Blood-Brain Barrier Damage and Neuroautoimmunity

Aristo Vojdani, PhD, MSc

The human brain contains more than ten billion capillaries, which translates to one vessel for each neuron. These capillaries form a circulatory network with over 400 miles (644 km) of blood vessels that nourish the brain, transporting as much as 20% of cardiac output. The entire length of this network is protected from toxins by the blood-brain barrier (BBB), a complex structure lining the capillaries throughout the brain.

When the BBB is damaged, circulating antibodies that cross-react with neurological tissues can infiltrate the brain and nervous system, with the potential destruction of neurologic tissues. The cycle of neuroautoimmunity begins with a breach of the gut and/or blood-brain barriers. BBB permeability and the effects of “leaky brain” contribute to many devastating neurological and autoimmune conditions, including traumatic brain injury (TBI).

New breakthroughs in laboratory testing make it possible to identify BBB permeability in trauma ranging from suspected concussions to TBI. The testing is also relevant to neurological disorders as advanced as Alzheimer’s dementia and as subtle as chronic headaches or memory loss. Testing for key antibodies makes it possible to diagnose many of these conditions at a preclinical stage, before frank symptoms of injury or disease are present. Increased permeability can manifest as CNS symptoms of neurotoxicity, autoimmunity, or even cancer.

Indications

Breaching or damage to this vast network is an underappreciated factor in a wide range of neurologic and autoimmune conditions, including:

• ADD, ADHD, autism spectrum disorder, developmental disorders

• Autoimmune conditions – celiac disease, type 1 diabetes, Hashimoto’s encephalitis, lupus with CNS involvement, rheumatoid arthritis, and sarcoidosis, as well as neuroimmune conditions such as CFS and FMS

• Concussions and traumatic brain injury

• Mental illness – obsessive-compulsive disorder, schizophrenia, and others

• Neurodegenerative disorders and diseases of aging – AIDS dementia, ALS, Alzheimer’s disease, epilepsy, multiple sclerosis, Parkinson’s disease, peripheral neuropathy, senile dementia, stroke

• Neurologic symptomology – cognitive impairment or decline, memory problems, chronic headaches, sleep disturbances, fatigue, dizziness, tremors, poor coordination, anxiety, depression

Barrier Dysfunction and Disease

Composed of highly specialized brain and endothelial cells, the BBB is fully differentiated to the neurovascular system, and present in all brain regions, except in those regulating the autonomic nervous system and the endocrine glands. Capillaries permit diffusion of blood-borne molecules, screening out molecules larger than 400 Daltons (Da), approximately the size of blood glucose. Larger molecules such as proteins, which can occur in the range of 50,000 to 100,000 Da, are denied access to the nervous system when the BBB is intact. Specific carriers, or transporters, in apical and basolateral membranes, control the influx of small polar solutes needed by the brain (nutrients such as glucose and amino acids) and the efflux of many waste products. Transporters also exclude many potentially toxic compounds, such as xenobiotics, food antigens, and peptides present in the circulation.

Absence of Immune Tolerance

Innumerable endothelial tight junctions in the BBB function to prevent the passage of large soluble molecules into the CNS. Consequently, the barrier also prevents developing B-cells from contact with brain antigens. During immune maturation, B cells are not exposed to the variety of unique brain antigens expressed on neurons, oligodendrocytes, microglia and astrocytes. The immune system apparently has no innate mechanisms for establishing tolerance to brain antigens or for preventing the production of antibodies against them. When exposure to an environmental trigger occurs that opens the BBB, the immune system, by producing antibodies against the CNS, attacks the CNS tissues.

Numerous exposures can trigger BBB permeability:

• Inflammatory responses associated with chronic or acute traumatic brain injury and concussions

• Environmental exposures – viral (CMV, Guillain-Barre, hepatitis B, herpes 6); bacterial (streptococcus, Chlamydia pneumoniae); molds and mycotoxins; EMF from power lines, computers, cell-phones, microwaves; and radiation

• Inhalation or touch contact – chemicals including formaldehyde, insecticides, pesticides; solvents such as benzene; heavy cigarette smoke.

• Intestinal permeability and exposures via the gut – dysbiosis, endotoxins from bacterial overgrowth; PCBs and other toxins ingested in food or water

• Other internal exposures and dysfunction – toxic metals such aluminum and heavy metals such as mercury; certain medications; silicone implants; shock, stress, and inflammation; hypertension; ischemia

Breaches of the BBB can result in the damaging effects of TH 1 and TH 17 lymphocytes, as well as antibodies that can target and damage neurons and tissues such as myelin basic protein. Autoantibodies can become pathogenic on penetration into the CNS through either BBBD (blood-brain barrier dysfunction) or extravasation into cerebrospinal fluid from the sub-arachnoid space.

Testing for Barrier Dysfunction

Advanced antibody testing now allows accurate evaluation of BBB competence. Patients with neuroautoimmunity frequently have high levels of antibodies against various components of the nervous system, particularly antibodies to S100-B and to occludin and zonulin.

BBB protein antibodies.

The blood-brain barrier includes endothelial cells, tight junctions, the capillary basement membrane (BM), pericytes (PCs), and astrocyte end-feet that tightly ensheath the vessel walls. Astrocytes located beneath the endothelial cells prevent the entry of unwanted molecules and produce a protein identified as BBB protein, which is now used in laboratory testing as a marker to identify the presence of neuroautoimmune reactivity. Elevated antibody titers for BBB protein and other brain proteins have been described in Alzheimer’s disease and epilepsy. In senile dementia, the presence of BBB protein antibodies is one of the early markers of cognitive decline. (Specific testing for BBB protein antibody is provided in Cyrex Laboratories’ Array 20.)

Occludin and zonulin antibodies.

Paracellular space between the endothelial cells of the BBB is protected by an extensive network of tight junctions, consisting of transmembrane proteins that include occludin, claudin, and junction adhesion molecule (JAM), as well as cytoplasmic proteins, specifically zonulin-1 (ZO1), zonulin-2 (ZO2), and zonulin-3 (ZO3). Many patients with autoimmune disorders produce antibodies against their own occludin and zonulin, which serve as useful laboratory markers in diagnostic testing. (This evaluation is provided in Cyrex Array 2.)

Lipophilic toxins.

Tight junctions between the cells of the BBB endothelium form a physical barrier, significantly reducing passive diffusion through the paracellular pathway, and forcing any molecular traffic to occur mainly across the endothelial transcellular pathway. However, gases such as oxygen and carbon dioxide can easily diffuse across the barrier via the lipid membranes. This provides a security breach through which toxic lipophilic chemicals including petroleum-based products and other fat-soluble chemicals can cross the BBB, invading the CNS. (Antibodies targeting lipophilic toxins are among the 12 substances evaluated in Cyrex Array 11.)

A growing body of research has implicated the effects of BBB dysregulation in acceleration of chronic neurodegenerative disorders, including:

• Faulty BBB clearance of potential brain toxins in Alzheimer’s and Parkinson’s disease

• Inefficient clearance of excitotoxins across the BBB after an ischemic insult or TBI

• Increased transport of leukocytes across the activated BBB in AIDS dementia, Alzheimer’s disease, multiple sclerosis, and during neuroinflammatory CNS responses

• BBB breakdown in Alzheimer’s disease, amyotrophic lateral sclerosis, epilepsy, and multiple sclerosis.

The mechanisms by which toxic chemicals disrupt the endothelial tight junctions are shown in Figure 1, through processes that result in damage to astrocytes, microglia activation, the production of autoreactive antibodies, free radical generation, and immunoexcitotoxicity.

[Figure 1. Immunoexcitotoxicity about here.]

Figure 1. Proposed mechanism for excitotoxicity from leaky BBB.

Environmental triggers, brain inflammation and disruption of the BBB leads to the release of free radicals and immunoinflammatory factors, causing acute microglia activation, which contributes to immunoexcitotoxicity.

Secondary Disorders

Chronic inflammation and deterioration.

Chronic inflammation in the brain can be caused by environmental triggers such as infection or toxic chemicals that activate neurons, astrocytes, and microglia to produce cytokines. As is shown in Figure 1, environmental triggers and inflammation in the brain cause brain cells to produce TNFα, IL-1β,and IL-6, which contribute significantly to neuronal cell destruction and neuroautoimmunity.

Cancer.

Polychlorinated biphenyls (PCBs) are a prime example of a toxin that can disrupt blood-brain barrier integrity and promote brain metastasis formation. When these exposures occur, the chemical opens the barrier, damaging the astrocytes, which then can no longer protect the barrier. The same process can occur with infections such as Borrelia burgdorferi, cytomegalovirus, Epstein-Barr virus, herpes type 6, and other environmental triggers which directly or indirectly damage the barriers, resulting in neuroautoimmunity and even cancer.

Specific Exposures

Traumatic brain injury

(TBI).

Injuries such as those occurring in accidents, military combat, or in many sports can lead to repeated disruption of the blood-brain barrier. It is estimated that in the United States alone, 4 million people experience sports and recreation-related concussions annually. In football, for example, approximately 40% of players experience concussions. The repeated head trauma and TBI associated with contact sports such as boxing, basketball, and soccer have also been shown to induce BBB permeability, followed by antibody production against BBB proteins through a number of processes:

• Autoantibodies targeting the BBB – indication of pathological alteration of the protective brain barrier.

• Neurotransmitter dysregulation – manifest in both pre- and post-synaptic dysregulation of neurotransmitters.

• Excitotoxicity – this pathology involves free radical formation, brain swelling, and the entry of locally produced molecules such as cytokines and chemokines, disrupting metabolism and contributing to neuroinflammation and oxidative stress.

These processes have been confirmed in clinical research such as a study of 57 football players, medically evaluated before and after games, measuring the level of S100-B proteins. Serum S100-B was detected in players who experienced the greatest number of sub-concussive hits. Since this is a large protein molecule (21 kDa), even trace amounts handled by cells involved in the immune system resulted in antibodies against S100-B. The presence of this protein in a blood sample means that the BBB has been breached or damaged, and antibodies produced against S100-B have invaded the CNS.

The high degree of sensitivity of this testing was confirmed in a French study involving 2,000 patients with minor head injuries, comparing CT scans with plasma S100-B levels. TBIs were identified by antibody testing with 99% sensitivity and 20% specificity, confirmed by CT scan with the conclusion that S100-B testing is a highly promising screening tool.

Environmental Exposures

Inhaled exposures.

Particulate matter due to environmental pollution can also cause breaching of the BBB and lead to the induction of neuroimmune disorders. Traces of toxic chemicals such as benzene and formaldehyde and heavy metals such as mercury proliferate in air, water, and soil. Particulates of these toxins can enter the human body through the lungs or digestive tract, activating lung and intestinal tissue enzymes and triggering inflammation. In the lungs, tight junctions normally seal the gap between epithelial cells of the airway preventing entry of antigens, microbial toxins, and particulate matter. The breakdown of tight junction protein complexes in the lungs (as well as the tight junctions in the gastrointestinal and BBB barriers) contributes to the entry of antigens, xenobiotics, and chemicals into the circulation, resulting in inflammatory responses followed by the formation of autoantibodies that readily cross the compromised BBB.

These autoantibodies can attack key nervous system components such as GAD-65, transglutaminase-6, and cerebellar tissue, potentially leading to neuroinflammatory and neurodegenerative disorders. Thus, detecting significant elevations in IgA, IgG, and IgM antibodies against barrier-forming proteins is key to understand the environmental pollutant mechanistic pathways affecting epithelial and endothelial barriers. The mechanisms by which environmental pollutants can affect the barriers and lead to neuroautoimmunity are shown in Figure 2.

[Figure 2. Environmental Pollution, about here.]

Figure 2. Proposed mechanism for the effect of environmental pollutants on the BBB, leading to neuroautoimmunity.

Environmental pollutants enter the system either through inhalation or consumption, activating enzymes and inducing inflammation. This opens the tight junctions, allowing pollutant molecules to enter the circulation, causing autoantibody production. Autoantibodies attack neuronal cells of the CNS, leading to demyelination and neuroautoimmunity.

Chemical toxins.

Simply measuring chemicals in urine or blood is not enough to indicate exposure. In 80% of patients, these chemicals are successfully excreted, but in an estimated 20% of patients, chemicals form a complex with human tissue and remain in the body, possibly for life. When these chemicals bind to human tissue (for example as deposits in fat cells), they induce antibody production against both the toxins and human tissue.

Whenever autoimmunity is present, it is vital to ask why the immune system is producing antibodies against the patient’s own tissue. The answer is usually because toxins or antigens of an infectious agent from the environment are binding to the patient’s tissues, causing antibody production.

Research was conducted measuring antibodies in blood samples from 400 so-called healthy subjects (200 males and 200 females of different ethnicities with a median age of 36.2 years). Researchers found that 25% of participants were producing antibodies against various chemicals, indicating chronic exposure to chemical haptens. The formation of chemical-protein adducts could be one of the mechanisms by which environmental chemicals induce autoimmune reactivity.¹

Exposure to molds and mycotoxins or infection.

A significant number of patients experience symptoms in response to molds and/or mycotoxin exposure. Various other types of infectious agents can also have these effects, including Lyme disease. Autopsy research has reported that brain tissue of Lyme patients evidenced plaque and tangles typically characteristic of patients with Alzheimer disease.

Viral hitchhiking.

The BBB works efficiently to prevent brain infections. However viruses can penetrate the barrier by attaching onto circulating cells of the immune system. Systemic LPS (lipopolysaccharides) enhance both immune cell and free virus transport across the intact BBB. In vivo and in vitro studies, for example, have found that free HIV-1 can be taken up by brain endothelial cells and cross the BBB. LPS act at the luminal surface of the brain microvascular endothelial cell monolayer, which induces abluminal secretion of cytokines and other factors that subsequently act on pericytes. The pericytes then secrete substances that enhance viral transcytosis across the BBB.

Electromagnetic fields.

Effects of cell phone radiation on the BBB were initially identified 20 years ago. In an animal study evaluating radio frequency radiation, Adlkofer found albumin leakage in 32% of the 184 animals evaluated. Human clinical trials have shown that exposure to radio frequency radiation results in the leakage of albumin from the brain and is also associated with symptoms of memory loss and forgetfulness. Radio frequency energy is transmitted by cell phones, tablets, and laptop computers through blue tooth and other wi-fi connections.

[Table 1 about here.]

Intestinal Permeability

Leaky gut.

The elegant barrier of epithelial tight junctions in the gut provides four or five different layers of protection that are not easily breached. However, environmental exposures of sufficient magnitude can trigger a cascade of events that open the tight junctions. When that occurs, undigested molecules, lipopolysaccharides (LPS), and other bacterial toxins enter the submucosa through the circulation.

The gut-brain connection has been well studied in the existing literature. Gut epithelium resembles the BBB in many respects, with perhaps 75% similarity in structural composition. Due to antigen similarity between tissue proteins in gut endothelium and in BBB proteins, “leaky gut” can result in “leaky brain.” If the gut barrier is not repaired, those molecules now open the BBB, resulting in inflammation and autoimmunity in the nervous system.

Dysbiosis.

Dysfunctional flora or overgrowth is a well-recognized trigger of leaky gut. Certain dietary components such as gluten, dairy, and other antigens can also open the BBB resulting in antibody production against nervous system antigens and brain receptor cells. Due to the similarity between gluten and casein (both α-casein and β-casein), and synuclein and oligodendrocytes in the brain, resulting antibodies can attack nervous system antigens, resulting in neuroautoimmunity.

Food antigens.

LPS is not the only molecule that has been shown to transport viruses across the BBB. In an animal study, wheat germ agglutinin (WGA), a lectin protein from wheat, was injected into subjects. WGA was found to bind to sialic acid and N-acetylglucosamine, inducing vesicle-mediated internalization of WGA by brain endothelial cells, a process called adsorptive endocytosis. Lectin-induced vesicles provide a mechanism by which enveloped viruses can be internalized by cells. These study results strongly suggest that glycoprotein gp120 or gp120/gp41 induces adsorptive endocytosis and the uptake of HIV-1 by brain endothelial cells. This action explains how free, blood-borne viruses can infect the CNS while the BBB remains intact.

Environmental factors, if they are powerful enough, can initiate the cascade of events that directly or indirectly opens the tight junctions. If we do not repair the gut barrier, those molecules can open the blood-brain barrier, triggering adverse effects that include inflammation, oxidative stress, neurotoxicity, autoimmunity, degenerative disease, or cancer.

Advanced Antibody Testing: Array 20

This array is relevant to the assessment, diagnosis, and management of disease risk, progression, and progress in healing for patients with suspected trauma, TBI, and neurodegenerative, and autoimmune disorders.

Evaluating preclinical symptomology.

The test has efficacy in the assessment of preclinical conditions to evaluate potential breaching of the blood-brain barrier due to trauma, environmental triggers, or dementia with useful findings even in the absence of a clearly defined diagnosis.

Assessment of minor injuries.

This lab work is useful for patients who play contact sports and/or have experienced trauma to determine whether a concussion or TBI has occurred. The test is cost-effective, so it can be used in situations involving minor injury such as high school athletics, for example, when an MRI would seem excessive and could be prohibitively expensive.

Rehabilitation.

Array 20 can also be used to monitor injuries, indicating when it will be safe to return to normal activities. One of the major concerns after a first injury is the prevention of recurrent injuries before the brain is completely healed. Repetitive brain injury that occurs before complete healing and repair of the neuroinflammatory response can accelerate and exacerbate chronic activation of the microglia, with serious consequences.

Management of complex neurological disease.

Antibody testing provides a noninvasive tool for assessing the effectiveness of interventions for neurodegenerative disorders such as Alzheimer’s disease, amyotrophic lateral sclerosis (Lou Gehrig’s disease), epilepsy, multiple sclerosis, Parkinson’s disease, stroke, and vascular dementia.

[Boxed sidebar 1 about here.]

Clinical Strategies

Antibody testing is an evaluation with high clinical utility. Identifying reactivity to foods or environmental exposures provides clear-cut action steps the patient can take to reduce immune activation. If the trigger is a food or a chemical, eliminate it—remove it from the environment of the patient. Once the exposure is reduced, typically patients experience major improvement in two to six months. If, for example, testing shows elevated lipopolysaccharides in the gut indicating leaky gut, then repairing the gut can also help to repair the brain barrier. Supplements that can be recommended include probiotics, curcumin, resveratrol, glutamine, n-acetyl-cysteine, vitamin A, vitamin D, omega 3, or omega 6 essential fatty acids. These are all nutrients that can help to repair the barriers. This evaluation also serves to motivate patients, since it provides the logic for specific interventions with scientific evidence to back up recommendations.

Rationale for Testing

The need for effective neurological evaluation today has never been greater, considering the millions of people who experience traumatic brain injury in any given year, including soldiers, accident and trauma victims, and athletes. Additionally, millions of individuals across the age spectrum suffer from cognitive disorders, ranging from children with ADD, ADHD, ASD, developmental disorders, or epilepsy to seniors with ALS, autoimmune conditions, MS, Parkinson’s, stroke, or various forms of dementia. The prevailing method of choice for detecting intracranial injury in patients with even minor injury is computed tomography (CT) scan or MRI (magnetic resonance imaging). Although the CT scan has high sensitivity in detecting intracranial injury, it is expensive, exposes patients to high doses of radiation, and identifies clinically relevant lesions in less than 10% of cases. In addition, neither MRI nor CT scan can detect autoimmune reactivities.

Antibody testing is an evaluation technique that has demonstrated 99% sensitivity in the clinical setting. This is a viable adjunct or alternative to imaging for the full range of neuroautoimmune disorders, including traumatic brain injury, progressive dementia, neurodegenerative disease, and autoimmunity.

[Photo of Dr. Vojdani page 1 or here.]

Aristo Vojdani PhD, MSc

Aristo Vojdani is presently a professor of neuroimmunology at the Carrick Institute for Graduate Studies and past associate professor at the UCLA School of Medicine and Science. He obtained his MSc and PhD in the field of microbiology and clinical immunology with postdoctoral studies in tumor immunology at UCLA. His ongoing research, spanning a 45-year career, focuses on the role of environmental factors in complex diseases. Dr. Vojdani’s research on predictive antibodies has resulted in the development of numerous antibody arrays for the detection of autoimmune disorders. He is the CEO and technical director of Immunosciences Lab in Los Angeles and chief scientific advisor for Cyrex Labs in Phoenix. Owner of 15 U.S. patents for laboratory assessments, Dr. Vojdani has published more than 140 scientific articles and sits on the editorial boards of six scientific journals. He is recipient of the Herbert J. Rinkel Award (2006) from the American Academy of Environmental Medicine, the Linus Pauling Award (2009) from the American College for Advancement in Medicine, and the Carrick Research Institute’s Lifetime Achievement Award (2012).

Resources

Cyrex™ Labs is an advanced clinical laboratory focusing on mucosal, cellular, and humoral immunology. Cyrex specializes in antibody arrays for the early detection and monitoring of complex conditions such as thyroid disorders, as well as gluten reactivity and other food-associated autoimmunities, and related neurodysregulation. Cyrex’s scientific advisory board, comprised of experts from a variety of medical and research disciplines, brings together the latest findings from medical research and the assessment needs of today’s healthcare professionals for the development of innovative arrays. For additional information, see www.CyrexLabs.com.

Cyrex Laboratories

5040 N. 15th Avenue, Suite 107

Phoenix, AZ 85015

Tel 602.759.1245

Fax 602.759.8331

NEW JOURNAL: Brain and Gut

Mary Ann Liebert, Inc., publisher, announces the launch of a new peer-reviewed journal,

Brain and Gut,

launching summer 2014. David Perlmutter, MD, a board-certified neurologist, will serve as Editor-in-Chief

Aristo Vojdani, PhD, Director of Immunosciences Lab, Inc., Los Angeles, CA, and Gerard E. Mullin, MD, Associate Professor of Medicine, Johns Hopkins University School of Medicine, will both serve as Editors. The journal will publish leading-edge research with a whole systems approach to digestive disorders, cognitive issues, mood disorders, autoimmune conditions, diabetes, and cancer, encompassing the intersection of neurology, gastroenterology and immunology.

EDITORIAL

This article is drawn from the writing of Aristo Vojdani, PhD, and also includes interview content. The editor, Nancy Faass, MSW, MPH, provides support for authors in the development of articles, books, manuals, white papers, and writing for the Web via her company, HealthWritersGroup.com

Our thanks to Jerry Stine, NC, for technical support on this article; see www.Lifespan-Institute.com.

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