Nimish K. Acharya

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.

Brain-reactive autoantibodies are nearly ubiquitous in human sera and may be linked to pathology in the context of blood-brain barrier breakdown

Previous studies have reported antibodies bound to cells in postmortem Alzheimers disease (AD) brains, which are only rarely observed in the brains of healthy, age-matched controls. This implies that brain-reactive autoantibodies exist in the sera of AD individuals and can gain access to the brain interstitium. To investigate this possibility, we determined the prevalence of brain-reactive antibodies in sera from AD patients, patients with other neurodegenerative diseases, age-matched, non-demented controls and healthy younger individuals via immunohistochemistry and western blot analysis. Surprisingly, western analyses revealed that 92% of all human sera tested contain brain-reactive autoantibodies. When sera were used to probe western blots of human, pig, or rat brain membrane proteins, a number of comparably-sized protein targets were detected, suggesting cross-species reactivity. While the presence of brain-reactive autoantibodies was nearly ubiquitous in human sera, some autoantibodies appeared to be associated with age or disease. Furthermore, the intensity of antibody binding to brain tissue elements, especially the surfaces of neurons, correlated more closely to the serums autoantibody profile than to age or the presence of neurodegenerative disease. However, while the blood-brain barrier (BBB) in control brains remained intact, BBB breakdown was common in AD brains. Results suggest a high prevalence of brain-reactive antibodies in human sera which, in the common context of BBB compromise, leads us to propose that these antibodies may contribute to the initiation and/or pathogenesis of AD and other neurodegenerative 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.

Neuronal expression of vimentin in the Alzheimer's disease brain may be part of a generalized dendritic damage-response mechanism

Early pathological features of Alzheimers disease (AD) include synaptic loss and dendrite retraction, prior to neuronal loss. How neurons respond to this evolving AD pathology remains elusive. In the present study, we used single- and double-label immunohistochemistry to investigate the relationship between neuronal vimentin expression and local brain pathology. Vimentin was localized to neuronal perikarya and dendrites in AD brain, with vimentin-immunopositive neurons prevalent in regions exhibiting intra- and extracellular beta-amyloid(1-42) (Abeta42) deposition. Neuronal co-localization of vimentin and Abeta42 was common in the cerebral cortex, cerebellum and hippocampus. Additionally, neurons in affected brain regions of AD transgenic (Tg2576) mice and in brain tissue subjected to mechanical injury expressed vimentin, while those in comparable regions of control mouse brain did not. Finally, we show that neurons in human fetal brain express vimentin concurrently with periods of rapid neurite extension. Overall, our results suggest that neurons express vimentin as part of an evolutionarily conserved, damage-response mechanism which recapitulates a developmental program used by differentiating neurons to establish dendrites and synaptic connections.

Plant Lectin Can Target Receptors Containing Sialic Acid, Exemplified by Podoplanin, to Inhibit Transformed Cell Growth and Migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

Brain-reactive autoantibodies prevalent in human sera increase intraneuronal amyloid-β(1-42) deposition.

Previous studies have reported immunoglobulin-positive neurons in Alzheimers disease (AD) brains, an observation indicative of blood-brain barrier (BBB) breakdown. Recently, we demonstrated the nearly ubiquitous presence of brain-reactive autoantibodies in human sera. The significance of these observations to AD pathology is unknown. Here, we show that IgG-immunopositive neurons are abundant in brain regions exhibiting AD pathology, including intraneuronal amyloid-β(42) (Aβ(42)) and amyloid plaques, and confirm by western analysis that brain-reactive autoantibodies are nearly ubiquitous in human serum. To investigate a possible interrelationship between neuronal antibody binding and Aβ pathology, we tested the effects of human serum autoantibodies on the intraneuronal deposition of soluble Aβ(42) peptide in adult mouse neurons in vitro (organotypic brain slice cultures). Binding of human autoantibodies to mouse neurons dramatically increased the rate and extent of intraneuronal Aβ(42) accumulation in the mouse cerebral cortex and hippocampus. Additionally, individual sera exhibited variable potency related to their capacity to enhance intraneuronal Aβ(42) peptide accumulation and immunolabel neurons in AD brain sections. Replacement of human sera with antibodies targeting abundant neuronal surface proteins resulted in a comparable enhancement of Aβ(42) accumulation in mouse neurons. Overall, results suggest that brain-reactive autoantibodies are ubiquitous in the blood and that a defective BBB allows these antibodies to access the brain interstitium, bind to neuronal surfaces and enhance intraneuronal deposition of Aβ(42) in AD brains. Thus, in the context of BBB compromise, brain-reactive autoantibodies may be an important risk factor for the initiation and/or progression of AD as well as other neurodegenerative diseases.

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.