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The Science Behind Treatment Strategies:
Underlying physiological conditions, biomarkers, and the Defeat Autism Now! approach
BY ELIZABETH MUMPER, MD
Dr. Mumper is the Medical Director of the Autism Research Institute and founder of the RIMLAND Center.
n the September issue of The Autism File, I wrote about vicious cycles in autism and how rewarding it can be to restore those cycles to better function. This month, I will share some of the scientific literature behind our concepts of how the gut, brain, and immune system are interconnected and how all are influenced by metabolic factors. Intestinal factors in autism Let’s start with the role of the gut. We coexist with a hundred trillion bacteria that live in our gut. These bacteria outnumber our own body cells ten to one! At birth, our guts are sterile, but quickly are colonized with our mother’s gut, vaginal, respiratory, and skin flora, as well as germs in the environment. During the first two years of life, our colonies of gut bacteria become established and play a major role in modulating our immune systems. At least 70% of our immune defenses are in our gut, so early disruption in the establishment of gut flora or early inflammation can have far-reaching consequences for our ability to fight infections and avoid allergies. Over time, our bodies develop oral tolerance to ingested foodstuffs (so our immune systems don’t attack the food we eat) yet develop appropriate immune responses to pathogenic bacteria. Dysbiosis exists when our good flora are outnumbered by the overgrowth of microorganisms (that may even be of low
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virulence) that induce pathology by altering the immune response or nutritional status of the host. Dendritic cells in the intestine are exposed to gut flora and ingested microorganisms and can be activated to induce T-cell responses and produce chemokines, which act as messengers that inflammation is present and other immune cells need to mobilize to help. Many clinicians utilizing the Defeat Autism Now! approach recommend probiotics early on in the treatment of children with autism: “…Probiotics possess the ability to modulate dendritic cell surface phenotype and cytokine release in granulocyte-macrophage colony-stimulating factor-stimulated bone marrow-derived dendritic cells. Regulation of dendritic cell cytokines by probiotics may contribute to the benefit of these molecules in treatment of intestinal diseases.” (Drakes, 2004) Fedorak enumerated several mechanisms of action for probiotics, including “(1) competitive exclusion, whereby probiotics compete with microbial pathogens for a limited number of receptors present on the surface epithelium; (2) immunomodulation and/or stimulation of an immune response of gut-associated lymphoid and epithelial cells; (3) antimicrobial activity and suppression of pathogen growth; (4) enhancement of barrier function; and (5) induction of T cell apoptosis in the mucosal immune compartment.” (Fedorak, 2004). Let’s examine some evidence that shows
that the gut flora in children with autism is different from that of neurotypical children. Finegold and colleagues have performed elegant studies demonstrating that both clostridial colony counts and the number of clostridia species were higher in the stools of children with autism than in controls. Children with autism had significant numbers of non-spore-forming anaerobes and microaerophilic bacteria in gastric and duodenal specimens, which were strikingly absent in control children. “These studies demonstrate significant alterations in the upper and lower intestinal flora of children with late-onset autism and may provide insights into the nature of this disorder.” (Finegold, 2002). Children with autism are demonstrated to have high rates of gastrointestinal disease. A study at the University of Maryland demonstrated by endoscopy that of 36 children with autism, 69.4% had reflux esophagitis, 41.6% had chronic gastritis, and 66.6% had chronic duodenal inflammation (Horvath, 1999). Further studies showed chemical messenger and white blood cell (in this case, lymphocytes) evidence of inflammation, with significant increases in CD3(+) and CD3(+)CD8(+) intraepithelial lymphocytes and CD3(+) lamina propria lymphocytes in children with autism compared to developmentally normal noninflamed control children (p<0.01). CD3(+) CD4(+) IEL (intraepithelial lymphocytes) and
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LP (lamina propria) CD19(+) B cells were dramatically increased in affected children compared to both non-inflamed and inflamed control groups, including inflammatory bowel disease (p<0.01) (Ashwood, 2003). This study demonstrated that some children with autism have evidence of inflammation and immunopathology anywhere from throat to anus. These children deserve to have their gastrointestinal problems investigated and treated and not written off as “just part of the autism.” Dr. Horvath went on to suggest that “treatment of the digestive problems may have positive effects on their behavior.” (Horvath, 2002). Other researchers demonstrated a lymphocytic colitis (inflammation of the intestine associated with lymphocytes, a type of white blood cell) in autistic children that was not as severe as classical inflammatory bowel disease (such as Crohn’s or ulcerative colitis). Mucosal gamma delta cell density and basement membrane thickness were significantly increased, more than in patients with inflammatory bowel disease. CD8(+) density and intraepithelial lymphocyte numbers were higher than those in the Crohn’s disease, lymphoid nodular hyperplasia (LNH), and normal control groups. Epithelial glycosaminoglycans were disrupted and the authors concluded they had found “increasing evidence for gut epithelial dysfunction in autism.” (Furlano, 2004). Pathologic changes observed in intestines of children with autism include: increased size and number of lymphoid follicles, particularly in the ileum (Wakefield, 2000; Ullman, 2002); crypt cell proliferation (Torrente, 2002); neutrophil and eosinophil infiltration of the intestinal lumen (Krigsman, personal communication); thickening of Gut Disorders in Autism
Endoscopy Submucosal hemorrhages of the colon Lymphoid Nodular Hyperplasia (LNH) Persistent Esophagitis with friability and cobblestoning of the mucosa and blunting of vascular markings
the basement membrane (Furlano, 2004); ulceration of the epithelium (Balzola, 2005); and decreased brush border digestive enzyme function (Kushak, personal communication). These pathology findings are consistent with the establishment of a chronic inflammatory condition. Evidence of autoimmunity includes IgG deposition on the “basolateral epithelial surface in 23/25 autistic children, co-localizing with complement C1q.” (Torrente, 2002). We will return to the issue of autoimmunity in children with autism and their families later. Food intolerance in autism Can gut dysfunction affect brain function? Dr. Hadjivassiliou investigated the frequency of antigliadin antibodies and celiac disease in neurological patients. Using ELISA techniques, he assessed serum IgG and IgA antigliadin antibodies in 53 patients with neurological dysfunction of unknown cause despite full investigation (25 ataxia, 20 peripheral neuropathy, 5 mononeuritis multiplex, 4 myopathy, 3 motor neuropathy, 2 myelopathy) and 94 patients with a specific
neurological diagnosis. They found that gluten sensitivity was common (30 of 53 of their cases) in patients with neurological disease of unknown cause (Hadjivassiliou, 1996). One of our teenage patients has only a few words, and does better neurologically when he stays on a casein-free, gluten-free diet. My staff can tell if he has gone off his diet by his ataxic gait when he comes to our office. Most children with cow’s milk intolerance have chronic diarrhea, but Iacano and colleagues hypothesized that cow’s milk intolerance could also cause painful perianal lesions and, therefore, constipation. A double blind placebo controlled crossover study comparing cow’s milk to soy confirmed their hypothesis and provided the treatment option of discontinuing cow’s milk in children with chronic constipation unresponsive to laxatives (Iacano, 1998). It seems like blasphemy to suggest that certain children should be taken off milk, but many infants with chronic diarrhea, constipation, allergies, recurring otitis media, and children with autism improve off dairy products. Children
Historical clues: Difficulty breastfeeding Food Gut Peristent Colic sensitivities inflammation Malabsorption Gastro-esophageal reflux Infantile eczema Food sensitivities Failure to thrive Abnormal intestinal Frequent antibiotics (abnormal flora) permeability Abnormal posturing Self injurious behavior Poor sleep
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Physical/Lab clues: Abnormal stools Abnormal cytokine profile Lymphonodular hyperplasia of ileum Esophagitis Gastritis
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need to have adequate protein, vitamin D and calcium when on a casein-free diet. Marked improvement in behavioral symptoms of patients with autism was noted after eight weeks on an elimination diet without cow’s milk and other foods to which the children had positive skin tests. High levels of IgA antibodies to casein, lactalbumin, and betalactoglobulin and IgG and IgM antibodies to casein were significantly higher in cases than controls (Lucarelli, 1995). The “hygiene hypothesis” suggests that children who are not exposed to enough germs in infancy are at risk for increased allergy. Infants with a family history of atopic allergy had a 100% higher prevalence of allergies and asthma at two years old than infants who received a Lactobacillus probiotic (Hanaway, personal communication). One way that maternal nutrition can affect offspring is that mothers who have low levels of omega-3 essential fatty acids and high levels of omega-6 essential fatty acids have infants more likely to develop problems with oral tolerance and, therefore, more likely to have food sensitivities. The gut brain connection There are several interesting animal models that demonstrate the intimate connections between the gut and the brain. In one set of experiments, a metabolite of gut flora (clostridia, which Finegold has demonstrated in several studies to be common in children who have autism) induced neuroinflammation and oxidative stress. MacFabe and colleagues infused propionic acid into the ventricles of rats and observed autistic behaviors such as obsessive compulsiveness and social withdrawal. These behaviors appeared and disappeared as the propionic acid infusion was turned on and off. Pathology Immune Dysregulation in Autism Clinical clues to immune dysregulation Allergic shiners Eczema Fungal skin infections Oral thrush Molluscum contagiosum Warts
Chronic Viral and fungal infections TH1 to TH2 shift
analysis of the rat brains showed evidence of neuroinflammation (MacFabe, 2008). Welch and her colleagues showed that experimentally induced inflammatory bowel disease in an animal model was associated with secondary neuropathological changes and that those changes could be treated with neuropeptides, such as secretin and oxytocin (Welch, 2005). Seizures in autism Seizures are a common disorder in children with autism; diagnosis requires a high index of suspicion. Some inattentive or “brain fog” behaviors may be due to absence seizures. Diagnosis of seizure disorders in children with autism may be missed on standard EEGs and may require 24-hour EEGs. Seizures may be a complicating manifestation of neurologic dysfunction in children who are slow to respond to nutriceuticals and standard therapies. Anticonvulsants of particular benefit in children with autism include gabapentin (neurontin) and lamotrigine (lamictal). Neurontin may help with self-stimulatory behaviors as well as seizures, and lamictal has the advantage of blocking glutamate in addition to stabilizing mood. Neuroinflammation in autism A watershed article that my colleagues at the Autism Research Institute have come to rely on heavily for our understanding of brain dysfunction in autism was done by Vargas and colleagues at Johns Hopkins. They examined the brains of 11 autistic patients aged 4–45 years and found degeneration in the cerebral cortex, white matter, and, particularly, the cerebellum. Their elegant neuropathologic studies, using cytokine profiles, immunocytochemistry, and enzyme linked immunosorbent assays,
demonstrated neuroglial activation and activation of the innate immune system (the nonspecific first responders of our immune systems), but not the adaptive immune system (which creates antibodies targeted specifically to the offending antigen). There was marked activation of the microglia and astroglia. Microglia are the immune cells in the central nervous system that exist throughout the brain in a resting state under normal conditions. With an insult to the brain, these resting cells assume an ameboid shape and are able to move and act as scavengers of the injured cells. Human microglia have receptors for many cytokines, including interleukins (IL-6, IL-8, IL-10, IL-12), tumor necrosis factor alpha (TNF alpha), macrophage chemotactic protein-1 (MIP-1), and monocyte chemotactic protein (MCP-1). Activated microglia express anti-inflammatory cytokines (such as IL-10), immunomodulatory cytokines (such as IL-5 and IL-12), and inflammatory cytokines (such as IL-6 and tumor necrosis factor alpha). In the Vargas study, the most prevalent cytokines in brain tissue were macrophage chemoattractant protein (MAP) and tumor growth factor beta 1, both of which are derived from neuroglia. Their study confirmed Purkinje cell loss, as noted on previous studies, and demonstrated a microvasculitis (Vargas, 2005). Autoimmunity in autism Other scientists have demonstrated autoantibodies to the brain in autism, Landau-Kleffner variant, and other neurologic disorders; anti-brain antibodies were present in 27% of the children with autism compared to 2% of control children (Connolly, 1999). Families of children with autism have a higher incidence of autoimmune disease than do families
Increased autoimmunity and allergy
Evidence of Immune Dysregulation Increased IgE Autoimmunity markers Abnormal natural killer cell function IgA deficiency Lymphopenia T cell abnormalities
Treatment of oxidative stress in autism, as in many chronic diseases, is absolutely crucial.
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of neurotypical children (Comi, 1999). Activation of cellular immunity in children with autism is supported by the observed increase in urinary neopterin in autism and suggests “involvement of autoimmunity in the pathogenesis of autism.” (Messahel 1998). The cholinergic nervous system The cholinergic anti-inflammatory pathway plays an important role in neuroimmunomodulation. The efferent vagus nerve plays an important role in controlling inflammation through interaction with nicotinic acetylcholine receptors expressed on macrophages (Pavlov, 2003). Galantamine and nicotine have been shown to have a synergistic effect on microglial activation (Gunta, 2004). Since chronic brain inflammation is a final common pathway in many neurodegenerative diseases, and Vargas and colleagues demonstrated microglial activation (which is central to the process of chronic brain inflammation) in the brains of patients with autism, novel therapeutic strategies may be developed to modulate microglial activation. As these strategies are tested, it will be prudent to respect the delicate balance between enough inflammation to protect the host and overactivity of cytokines in ways that lead to chronic inflammation. Anti-inflammatory strategies Therapeutic strategies to address the ongoing inflammation that, based on the evidence, seems to be occurring in the brains of children with autism include nonsteroidal anti-inflammatories (NSAIDS), which carry side effects such as abdominal pain and diarrhea, and which may worsen existing pathologies like gastritis and esophagitis. Steroids may be effective, but the myriad side effects when used long-term often prohibit their use. Practitioners associated with Defeat Autism Now! have used novel agents such as pioglitazone (Actos), minocycline, and spironolactone (aldactone). Actos (pioglitazone) inhibits NFkappaB, which is involved in immune and inflammatory responses, cellular growth, and apoptosis. NF-kappaB proteins are transcription factors persistently active in chronic inflammation and neurodegenerative diseases. One therapeutic approach to the inflammatory-autoimmune conditions in children with autism would be the use
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Dramatically, symptoms were reversed in each case by various molecular interventions, even in older animals! These experiments must be recognized as changing the paradigm that such conditions are beyond intervention; potentially, the quality of life for the child and family can be significantly improved.
of agents, such as Actos, that inhibit nuclear factor kappa B (NF-KB) or antileukotrienes. Boris and colleagues have studied pioglitazone at doses of 30 mg/day for children 10-20 kg, 45 mg/day for 20-30 kg, and 60 mg/day for children over 30 kg (Boris, 2007). A new black box warning for Actos will warn that this class of drugs increases a person’s risk of congestive heart failure and fluid retention. Minocycline is an antibiotic that also has immunomodulating effects on microglia. Anecdotal experience suggests improvements in a subset of children with autism; this therapy for neuroinflammation is currently being evaluated in children with autism by the NIH (National Institutes of Health). Interferon gamma, testosterone, TNF alpha, MCP 1, in addition to inflammation and oxidative stress, have been elevated in children with autism. Spironolactone has been shown to decrease all of the above, so it has been proposed as a potential hormonal and immunologic intervention for a subset of children with autism (Bradstreet, 2007). Mitochondrial dysfunction in autism Mitochondrial dysfunction is more common than initially recognized in children with autism; in addition to classic congenital mitochondrial disorders associated with low tone, it behooves us to consider acquired mitochondropathies. You may remember mitochondria being described in high school biology as the powerhouses or batteries of the cell. Their primary role is to make ATP (adenosine triphosphate) for energy, which is used to build and maintain our bodies. Mitochondria have their own DNA. Mitochondria are important in regulating apoptosis, which is programmed cell death, a crucial process in living beings where cells are constantly turning over. Mitochondria in the liver help detoxify ammonia, which can accumulate during the metabolism of protein. Mitochondria also help produce heme via the porphyrin pathway. Carnitine levels in 100 children with autism showed significantly low levels of carnitine, slightly elevated lactate, and significantly high levels of ammonia and alanine levels, all of which are consistent with mitochondrial dysfunction (Filipek, 2004). Potential strategies for helping mitochondrial function include: antioxidants, L-carnitine, CoQ10 (UBHQ), and alpha-ketoglutarate. Genetics and autism Autism has classically been considered a genetic disease, with the implication that it is therefore incurable. Typically coded as a “static encephalopathy” by developmental pediatricians, traditional therapies have included speech, physical, and occupational therapies and applied behavior analysis (ABA). Three recent papers described mouse models of developmental disorders, specifically Fragile X (Hayashi, 2007), Rett syndrome (Guy, 2007), and tuberous sclerosis (Ehninger, 2008), all previously considered beyond interventions that could change the trajectory of those conditions. Dramatically, symptoms were reversed in each case by various molecular interventions, even in older animals! These experiments must be recognized as changing the paradigm that such conditions are beyond intervention; potentially, the quality of life for the child and family can be significantly improved. Genetic susceptibilities play a role in determining a child’s vulnerability to environmental stressors. Single nucleotide polymorphisms (SNPs) are variations in an individual’s genetics affecting enzymes and body metabolism that might make one person prone to heart disease, hypertension, and stroke and another person more prone to cancer. SNPs of interest in autism include: PON-1 (paraoxonase 1), which makes children more susceptible to pesticide toxicity; ALAD (aminolevulinate, delta-, dehydratase), which increases risk of lead toxicity; and GST M1-null (Glutathione S-transferases), which increases a child’s
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Vicious Cycles - metabolic The vital role of Methylation and Transulfuration When this cycle does not function, downstream effects are huge: cannot make normal neurotransmitters cannot make creatine cannot control gene expression cannot make cell membranes susceptibility to mercury and xenobiotic toxicities. SNPs that are associated with interference in proper methylation and transulfuration biochemistry include: MTHFR (methylene tetrahydrofolate reductase), MTRR (methionine synthase reductase), and adenosine deaminase weakness. The HLA-DR4 allelle is associated with increased allergies and poor handling of heavy metals. Children with autism are more likely than controls are to have problems with the function of reduced folate carrier (associated with entry of folate into the cell where it can perform its jobs, which include promoting proper neurologic functions) and transcobalamin II (associated with the transport of methylcobalamin into the cell, where it performs a crucial role in the re-methylation of methionine from homocysteine) (James, 2006). Glutathione is the body’s major intracellular anti-oxidant, and it also has important roles in detoxification, T cell regulation, maintenance of the gut epithelium, and mitochondrial function. Total reduced glutathione levels (the reduced form is the active form) were 46% lower in children with autism compared to neurotypical controls, and oxidized glutathione (the bad stuff) was 72% higher in the children with autism. Cysteine levels were 19% lower in autism (James, 2004). Cysteine is the rate limiting amino acid for glutathione synthesis. Furthermore, the parents of children with autism showed abnormal methylation and transulfuration metabolism and DNA hypomethylation, which puts those parents at risk for diseases like heart disease and cancer (James, 2008). This recent research has opened doors of opportunity for treating dysfunctional metabolic pathways in children with autism via nutriceutical means. No longer should we only consider classic inherited inborn errors of metabolism; managing children
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Increased oxidative stress
Abnormal Methylation biochemistry
with autism in the new millennium requires consideration of acquired and potentially treatable metabolic dysfunctions. Oxidative stress in autism Oxidative stress is common in children with autism, as demonstrated by increased lipid peroxidation markers. Transferrin (which binds iron) and ceruloplasmin (which binds copper) are both reduced in children with autism compared to their non-autistic siblings. Low levels of these two antioxidant proteins were strikingly correlated with loss of previously acquired language in the children with autism (Chauhan, 2004). Environmental factors that act as prooxidants include toxins, pollution, heavy metals, viral infections, pathogenic bacteria, thalidomide, and valproic acid. Endogenous (in the body) pro-oxidants include homocysteine, xanthine oxidase, and nitrous oxide. Decreased antioxidant capabilities in children with autism are related to low levels of the major intracellular antioxidant, glutathione, poor function of antioxidant enzymes such as catalase and superoxide dismutase, and low levels of the major antioxidant proteins, transferrin and ceruloplasmin. Treatment of oxidative stress in autism, as in many chronic diseases, is absolutely crucial. Ongoing oxidative stress makes metabolic abnormalities worse. Foods and supplements are rich sources of antioxidants and come in sufficient varieties so that appropriate forms can be found for virtually any child. Biomarkers in autism Clinicians caring for children with autism can use various biomarkers to work up immune function, evaluate oxidative stress, and assess mitochondrial function. Of particular value are neopterin, which signifies immune activation leading to oxidation, and
biopterin, which signifies oxidation due to inflammation. Isoprostane elevations imply oxidized fatty acid membranes, and 8 OHG elevations imply oxidized RNA. Low cysteine and glutathione are nearly ubiquitous in children with autism, and these reflect abnormalities in methylation and transulfuration biochemistry. Elevations in ammonia and lactic acid may be a result of mitochondrial dysfunction, although the differential diagnosis of each includes many other potential causes. Low L-carnitine levels are associated with mitochondrial problems. Quantitative immunoglobulins with IgG sub-classes may reveal immune dysfunction, and assessment of natural killer cell function may reveal impaired ability to fight bacterial and fungal infections. At the Autism Research Institute, we believe that the use of biomarkers in the evaluation of children with autism will help us to categorize our patients into different phenotypes and determine specific therapies influenced by the metabolic and inflammatory profiles of subgroups of patients. A call for urgent action Clinicians, researchers, and parents who are part of the Defeat Autism Now! initiative have spoken of the autism epidemic for over a decade now. Dr. Martha Herbert has articulated the paradigm shift from considering autism to be a brain disorder, to seeing it as a disorder with many potential etiologies that affect the brain and must include environmental factors (Herbert, 2006). The mantra of the Autism Research Institute is that autism is treatable. Our children deserve nothing less than heroic efforts on the part of their governments, medical establishments, and educational institutions. It is crucial that we apply the currently available science in ways that are designed to help the many families affected by this catastrophe. It is critical that we work within a paradigm that acknowledges the reality that many of the changes affecting the brains of children with autism are metabolic or inflammatory and, therefore, subject to intervention. It is imperative that we use what we know now to organize treatment-oriented research initiatives to help as many children as possible. Many children in the autism spectrum have so much more potential than they currently are able to achieve.
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