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According to the Autoimmune Disease Society, 53 million Americans suffer from some form of autoimmune disease or associated disorder. A 2005 National Institutes of Health (NIH) report1 estimates that 23.5 million Americans suffer from autoimmune disease alone; by now this number may have increased to 30 million. An estimated 10% of the world population experiences some form of autoimmune disease, and another 10% is in the process of developing one of these disorders. Over the past four decades, rates of autoimmune diseases such as multiple sclerosis (MS), type 1 diabetes, and lupus have tripled in Western countries. There is a significant need for biomarkers for the early detection of autoimmune disorders, so clinicians will be forewarned and guide their patients in preventive measures.
Predictive Antibodies in Autoimmunity
Researchers have identified more than 80 autoimmune conditions and suspect at least 40 more disorders of having an autoimmune basis. These conditions are chronic and many can be life-threatening.
The predictive function of antibodies. Research was conducted by the author several decades ago under an NIH grant to establish a test for measuring antibodies predictive of type 1 diabetes. We found that children developed antibodies against one or more components of islet cell antigens at least five to ten years before the onset of the disease. From this we learned that detection of tissue-specific antibodies could be used as an early biomarker of autoimmune disease.
Increasing predictive accuracy. A research protocol developed by A. L. Notkins, MD, an NIH immunologist, evaluated patients for diabetes risk by measuring antibodies against insulin, GAD-65 (glutamic acid decarboxylase), and islet cell antigens (protein tyrosine phosphatase).2 He found that measuring antibodies against just one antigen, such as insulin, the probability of predicting the disease was only 12%. When measuring antibodies against two antigens such as GAD-65 and PTP, the predictive accuracy increases to 50%. By measuring antibodies against all three of these antigens, predictive ability approaches 75%. The insight from this study is that the more tissue-specific antibodies we measure and the more antigens we identify, the better we can predict the risk of the disease.
This research provided further evidence of the "early warning" function of antibodies, concluding, "…predictive autoantibodies appear in the blood years before people show symptoms of various disorders. Tests that detected these molecules could warn of the need to take preventive action."2
The potentiating effect of antigens. Several articles published in scientific journals have reported that children with type 1 diabetes develop the disease much faster if put on baby formula containing cow's milk.3,4 While this does not constitute evidence that milk causes type 1 diabetes, the consensus in the literature is that the presence of dairy antigens potentiates the disease. If we remove that antigen from the diet, and immune potentiation does not occur, perhaps the development of autoimmune disease in that child will be delayed or avoided.
Correlation of antibodies and autoimmune reactivity. In patients with celiac disease (CD) or non-celiac gluten sensitivity (NCGS), it has been established that the probability of having one (or several) other autoimmune diseases is 30 times higher than in those who do not have these conditions. The incidence of gluten ataxia, for example, which manifests in disorders such as autism, is increased up to 30-fold in patients with celiac disease.5
In order to implement preventive measures, it is extremely important to identify antibody reactivity before significant tissue damage has occurred. If we detect autoimmunity at an early stage, we can help millions of people reverse the course of this disease.
Cross-Reactivity in Foods
The concept of cross-reactivity enables us to address factors beyond gluten sensitivity with targeted therapies. Cross-reactivity explains how immune sensitization to an antigen such as alpha-gliadin can trigger an immune attack against tissues and organs.
Immune sensitization and cross-reactivity. Gliadin is a prime example of an antigen that cross-reacts with other foods, as well as with human tissue antigens, causing symptoms of celiac disease beyond the gut.6 Food cross-reactivity is a sinister phenomenon that occurs as a result of the similarity in the sequence of amino acids in food proteins and in tissues and organs. Surprisingly, cross-reactivity can occur in a duplicate sequence involving as few as ten or so amino acids out of possibly hundreds.
Limitations of the gluten-free diet. It is widely reported in journal articles that patients with celiac disease frequently have abnormal villous atrophy,7 indicating complete destruction of certain gut tissues. Many clinicians have the impression that removing gluten from the diet will make a significant difference, reversing villous atrophy. However, research has found that when CD patients were put on a gluten-free diet, after six months only 8% of these patients had reversal of villous atrophy. An additional 65% of patients showed 50% improvement. The remaining 27% experienced no improvement in their clinical condition.8
Evaluating food cross-reactivity. We conducted a study to evaluate cross-reactivity between alpha-gliadin and extracts of numerous foods, including grains such as corn, millet, oats, and rice; dairy products; and other foods that are often recommended to patients on a gluten-free diet.9 The author tested this premise using two different sets of highly specific, purified antibodies made against alpha-gliadin 33-mer peptide, the major antigen causing celiac disease. One set of these antibodies consisted of polyclonal antibodies that were affinity purified, indicating that approximately 99% reacted with alpha-gliadin. All other reactive components had been removed to ensure that the antibodies were only reacting to the amino acid sequences of alpha-gliadin. In addition, highly specific monoclonal antibodies prepared against alpha-gliadin were utilized.
We tested the reactions of both the polyclonal and monoclonal antibodies against alpha-gliadin in conjunction with 24 different food antigens and found significant immune reactivity when these antibodies were applied to dairy products and their components such as cow's milk, casein, whey, butyrophilin (a protein in milk found to cause an MS-like syndrome in an animal model), and milk chocolate. When various grains were tested, we found that both polyclonal and monoclonal antibodies prepared against alpha-gliadin reacted significantly with corn, millet, rice, and oats.
We concluded that for patients who do not show improvement in digestion or other symptoms on a gluten-free diet, attention should be given to various cross-reactive foods, such as dairy products, grains, and yeast. This intervention is ideally used in a phased approach. If patients do not initially improve on a gluten-free diet, they should be advised to remove cross-reactive foods. If they still do not improve after the elimination of dairy products, grains, and other reactive foods, it is important to check for other food intolerances. In some cases, symptoms may be associated with factors beyond cross-reactivity. Based on my communications with many clinicians, indications are that the majority of patients who had been diagnosed with non-celiac gluten sensitivity (NCGS) showed significant improvement in their symptomatologies after a gluten-free diet that also eliminated cross-reactive foods.
Testing for food cross-reactivity. Based on our research on cross-reactivity, the following lab panel was developed, which is available through Cyrex Laboratories (see www.CyrexLabs.com).
Array 4. Gluten-Associated Cross-Reactive Foods and Food Sensitivity
Rye, Barley, Spelt, Polish Wheat Sorghum IgG + IgA Combined
IgG + IgA Combined
Cow's Milk IgG + IgA Combined Millet IgG + IgA Combined
Alpha-Casein & Beta-Casein Hemp IgG + IgA Combined
IgG + IgA Combined
Casomorphin IgG + IgA Combined Amaranth IgG + IgA Combined
Milk Butyrophilin IgG + IgA Combined Quinoa IgG + IgA Combined
Whey Protein IgG + IgA Combined Tapioca IgG + IgA Combined
Chocolate (Milk) IgG + IgA Combined Teff IgG + IgA Combined
Oats IgG + IgA Combined Soy IgG + IgA Combined
Yeast IgG + IgA Combined Egg IgG + IgA Combined
Coffee IgG + IgA Combined Corn IgG + IgA Combined
Sesame IgG + IgA Combined Rice IgG + IgA Combined
Buckwheat IgG + IgA Combined Potato IgG + IgA Combined
This testing provides the opportunity to identify not only cross-reactive foods, but also additional reactive foods which are often recommended as substitutes or "safe" foods but which can be problematic to some gluten-sensitive patients (both celiac patients and non-celiac gluten-sensitive patients).
Dairy products. Research indicates that approximately 50% of all patients who have CD or NCGS are also dairy sensitive.10 Dairy sensitivity can generate symptoms similar to those of gluten sensitivity and can be another major factor in autoimmune disorders. Milk, for example, cross-reacts with islet cell antigens, which are involved in type 1 diabetes.
Coffee. Many people find it difficult to give up coffee. In one of our earlier experiments we had found that gliadin antibodies cross-reacted with instant coffee. Therefore, for this study we purchased coffee from Brazil, Colombia, Turkey, Israel, and Hawaii, and espresso from different coffeehouses.9 We were surprised to find that pure coffee did not cross-react with gluten. In repeat experiments we found that these antibodies did not react with pure coffee, but they did react with instant coffee. However, we do not know whether reactivity with instant coffee was a result of the manufacturing process or due to contaminants such as gluten or dairy at the manufacturing site.
Alcoholic beverages. Many patients are interested to know whether they can have whiskey or beer on a gluten-free diet. Our research has shown that distilled alcoholic beverages do not contain gluten or cross-reactive antigens. However, beer contains hundreds of gluten peptides. An Italian research team performed an elegant study evaluating various beers and found more than 150 different gliadin peptides in a single beer.11 Our own research included the evaluation of beers that claimed to be gluten-free, and we found some contamination. In short, distilled alcoholic beverages are gluten-free, but beer is not. This finding is in no way an endorsement of the consumption of distilled alcoholic beverages.
Wheat grass juice. It is important not to confound the issue of wheat germ and agglutinin with wheat grass. Approximately 2% of the dry protein of the wheat kernel consists of wheat germ agglutinin. In contrast, wheat grass juice is made from the green sprouts of the grain. We purchased wheat grass juice from three different sources and found that one of them was cross-reactive and two of them were not. Wheat grass in a pure form is not cross-reactive with alpha-gliadin. However, if one or two kernels of wheat are crushed into the juice during preparation, that can react with the antibodies. Only when the juice is contaminated does it cause immune reactivity with alpha-gliadin antibody.
Oats. The reactivity of oats depends on the variety of the grain. Some have an epitope (amino acid sequence in the DNA) that cross-reacts with alpha-gliadin, and other variants do not. How can we determine whether every food that we purchase contains gluten or not? In the past, the supposition was that reactivity was the result of contamination during manufacturing.
Research by an Italian group has shown that certain varieties of oats contain components of alpha-gliadin and others do not.12 This study indicated that reactivity is not just a matter of contamination, but occurs because of cross-breeding. Some varieties of oats carry a cross-reactive epitope that resembles the epitope of alpha-gliadin. Therefore, antibodies against alpha-gliadin cross-react with certain cultivars of oats, but not with others. Since there is currently no way to screen every variety of oats on the market for alpha-gliadin-type epitopes, the best approach for patients with CD or NCGS would be to consider oats cross-reactive and eliminate them from their diet.
Cross-Reactivity with Tissue
In our research, antibodies found to cross-react against alpha-gliadin also reacted significantly with a number of human tissues, including asialoganglioside, hepatocytes in the liver, glutamic acid decarboxylase (GAD-65) in the pancreas, adrenal 21 hydroxylase in adrenal glands, and various neuronal antigens (such as synapsin, myelin basic protein, and cerebellar).
Autism and cross-reactivity. The author performed a study eight years ago comparing antibody levels in children with autism with those of healthy controls, measuring antibodies against 12 different neural cell antigens including cerebellar antigen.13 Close to 25% of children with autism had antibodies against cerebellar and other neural antigens, while only 2% of children serving as controls had these antibodies. In another study, we demonstrated that alpha-gliadin cross-reacts with cerebellar due to a specific sequence of eight amino acids in cerebellar and in alpha-gliadin that are identical.6 This is the key mechanism of action in gluten ataxia and other forms of cerebellar degeneration. Although we do not know whether all the autistic children had cerebellar ataxia, we do know that if antibodies made against alpha-gliadin manage to cross the blood-brain barrier, the children may be at risk of developing ataxia in the future.
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