Cassandra DeMarshall

Diagnosis of Alzheimer's Disease Based on Disease-Specific Autoantibody Profiles in Human Sera

After decades of Alzheimers disease (AD) research, the development of a definitive diagnostic test for this disease has remained elusive. The discovery of blood-borne biomarkers yielding an accurate and relatively non-invasive test has been a primary goal. Using human protein microarrays to characterize the differential expression of serum autoantibodies in AD and non-demented control (NDC) groups, we identified potential diagnostic biomarkers for AD. The differential significance of each biomarker was evaluated, resulting in the selection of only 10 autoantibody biomarkers that can effectively differentiate AD sera from NDC sera with a sensitivity of 96.0% and specificity of 92.5%. AD sera were also distinguishable from sera obtained from patients with Parkinsons disease and breast cancer with accuracies of 86% and 92%, respectively. Results demonstrate that serum autoantibodies can be used effectively as highly-specific and accurate biomarkers to diagnose AD throughout the course of the disease.

Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera, and Their Number Is Influenced By Age, Gender, and Disease

The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.

Diabetes and Hypercholesterolemia Increase Blood-Brain Barrier Permeability and Brain Amyloid Deposition: Beneficial Effects of the LpPLA2 Inhibitor Darapladib

Diabetes mellitus (DM) and hypercholesterolemia (HC) have emerged as major risk factors for Alzheimers disease, highlighting the importance of vascular health to normal brain functioning. Our previous study showed that DM and HC favor the development of advanced coronary atherosclerosis in a porcine model, and that treatment with darapladib, an inhibitor of lipoprotein-associated phospholipase A2, blocks atherosclerosis progression and improves animal alertness and activity levels. In the present study, we examined the effects of DM and HC on the permeability of the blood-brain barrier (BBB) using immunoglobulin G (IgG) as a biomarker. DMHC increased BBB permeability and the leak of microvascular IgG into the brain interstitium, which was bound preferentially to pyramidal neurons in the cerebral cortex. We also examined the effects of DMHC on the brain deposition of amyloid peptide (Aβ42), a well-known pathological feature of Alzheimers disease. Nearly all detectable Aβ42 was contained within cortical pyramidal neurons and DMHC increased the density of Aβ42-loaded neurons. Treatment of DMHC animals with darapladib reduced the amount of IgG-immunopositive material that leaked into the brain as well as the density of Aβ42-containing neurons. Overall, these results suggest that a prolonged state of DMHC may have chronic deleterious effects on the functional integrity of the BBB and that, in this DMHC pig model, darapladib reduces BBB permeability. Also, the preferential binding of IgG and coincident accumulation of Aβ42 in the same neurons suggests a mechanistic link between the leak of IgG through the BBB and intraneuronal deposition of Aβ42 in the brain.

Neuronal PAD4 expression and protein citrullination: Possible role in production of autoantibodies associated with neurodegenerative disease

Peptidyl arginine deiminases (PADs) catalyze a post-translational protein modification reaction called citrullination, where arginine is converted to citrulline. This modification has been linked to the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA). More recently, several studies have suggested that Alzheimers disease (AD), a devastating neurodegenerative disorder, may have an autoimmune component. In the present study, we have investigated the possibility that expression of PADs and protein citrullination plays a role in the production of brain-reactive autoantibodies that may contribute to Alzheimers-related brain pathology. Here, we report the selective expression of the PAD isoforms, PAD2 and PAD4, in astrocytes and neurons, respectively, and the concomitant accumulation of citrullinated proteins within PAD4-expressing cells, including neurons of the hippocampus and cerebral cortex. Expression of PADs and citrullinated proteins is prominent in brain regions engaged in neurodegenerative changes typical for AD pathology. Furthermore, we also demonstrate that the pentatricopeptide repeat domain2 (PTCD2) protein, an antigen target of a prominent AD diagnostic autoantibody, is present in a citrullinated form in AD brains. Our results suggest that disease-associated neuronal loss results in the release of cellular contents, including citrullinated proteins, into the brain interstitium. We propose that these citrullinated proteins and their degradation fragments enter into the blood and lymphatic circulation, and some are capable of eliciting an immune response that results in the production of autoantibodies. The long-term and progressive nature of AD and other neurodegenerative diseases results in chronic exposure of the immune system to these citrullinated products and may drive the continual production of autoantibodies.

Sevoflurane and Isoflurane induce structural changes in brain vascular endothelial cells and increase blood−brain barrier permeability: Possible link to postoperative delirium and cognitive decline

A large percentage of patients subjected to general anesthesia at 65 years and older exhibit postoperative delirium (POD). Here, we test the hypothesis that inhaled anesthetics (IAs), such as Sevoflurane and Isoflurane, act directly on brain vascular endothelial cells (BVECs) to increase blood-brain barrier (BBB) permeability, thereby contributing to POD. Rats of young (3-5 months), middle (10-12 months) and old (17-19 months) ages were anesthetized with Sevoflurane or Isoflurane for 3h. After exposure, some were euthanized immediately; others were allowed to recover for 24h before sacrifice. Immunohistochemistry was employed to monitor the extent of BBB breach, and scanning electron microscopy (SEM) was used to examine changes in the luminal surfaces of BVECs. Quantitative immunohistochemistry revealed increased BBB permeability in older animals treated with Sevoflurane, but not Isoflurane. Extravasated immunoglobulin G showed selective affinity for pyramidal neurons. SEM demonstrated marked flattening of the luminal surfaces of BVECs in anesthetic-treated rats. Results suggest an aging-linked BBB compromise resulting from exposure to Sevoflurane. Changes in the luminal surface topology of BVECs indicate a direct effect on the plasma membrane, which may weaken or disrupt their BBB-associated tight junctions. Disruption of brain homeostasis due to plasma influx into the brain parenchyma and binding of plasma components (e.g., immunoglobulins) to neurons may contribute to POD. We propose that, in the elderly, exposure to some IAs can cause BBB compromise that disrupts brain homeostasis, perturbs neuronal function and thereby contributes to POD. If unresolved, this may progress to postoperative cognitive decline and later dementia.

Potential utility of autoantibodies as blood-based biomarkers for early detection and diagnosis of Parkinson's disease.

INTRODUCTION There is a great need to identify readily accessible, blood-based biomarkers for Parkinsons disease (PD) that are useful for accurate early detection and diagnosis. This advancement would allow early patient treatment and enrollment into clinical trials, both of which would greatly facilitate the development of new therapies for PD. METHODS Sera from a total of 398 subjects, including 103 early-stage PD subjects derived from the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) study, were screened with human protein microarrays containing 9,486 potential antigen targets to identify autoantibodies potentially useful as biomarkers for PD. A panel of selected autoantibodies with a higher prevalence in early-stage PD was identified and tested using Random Forest for its ability to distinguish early-stage PD subjects from controls and from individuals with other neurodegenerative and non-neurodegenerative diseases. RESULTS Results demonstrate that a panel of selected, blood-borne autoantibody biomarkers can distinguish early-stage PD subjects (90% confidence in diagnosis) from age- and sex-matched controls with an overall accuracy of 87.9%, a sensitivity of 94.1% and specificity of 85.5%. These biomarkers were also capable of differentiating patients with early-stage PD from those with more advanced (mild-moderate) PD with an overall accuracy of 97.5%, and could distinguish subjects with early-stage PD from those with other neurological (e.g., Alzheimers disease and multiple sclerosis) and non-neurological (e.g., breast cancer) diseases. CONCLUSION These results demonstrate, for the first time, that a panel of selected autoantibodies may prove to be useful as effective blood-based biomarkers for the diagnosis of early-stage PD.

Detection of Alzheimer's disease at mild cognitive impairment and disease progression using autoantibodies as blood-based biomarkers

There is an urgent need to identify biomarkers that can accurately detect and diagnose Alzheimers disease (AD). Autoantibodies are abundant and ubiquitous in human sera and have been previously demonstrated as disease‐specific biomarkers capable of accurately diagnosing mild‐moderate stages of AD and Parkinsons disease.

Autoantibodies as diagnostic biomarkers for the detection and subtyping of multiple sclerosis

The goal of this preliminary proof-of-concept study was to use human protein microarrays to identify blood-based autoantibody biomarkers capable of diagnosing multiple sclerosis (MS). Using sera from 112 subjects, including 51 MS subjects, autoantibody biomarkers effectively differentiated MS subjects from age- and gender-matched normal and breast cancer controls with 95.0% and 100% overall accuracy, but not from subjects with Parkinsons disease. Autoantibody biomarkers were also useful in distinguishing subjects with the relapsing-remitting form of MS from those with the secondary progressive subtype. These results demonstrate that autoantibodies can be used as noninvasive blood-based biomarkers for the detection and subtyping of MS.

UTILITY OF AUTOANTIBODY BIOMARKERS FOR DETECTION OF ALZHEIMER'S DISEASE

misfolded proteins. Currently there is no consensus for blood based tests for these diseases. Expression profiling of small non-coding RNA’s, microRNA (miRNA), has revealed diagnostic potential in human disease such as cancer. miRNA’s can be found in the blood stream and other body fluids encapsulated in small extracellular vesicles known as exosomes.Methods: Using deep sequencing we have analysed the RNA content of exosomes from a cohort of healthy aged and Alzheimer’s patients (n1⁄449) using samples from the Australian Imaging, Biomarkers and Lifestyle Flagship Study (AIBL). miRNA associating with disease were validated using qPCR(N1⁄460), with predictions performed using RandomForest. Additional risk factors collected during the 4.5 year AIBL study were included, which consisted of baseline and 54 month clinical, medical and cognitive assessment including Amyloid-PET imaging. Results:We developed a workflow for the analysis of exosomal miRNA from human blood serum that has diagnostic potential for AD. We have identified an AD specific, 16 miRNA signature, derived from the deep sequencing data and validated in an additional set of samples. Adding established risk factors including age, sex, and APOE allele status to the panel of deregulated miRNA resulted in a sensitivity and specificity of 87% and 77% respectively for predicting AD. Furthermore, Amyloid-PET information for those HC participants incorrectly classified with AD suggested progression towards AD. Conclusions: These data suggest that an exosomal miRNA signature may be a suitable peripheral screening tool for AD and other neurodegenerative disorders such as Prion and Parkinson’s diseases.

S12. THE ‘AUTOANTIBODYOME’ IN PSYCHOSIS: A PILOT STUDY AND BLUEPRINT FOR BIOMARKER DISCOVERY

Abstract Background Recent studies seeking to describe the prevalence and significance of autoantibodies in psychotic disorders can be characterized as ‘top-down’ in theoretical approach; that is, autoantibodies to specific (usually CNS) antigens are sought based on a) the known function of the antigen (e.g. NMDAR) and its putative role in psychosis or b) the clinical observation that these autoantibodies can cause psychosis as part of a more complex neuropsychiatric presentation (e.g. autoimmune encephalitis). No candidate autoantibodies with a clear diagnostic or prognostic role have been definitively established. We sought to take an alternative, ‘bottom-up’ approach to the significance of autoantibodies in psychosis that remains agnostic as to the potential significance of any one antigen. Every individual harbours autoantibodies directed against many thousands of self-antigens and the vast majority are not disease-causing. Indeed production of autoantibodies may represent a physiological, antigen-specific ‘debris-clearing’ response to tissue destruction or damage. It follows that the autoantibody profile of any individual reflects any pathological process that is ongoing in that individual and can thus serve as a ‘readout’ of the disease state in question. Methods Sera from 20 patients with a first episode of psychosis (FEP) (males: n=16; mean age: 29.35 s.d.: 7.07) from the Genetics and Psychosis (GAP) study and 20 matched controls were analysed, using Invitrogen’s ProtoArray v5.1 Human Protein Microarrays, for the presence of IgG to 9486 unique human protein antigens which had been expressed as GST fusion proteins in insect cells, purified and spotted onto slides. Following application of a fluorescent secondary IgG, reactivity patterns were automatically read using a fluorescence scanner. Samples were split into testing and training sets, and the top 50 most differentially expressed and differentially depleted antibodies were then chosen as biomarkers. Results The top 50 expressed biomarkers from the training set correctly identified 100% of psychosis subjects from the testing set, and 80% of healthy controls (OOB estimate of error rate 10%). When training and testing sets were swapped, biomarker overlap was 46% and 90% of psychosis subjects and 90% of controls were correctly identified (OOB estimate of error rate 10%). The top 50 depleted biomarkers from the training set correctly identified 90% of psychosis subjects from the testing set, and 90% of healthy controls (OOB estimate of error rate 10%). When training and testing sets were swapped, depletion biomarker overlap was 2% and 70% of psychosis subjects and 40% of controls were correctly identified (OOB estimate of error rate 45%). Discussion The autoantibodyome in FEP differs from that of healthy individuals. In this novel pilot study, a panel of 50 differentially expressed autoantibodies allowed confident discrimination between patients and controls, potentially paving the way for development of antibody-based diagnostics for psychosis using a simple blood test and fewer autoantibodies. Depletion biomarkers, thought to represent antibodies selectively depleted from the blood due to target binding in tissues, had less replicability and utility than expression biomarkers, which may offer insights into the active role of the adaptive immune system in psychosis. Further work will attempt to validate this approach in larger samples, using psychiatric disease controls. This autoantibodyomic approach may also show promise for the identification of predictive and prognostic biomarkers in psychotic disorders.