5 prior to death was associated with a suggestive increase in the odds of worse neuropathology as indicated by CERAD score (OR: 1.35 (0.90, 1.90)) but a suggestive decreased odds of neuropathology as defined by the ABC score (OR: 0.79 (0.49, 1.19). There was no association with Braak stage (OR: 0.99 (0.64, 1.47).
Conclusion:
We report inconclusive associations between PM2.5 and AD neuropathology at autopsy among a cohort where 94% of individuals experienced 10-year exposures below the current EPA standard. Prior studies of AD risk factors and AD neuropathology are similarly inconclusive, suggesting alternative mechanistic pathways for disease or residual confounding.
Alzheimer disease (AD), the most common form of dementia globally, classically defined a clinicopathological entity, is a heterogenous disorder with various pathobiological subtypes, currently referred to as Alzheimer continuum. Its morphological hallmarks are extracellular parenchymal β-amyloid (amyloid plaques) and intraneuronal (tau aggregates forming neurofibrillary tangles) lesions accompanied by synaptic loss and vascular amyloid deposits, that are essential for the pathological diagnosis of AD. In addition to “classical” AD, several subtypes with characteristic regional patterns of tau pathology have been described that show distinct clinical features, differences in age, sex distribution, biomarker levels, and patterns of key network destructions responsible for cognitive decline. AD is a mixed proteinopathy (amyloid and tau), frequently associated with other age-related co-pathologies, such as cerebrovascular lesions, Lewy and TDP-43 pathologies, hippocampal sclerosis, or argyrophilic grain disease. These and other co-pathologies essentially influence the clinical picture of AD and may accelerate disease progression. The purpose of this review is to provide a critical overview of AD pathology, its defining pathological substrates, and the heterogeneity among the Alzheimer spectrum entities that may provide a broader diagnostic coverage of this devastating disorder as a basis for implementing precision medicine approaches and for ultimate development of successful disease-modifying drugs for AD.
