Celiac, Gluten and Trypsin Inhibitor

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Wheat

Summary

Forget the gluten.  Celiac is caused by trypsin inhibitors (ATI) that were increased in wheat fifty years ago to combat pests.  Immune response to ATI spreads to include gluten and transglutaminase that perpetuates the disease.  Celiac is an unexpected consequence of traditional plant breeding that could be fixed with GMO approaches.

Plants Protect Themselves with Antibiotics, Pesticides and Trypsin Inhibitors.

Plants respond to pathogens and pests by making themselves toxic.  Thus, plants produce natural antibiotics, phytoalexins, a.k.a. phytochemicals, polyphenolics or antioxidants, to kill bacteria and fungi.  They also produce chemical pesticides and proteins, e.g. trypsin inhibitor, that block the digestion and utilization of plant proteins by insects.  One of these trypsin inhibitors makes ground soybeans inedible until it is removed in water rinses during the production of tofu.  Another of these trypsin inhibitors, in wheat, is the cause of celiac.

Plants Target the Nerves, Immune Cells and Intestines

Plants have evolved chemicals and proteins that attack and punish plant-eating animals.  A single molecule of caster bean toxin protein, for example, can kill a human cell.  Plants produce some of the most toxic molecules on earth.  The nervous system of insects and other herbivores is typically targeted by plants.  Many recreational drugs, e.g. opioids, THC, nicotine, caffeine, etc., for example, are made by plants in self defense.  Human nerves respond to these natural pesticides and the bitter taste and the vomit reflex help us to detect and avoid toxic phytochemicals.  Gluten proteins contain polyglutamine stretches of amino acids that resist digestion and bind to intestinal cells.  Seed lectins bind to the glycoproteins on the surface of the intestines and inhibit digestion.  Wheat seeds also contain an inhibitor of starch and protein digestion, the amylase/trypsin inhibitor, ATI.  ATI binds to the receptors on immune cells that trigger general inflammatory responses to pathogens, e.g. TLR4.  It is the ATI in wheat that starts an immune response to gluten and celiac.

Wheat trypsin inhibitor causes celiac and autoimmunity

ATI Increased to Make Wheat Resistant to Pests

More than fifty years ago, plant breeders began to screen wheat varieties for resistance to pests.  Breeding ultimately resulted in enhanced pest resistance that resulted from increased production of ATI in wheat kernels.  Modern wheat flour contains modest changes in gluten and other components over the last century with the singular exception of ATI, which has increased about 50 fold.  It is also interesting that ATI is a major wheat allergen.  This suggests that celiac starts as an allergy to ATI present in wheat flour.

Celiac Results from Superfine Milling of High-ATI Wheat

Wheat has been milled more and more finely to improve the shelf-life of bread flour.  The inedible bran and the germ are first removed from the wheat kernels and then the endosperm is ground so finely that the starch granules are broken.  Even "whole wheat flour" is ground in the same way and the bran and germ are simply added back to make it “whole.”  The important point here is that superfine milling results in starch that is readily digested by amylase in the small intestines, instead of acting as soluble fiber to feed gut flora.  The result of eating bread from superfine flour is that gut flora are starved for soluble fiber and the immune system is depleted of Tregs that would otherwise suppress allergy and autoimmunity.  Superfine milling of high-ATI wheat presents ATI to an immune system that is primed for allergy.

ATI is a Good Immunogen

Allergy development requires 1) inflammation, 2) an appropriate immunogen and 3) lack of Tregs (immune system cells that develop in the lining of the intestines and block allergies and autoimmunity.)  The modern milling of wheat flour eliminates a major source of soluble fiber, starves gut flora and reduces Tregs, but allergy development still requires inflammation and an appropriate immunogen.  An immunogen is a protein that will interact with cells of the immune system to produce antibodies and activate aggressive attacks.  I have found that all proteins of food or the environment, i.e. allergens, or of the body, i.e. autoantigens, that act as immunogens to initiate allergies or autoimmunity have the same sequence of three amino acids, a "basic triplet."  ATI has a characteristic basic triplet in its protein amino acid sequence and that is why it is a good immunogen to initiate allergies.

Allergy to ATI is Aggrevated by TLR Recognition of ATI

ATI enriched, superfine flour Is a powerful initiator of allergies, because it starves gut flora to block Treg production and is a good immunogen, but the immune system will still ignore ATI in the gut, unless inflammation is also activated.  Unfortunately, ATI actively stimulates inflammation of the intestines by specifically binding to TLR4, which is the receptor that also binds/recognizes the LPS of bacteria.  Thus, ATI is a way for the wheat plant to defend its seeds by triggering excessive Intestinal inflammation.  Inflammation, immunogen and Treg insufficiency is the ATI allergy trifecta.

Wheat ATI Allergy Leads to Celiac

First exposure to ATI and development of an allergy will make subsequent expose to wheat proteins more immunologically intense.  I discussed the response of the intestinal lining to gluten in previous posts.  Wheat gluten proteins are adapted to provide nutrients for growing wheat embryos and to provide defense against pathogens and herbivores.  Gluten proteins contain long stretches of amino acid glutamine, which is poorly digested by gut enzymes.  The glutamine is also converted into glutamate by the gut enzyme, transglutaminase, tTG.  Unfortunately, during the process, the enzyme is covalently connected to the undigested gluten fragments.  The allergic ATI reaction combined with gluten/tTG conjugates, leads to presentation of the gluten/tTG to the immune system and antibody production agains both gluten and tTG.  Subsequent exposure to gluten results in the autoimmune disease of celiac.

Celiac is Self-Perpetuating

The aggressive immune attack on the intestines in response to eating gluten-containing grains, is bad in itself, but it also causes a series of related autoimmune diseases.  Attack on the intestines also disrupts the development of the lining of the intestines, which in turn disrupts the community of bacteria and fungi, gut flora, that are essential for digestion of plant polysaccharides, soluble fiber, and the development of the immune system.  Gut flora dysfunction results in vitamin deficiencies, food intolerances and autoimmunity.  Thus, celiac is self-perpetuating, because it causes inflammation, immunogen presentation and Treg deficiency.

Celiac Causes Numerous Autoimmune Diseases

Celiac is often associated with other autoimmune diseases, because it causes them.  Antibodies to tTG are diagnostic for celiac and the autoimmune attack on the intestines is mediated by anti-tTG antibodies.  But anti-tTG antibodies of celiac don’t just attack the intestines, they attack any other tissues that have tTG, such as the thyroid gland and hair follicles.  Thus, it should not be a surprise that celiacs are at high risk for autoimmune disease, e.g. Hashimoto’s thyroiditis, of the thyroid gland, including both hypothyroid and hyperthyroid diseases, depending on which region of the thyroid is attacked.  Some forms of hair loss, alopecia, are also initiated by autoimmune attack on the tTG in hair follicles.  Persistent exposure of celiacs to gluten will result in a cascade of autoimmune diseases as other body antigens are presented to the immune system and tissues with those antigens are targeted and attacked to produce arthritis, vitiligo, etc.

Pest Resistance, Plant Breeding and GMO Solutions

Genetic modification of plants occurs every time seeds are planted.  Traditional plant breeding by selecting desirable individual plants grown from crosses of selected parents is one form of genetic modification.  Specifically introducing desired genes using recombinant DNA techniques is another, more controlled method.  Traditional plant breeding has systematically destroyed the diversity of crop plants by loss of genes that are not selected, but even the traits, such as pest resistance, that provide benefit, have also brought unintended consequences.  We now have grains with many desirable features of high yield and disease resistance, but they also provide increased risk of celiac, gluten intolerance and associated autoimmune diseases.  Maybe it is time to consider GM techniques as a safer alternative to fix modern wheat and to examine milling approaches to save our gut flora.

Health Diagrams II — Curing Autoimmunity and Allergies

Cure for Celiac and Autoimmunity

Celiac and other autoimmune diseases are perpetuated by the presence of the corresponding autoantigen/allergen, in this case tTG and gluten proteins, and a deficiency of Tregs.  Oddly enough, some pathogens (Helicobacter pylori) and parasites (Helminth worms) stimulate Treg development in the lining of the intestines, in addition to normal gut flora, Clostridium spp.  It may be the relative absence of pathogens and parasites in affluent societies that reduces Tregs and enhances the incidence of allergies and autoimmunity.  Antibiotics and the antibiotic activity of pharmaceuticals in general may also contribute to Treg deficiencies by damage to gut flora.  Clearly, the repair of gut flora and reestablishment of the associated immune system will go a long way toward curing autoimmune diseases such as celiac.  Celiac, however, provides the added complexity that it damages the ability of the intestines to maintain a functional gut flora.  Thus, the cure for celiac would require simultaneous repair of both the gut and its flora, e.g. by a  fecal transplant and supportive diet containing numerous soluble fibers to which the donor flora have been previously adapted, i.e. lacking antigenic triggers.

Peanut Allergy Cause and Cure

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Summary:  The cure for peanut allergy should follow naturally from knowledge of the cause.  Since most allergies and autoimmune diseases result from the combination of 1) inflammation, 2) breakdown of immunological tolerance and 3) presentation of a primary immunogen, it follows that some types of peanut allergy are based on a continued problem with immune tolerance and fixing that defect should eliminate an allergic response to peanuts.  The current cure to resurrect immune tolerance is by enhancing regulatory T cells (Tregs) in the gut using resistant starch to improve the growth of Clostridia in the gut.

Peanut allergies are dangerous and this post does not advocate any medical treatments, but rather attempts to explain the cause and cures of allergies.

Just Treat the Immunological Tolerance Problem Instead of Mast Cells

Most people in fear of anaphylaxis from peanut dust, just try desperately to avoid peanuts in any guise.  That avoids the problem, but why not cure the allergy?  Recent research shows that peanut allergens can be prevented from establishing an allergic response in mice by addition of Clostridium species of bacteria in the gut flora.  It was shown that the Clostridia increased Tregs (regulatory T cells responsible for immune tolerance) in the lining of the intestines via interleukin 22 production.  So the cure to some peanut allergies may be increasing Tregs and fixing tolerance.

I Said It All Before

It is not a large step to combine my previous posts covering potato resistant starch for treatment of deficiencies of immunological tolerance with my explanation of the cause of allergies and autoimmunity to provide a simple explanation of the cause and cure for some peanut allergies.

Peanut Allergen is a Typical Bean Storage Protein Except for the Basic Triplet

It is not difficult to find out why peanuts are allergenic.  I just went to the National Center for Biotechnology Information (NCBI) web site and queried the protein sequence databases for “peanut allergen.”  Here is the complete amino acid sequence (each of the 20 amino acids of the protein is assigned a letter) of the major peanut [Arachis hypogaea] allergen:

MMVKLSILVALLGALLVVASATRWDPDRGSRGSRWDAPSRGDDQCQRQLQRANLRPCEEHMRRRVEQEQEQEQDEYPYSRRGSRGRQPGESDENQEQRCCNELNRFQNNQRCMCQALQQILQNQSFWVPAGQEPVASDGEGAQELAPELRVQVTKPLRPL

The triplet of basic amino acids (R=arginine, K=lysine), RRR in this case, which is found in all allergens and autoantigens, is highlighted in red.  If you eat peanuts with an inflamed gut and you have wiped out your Clostridia and associated Tegs with antibiotics, you have a good chance of developing autoimmunity, as well as a peanut allergy.  The cause of allergies is that simple and the cure is equally simple.

Shellfish Allergy Shows the Relationship between Allergy and Autoimmunity

I ran across a list of other food allergens when I was checking up on peanuts.  Shellfish was listed as another of the big allergies.  I looked up “shellfish allergen” and ran into thousands of entries.  The first couple of dozen proteins lacked the characteristic basic triplet, so I had to step back and try to guess the most typical shellfish for first exposure, i.e. the primary immunogen.  All of the other shellfish allergens were various versions of the muscle protein, tropomyosin, so I looked up “shrimp allergen.”

MDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKSEEEVHNLQKRMQQLENDLDQVQESLLKANIQLVEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY

Note the predicted basic triplet in red.  Since I was on a roll, I also checked out related tropomyosin sequences in humans:

MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEDELVSLQKKLKGTEDELDKYSEALKDAQEKLELAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTNNLKSLEAQAEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLEKSIDDLEDELYAQKLKYKAISEELDHALNDMTSM

Once again the basic triplet indicated that there was a related human tropomyosin that could interact with antibodies to the shellfish allergen or could be an autoantigen participating in autoimmune diseases.  So I checked PubMed for “tropomyosin autoantigen” and quickly found that antibodies to tropomyosin are important in ulcerative colitis (UC).  Thus, shellfish allergy may be an indication of an underlying predisposition to UC.  And, the traditional cure for allergy by injection with small amounts of the allergen to convert from IgE to IgG, would convert a shellfish allergy into UC.

Avoiding Allergens Makes No More Sense Than Trying to Avoid Autoantigens

To fix allergies, it is necessary to eliminate the cause and block perpetuation of the condition.  The cause is based on 1)inflammation, 2) broken immune tolerance and 3) primary immunogen.  Peanuts are the primary immunogen, but that is unimportant if the causing conditions are eliminated and tolerance is reestablished.  Clearly, if immunological tolerance is reestablished, then it's just a matter of time before peanuts are no longer a problem, because increasing Tregs will silence the dramatic immunological response to peanuts.  Tolerance is based on Tregs and Tregs develop in the intestines in response to Clostridia feeding on soluble fiber/resistant starch.

Curing Peanut Allergies is Based on Repairing Gut Flora

There are a couple of hundred different species in the pounds of bacteria in the healthy human gut.  Most of those bacteria require soluble fiber that is systematically removed during food processing.  For most people, the cure for peanut allergies will be resistant starch/Clostridium therapy, followed by further repair with fermented foods that provide the typical lactic acid bacteria and soluble fiber along with companion bacteria that can recolonize the gut.  The cure for many allergies and autoimmune diseases is just to eat a couple of tablespoons of resistant starch each day and if needed, supplement with probiotics containing Clostridium butyricum.  If there is severe dysbiosis, as indicated by constipation, then fixing the gut flora is a little more difficult, but for most people cures are much cheaper and effective than just treating symptoms.

A guide for the use of resistant starch is provided by Richard Nikoley, et al. at Free the Animal.

Health Diagrams II — Curing Autoimmunity and Allergies

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In this second in a series of posts explaining the concepts that I think are central, but misunderstood, about health, I am focusing on how diet and gut flora impact the immune system and cause autoimmunity and allergies.  This cause also suggests a simple cure.

Health in Diagrams I -- Gut Flora and Diet
Health in Diagrams III -- Inflammation from Cell to Tissue

Gut Flora to Tregs to Suppression of Autoimmunity

It is important to understand at the outset that autoimmunity and allergies are caused by a damaged immune system, and repairing the damage cures the diseases.  Damage to the immune system typically represents a break in the continual development of immune cells in the lining of the intestines.  Immune cell development in the gut is dependent on bacteria, the gut flora.  Damage to the gut flora, e.g. by antibiotics, processed foods that lack flora feeding fiber or extreme diets, disrupts development of immune cells.  Typically, loss of the immune cells that keep the aggressiveness of the immune system in check, regulatory T cells or Tregs, results in autoimmunity.  Fix the gut flora and autoimmunity recedes.  

Health Requires Suppression of the Aggressive Immune System

For simplicity, I am focusing on the T cells of the immune system that develop in the intestines and either kill other human cells that are dangerous, e.g. virus-infected or cancer cells, or provide protection by regulating the aggression, Tregs.  Normal functioning of the immune cells permits elimination of damaged or dangerous human cells, while at the same time avoiding rampages of lethally armed T killers.  Examples of untamed T killers in action are degenerative autoimmune diseases, such as arthritis, asthma, prostatitis, celiac, Hashimoto’s thyroiditis, type I diabetes, inflammatory bowel diseases and atherosclerosis. 

Milk Births Baby Immune System

It should not be surprising that the focus of immune system development is the gut.  We start as babies with explicit links between nourishment and immunological protection.  Milk connects the immune systems of mother to baby.  Immune cells from the mother are transferred in milk and colonize the respiratory and digestive system of the baby — the mother’s immune system coats and buffers the baby’s exposure to the world.  Milk hormones close the baby’s gut and milk bacteria are the first probiotics that exploit the milk prebiotics (bifidus factor, human milk oligosaccharides) to produce a gut flora.  [Also note that most commercial probiotics are adapted to grow on cow’s milk and hence these dairy probiotics do not survive in adults.]  The lymphatic system of the breast terminates at the nipple and samples antigens/pathogens from the baby’s mouth, resulting in baby-specific secretory antibodies that return in the milk.  Milk supports a starter set of gut flora, essentially dairy probiotics, that stimulates development of the baby immune system, but inhibits adult gut flora that would digest the protective components of milk.  Formula, on the other hand, is inflammatory to the baby gut, because it supports adult gut flora before the immune system is ready.  Inflammation and stimulation of innate immunity is sufficient, if supported with high levels of sanitation, to permit survival of babies fed formula.  Milk of any type is incompatible with adult gut flora, so breast milk will attack adult gut flora and adult gut flora will digest and inactivate the otherwise beneficial components of the milk.

Milk, Kefir and Gut Flora

Aggressive and Suppressive Cells of Immune System Develop in Intestines

Gut bacteria are required for the development of immune T cells in the lining of the intestines.  Mice grown without gut flora do not have functional immune systems.  In humans, extensive antibiotic treatment produces defective immune systems that are either overly aggressive, i.e. autoimmune, or susceptible to infection and cancer.  They can’t be both.  Aggressive T killers are stimulated to develop by filamentous bacteria and Tregs develop in response to members of the Clostridium family.  In a healthy body, there is a balance between aggression and suppression; there are functional defenses against infection and cancer, while also avoiding autoimmune disease and allergies.

Suppressive Tregs are Deficient in Autoimmunity

Immune cells result from replicative divisions of stem cells.  Antibody producing B cells are produced through a million random rearrangements of antibody genes and those B cells producing antibodies against common self proteins are killed (clonal deletion).  Similarly, T cells are produced by rearrangements of receptors and those that would recognize self are eliminated.  The T cells then migrate to the intestines where they can develop into killer T cells or Tregs, in response to gut flora.  The Tregs act to suppress killer T cells that mistakenly recognize healthy self cells.  Thus, the initial elimination of self-attacking T cells or for B cells that produce antibodies that bind to normal cells, is not perfect and the Tregs are needed to avoid the mistakes.  Tregs are necessary to avoid the immune attack on healthy cells that is the basis of autoimmunity.

Autoimmunity Starts with Inflammation, but Requires Deficient Tregs

Bacterial or viral infections, or physical damage causing inflammation is the first step in autoimmunity.  It is the inflammation that initiates the interactions between proteins, autoantigens, of normal cells and cells of the immune system that bind, internalize, fragment and present the antigen fragments/peptides to activate B or T cells with corresponding receptors.  The activated B cells make antibodies specific for the antigen and the T cells will kill cells displaying the antigen.  It is interesting that most proteins are not autoantigens and are never involved immune reactions.  Only proteins with an unusual triplet of basic amino acids, similar to the quartet of basic amino acids used to transport proteins into the cell nucleus, are candidates to be autoantigens or allergens.  In fact, since nuclear proteins already have a quartet, i.e. the nuclear localization signal, they are common autoantigens.  The last requirement for autoimmunity is a deficiency in Tregs, because if the Tregs are functioning, they will block attack on healthy cells.  Treg deficiency usually results from loss of the type of gut bacteria that stimulate Treg production in the lining of the intestines, i.e. species of Clostridium.

Hospitals are Notorious for Clostridium difficile Infections

Fecal transplants are now recommended as a safe and efficacious treatment for C. diff hospital infections.  That makes sense, because hospitals are where antibiotics are routinely used and C. diff can only infect people missing their healthy species of Clostridium.  Thus, the hospitals wipe out the gut flora with antibiotics and then recolonize them with their own antibiotic resistant C. diff.  More antibiotics can’t fix it, but providing healthy gut flora (transplant) can.

Autoimmune Diseases are Treated/Exacerbated with Antibiotics

Both the aggressive and the suppressive immune cells require gut flora, so after initial antibiotic treatment wipes out bacteria required for suppression and results in autoimmunity, the remaining aggressive half of the immune system can be eliminated by blasting the remaining gut flora with more antibiotics.  Of course this will leave a highly compromised, incompetent immune system that will ultimately yield more extreme symptoms.  This is the typical medical progression for Crohn’s disease, for example.  The alternative is just fixing the gut flora to begin with and curing autoimmunity.

Cure Autoimmunity by Feeding Clostridium Resistant Starch

Autoimmune diseases, by their symptoms, show that sufficient gut flora to stimulate the aggressive half of the immune system is still present.  What is missing are the Clostridium species that convert soluble fiber, such as resistant starch, into short chain fatty acids, e.g. butyrate.  Patients treated with antibiotics usually walk away from the hospital with a suggestion to eat some yogurt to repopulate their missing gut flora.  Unfortunately, dairy probiotics don’t survive in the gut and cannot repair the gut flora and immune system.  The result, after the gut fails to repair and the immune system crashes, is autoimmunity.  There is a more appropriate possibility to avoid or fix autoimmunity.  Some people suffering from autoimmunity (and with remnants of their gut flora intact) have simply fed their gut flora on resistant starch and achieved complete recoveries.  Others fail to respond, because their gut flora is too severely damaged and necessary bacterial species are gone.  Those individuals need to eat the missing species of bacteria and some probiotics (more common in Asia) contain Clostridium species.  Consistent with this use of soluble fiber to feed gut bacteria that produce butyrate and stimulate the suppressive immune system are reports of healing by combining potato starch (RS) and probiotics with Clostridium butyricum (Probiotic-3).  Repair of the suppressive immune system by repair of gut flora (including fecal transplants) and feeding gut flora with appropriate soluble fiber, may be a general approach to the cure of most autoimmune diseases and allergies.

Paleo Gut Flora Repair

Antibiotics, Gluten, Hashimoto's Thyroiditis and Baldness

My impression is that Hashimoto's is caused by a combination of an initial immune attack on the thyroid and incompetent regulatory T cells.  In most cases the immune attack on the thyroid is a secondary consequence of celiac/gluten intolerance, in which anti-transglutaminase antibodies attack transglutaminase bound to gluten in the intestines.  Transglutaminase  is an enzyme that is also produced by the thyroid (and hair follicles) and attack by celiac antibodies can enhance or inhibit thyroid hormone production (or baldness.)  Both Hashimoto's and celiac do not occur if the suppressive part of the immune system, i.e. regulatory T cells, is functioning.  

Antibiotics Compromise the Immune System

The major point here is that antibiotics disrupt normal bacterial biofilms that line the intestines and these healthy gut bacteria are required for development of regulatory T cells.  Compromise of Tregs leads to autoimmune diseases, e.g. celiac, Hashimoto’s and baldness, and also allergies.

Antigens/Allergens Have Basic Amino Acid Triplets

The antigens targeted in autoimmune diseases, e.g. tTG, anti-nuclear, TPO, and allergies form an obvious pattern.  All of these antigens and allergens have simple amino acid sequences (rare patches of three basic/positively charged amino acids) that enhance their presentation to the immune system to produce antibodies.  Nuclear proteins, for example, are frequent autoantigens and most of these proteins interact with nucleic acids (negatively charged) and have predictable patches of positively charged amino acids (arginine and lysine).  Other common autoantigens have basic amino acid (arg/lys) patches, because they interact with phospholipids (also negatively charged.)  Proteins with basic patches, e.g. HIV-TAT or heparanase, are also readily transported into cells and nuclei.  Peptides with these sequences are produced by action of stomach enzymes on proteins, e.g. milk lactoferrin, and are antimicrobial.

Allergies / Autoimmune Diseases Are a Predictable Consequence of Antibiotics

Doctors treat with antibiotics, but they fail to repair damage that they cause to gut flora.  The gut flora of most patients treated with antibiotics, especially those who are most fastidiously hygienic, never fully recover.  Constipation is a common symptom of severe dysbiosis and related immunoincompetence.  Probiotics are gut flora bandaids and do not survive as components of gut flora.

Gut bacteria are also needed for development of the aggressive part of the immune system.  Thus, autoimmune diseases can be treated with even more intense use of antibiotics, that will eliminate the rest of the immune system.  Since all vitamins are produced by gut flora as quorum sensing signals, antibiotics can also produce the exotic symptoms of vitamin deficiencies.

Antibiotics are essential to many therapeutic approaches, e.g. surgical procedures or therapy for chronic Lyme disease, but they must be used responsibly and treated patients must be subsequently tested to ensure a repaired gut flora and a functional immune system have been reestablished after antibiotics.  Long term antibiotic use needs special attention, e.g. deliberate Repair of Gut Flora or a fecal transplant.

Thus, I think that it is most likely that ever increasing antibiotic exposure and processed foods, coupled with obsessive hygiene have led to crippled gut flora (as observed in the simplified gut microbiomes of Americans), a net decline in suppressive Tregs and the observed increase of autoimmunity and allergies.  The competence of the immune system may be a major determinant in the course of infection with a pathogen that can produce chronic infections.

Autoimmune Diseases, Bacteria and GALT (Gut Associated Immune System)

Celiac, Oxidative Stress, Peroxiredoxin, Alopecia

Grain/gluten intolerance, celiac is an immunological attack on the small intestines with increased risk for numerous autoimmune diseases.  Hashimoto’s thyroiditis is a common sequela of celiac and the two diseases share the same autoantigen, tissue transglutaminase (tTG).  Thus, the development of celiac and the production of antibodies to the tTG produced in the intestines, results in a subsequent immunological attack on other tissues that produce lots of tTG, e.g. the thyroid.  Gluten intolerance, because of its attack on the intestines and the proximity of a major part of the immune system (GALT), may play a major role as the foundation for autoimmune diseases.

Gluten Intolerance First Step in Autoimmune Diseases

Celiac may also lead to herpatic lesions of the skin, dermatitis herpetiformis and loss of hair, alopecia.  In these cases, the autoantigen is peroxiredoxin, an enzyme that eliminates hydrogen peroxide produced as a result of accumulation of reactive oxygen species, e.g. superoxide, associated with inflammation.  Peroxiredoxin is also implicated as an autoantigen in periodontal disease, suggesting that celiac may also contribute to dental gum inflammation.

Peroxiredoxin 5 Gene Associated with Alopecia Risk

A recent study (see ref. below) of genes associated with alopecia identified genes involved in Treg and Th-17 development, as well as peroxiredoxin 5 as contributors.  As expected, several genes involved in antigen presentation (HLA-DRA, HLA-DQA)  were also identified.  Th-17 lymphocytes are involved in immune attacks on self tissue, i.e. autoimmune diseases, such as alopecia, in which hair follicles are attacked by the immune system.  Tregs control immune attacks on self tissues.  Peroxiredoxin is an autoantigen and is produced in elevated amounts around hair follicles attacked in alopecia.

Basic Amino Acids of Peroxiredoxin as Expected for Autoantigen

I checked the amino acid sequence of human peroxiredoxin 5 and found an alternative (-nrrlkrfsmv-) to the triplet of basic amino acids that I expect for an autoantigen.  In this case there are two adjacent pairs of basic amino acids (blue rr and kr) that I think precipitate immunological presentation of peroxiredoxin.  Peroxiredoxins are produced in response to oxidative stress at sites of  inflammation and the presence of celiac compromises the gut associated immune system (GALT) that provides Tregs to restrict autoimmunity, so celiac sets the stage for peroxiredoxin presentation to the immune system and for subsequent production of anti-peroxiredoxin antibodies, autoimmunity and destruction of hair follicles, alopecia.

Anti-Inflammatory Diet Should Avoid and Treat Autoimmunity

Control of autoimmune diseases mediated by peroxiredoxin should benefit from a reduction in the conditions that spawned the diseases:

• Th-17 elevation -- celiac inflammation stimulated by grain/gluten
• Treg loss -- GALT inactivation due to inflammatory diet and inappropriate gut flora
• Autoantigen (basic amino acid concentration) presentation -- oxidative stress stimulation of peroxiredoxin

Treatment would be supported by dietary changes:

• anti-inflammatory diet to control gut inflammation and minimize celiac symptoms (vitamin D, low carb/high saturated fat, high omega-3 to -6 fatty acid ratio, no grains)
• probiotics and soluble fiber (e.g. pectin, inulin) to re-establish gut flora (cure dysbiotic constipation) and GALT function, and development of Tregs
• supplements to compensate for depletion of vitamin C and glutathione by oxidative stress, e.g. vitamin C and acetylcysteine (NAC)

Th-17 and Tregs in HIV Infections

Th-17 cells are also reduced by HIV infection, producing susceptibility to infection, but this infection should also reduce autoimmune disease.  The reduction in Th-17 also may be a consequence of problems in the GALT.  Therapy for HIV infection should also include diet considerations to increase Th-17 and also Tregs to reduce autoimmune diseases due to unbalanced Th-17.

ref.

Petukhova L, Duvic M, Hordinsky M, Norris D, Price V, Shimomura Y, Kim H, Singh P, Lee A, Chen WV, Meyer KC, Paus R, Jahoda CA, Amos CI, Gregersen PK, Christiano AM.  2010. Genome-wide association study in alopecia areata implicates both innate and adaptive immunity.  Nature. 466(7302):113-7.

Helminths, Oligosaccharides and Immunotolerance

Parasitic worms reverse allergies and autoimmune diseases using oligosaccharides to mimic self and silence immune over-responsiveness.

Helminth therapy, i.e. infection with parasitic intestinal worms to provide remission from allergies, inflammatory bowel and other autoimmune diseases, has been examined as a potential therapeutic model to rehabilitate immunological dysfunction.  The surface oligosaccharides of these worms have been found to mimic human oligosaccharides and alter immune responses by binding to carbohydrate-binding, i.e. lectin, receptors.

Immune Tolerance
The essence of allergic and autoimmune diseases is a defect in distinguishing between pathogen, innocuous and self molecules.  Heightened immune reactions as a result of inflammation move the immune system toward production of antibody and T cell receptors specific for antigens.  Those antigens respond to unique receptors on the surface of each B and T lymphocyte.  The lymphocyte population has been previously depleted of cells that can produce receptors that will bind to most self antigens.  This depletion makes the lymphocyte population generally non-responsive, or tolerant to self antigens.  Thus, the immune system is blind to the body.

Regulatory T Cells and Tolerance
Most of the immune cells of the body are present in the lining of the gut.  It is in the gut that various immune cells continue to develop for their various roles, including controlling immune reactions to self antigens and to common food molecules.  Immune cells in the gut are exposed to some food molecules and bacteria that leak through the cells of the intestinal villi.  Responding to these common antigens by inflammation can lead to inflammatory bowel disease.  This pathological over-responsiveness is normally avoided by development of regulatory T cells, Tregs, that suppress immune responses to common food molecules and to surface antigens of common bacteria.

Treg Development Depends on Gut Flora
Gut bacteria are needed for the normal function of the immune system.  Oddly, Helicobacter pylori, Hp, the cause of stomach ulcers and cancer, also stimulates the development of Tregs.  Thus, the pathology of Hp may result not from its presence, but rather from how it is growing.  Since Hp uses hydrogen gas produced by Klebsiella in the lower bowel and hydrogen production is dependent on dietary starch, then it follows that the pathological behavior of Hp may be dependent on dietary starch.  A low starch diet may actually result in Treg stimulation from Hp and a reduction in allergies and autoimmune diseases.

Tregs Enhanced by Heliminths
Immunological tolerance is also stimulated by parasitic worms, Helminths.  Helminth infestations, therefore, reduce allergies and autoimmune diseases and may contribute to the hygiene hypothesis to explain the prevalence of allergies, autoimmune and other inflammation-based degenerative diseases in modern societies.  Examination of worms to find the molecules responsible for inducing immunological tolerance has identified complex surface and secreted oligosaccharides (small sugar chains) as the active molecules.  Helminth oligosaccharides mimic human cell surface oligosaccharides and bind to carbohydrate-binding, lectin, receptors on immune cells to stimulate Treg development.

Lectin Receptors Control Tolerance
There are many implications of the modulation of the immune system via oligosaccharides.  Note that related oligosaccharides are components of human milk and prepare the gut and develop the immune system.  This explains why formula, which lacks these unique oligosaccharides, results in aberrant gut flora, contributes to neonatal necrotizing colitis and supports the development of allergies and autoimmune diseases.  In contrast, judicious use of self or Helminth oligosaccharides may provide a means of restoring the function of damaged immune systems and therapy for allergies and autoimmune diseases.  Also note that the critical use of lectins, which have oligosaccharide-binding sites rich in aromatic amino acids to bind the hydrophobic faces of the sugars, will also bind and provide entry into immune cells for allergens and autoantigens that have triplets of basic amino acids.  The binding sites of lectins should also bind many aromatic phytochemicals.  Immunomodulation by phytochemicals may result from interference with or mimicking the binding of oligosaccharides to lectin receptors.

reference:
van Die I, Cummings RD.  Glycans modulate immune responses in helminth infections and allergy.  Chem Immunol Allergy. 2006;90:91-112.

Arthritis, Autoimmunity and Arginine Deimidation

Celiac and Antibody Production Against Tissue Transglutaminase as a Model

Arthritis is an autoimmune disease in which the immune system attacks and degrades the connective tissue of joints.  Antibodies against modified amino acids, arginine converted to citrulline, and proteins commonly found in joints, mediate the arthritis disease process.  The development of arthritis mimics the development of gluten intolerance, celiac, in which another enzyme, transglutaminase ( tissue transglutaminase, tTG or TG2) modifies the major gluten protein, gliadin, and antibodies are produced against both modified gliadin and TG2 autoantigen.

Arthritis of Joints Is Like Coeliac of Intestines;  Autoantibodies to Protein Modifying Enzymes

In other articles, I outlined the pathology of gluten intolerance:

• The major protein of wheat gluten, gliadin, contains long stretches of glutamines.
• An intestinal enzyme, TG2, converts the glutamines to glutamates by deamination.
• As TG2 works it binds to gliadin.
• In celiac, the TG2-gliadin complexes are internalized and fragmented to stimulate antibody production against both TG2 and gliadin.
• I think that the internalization and processing for antibody stimulation is dependent on the basic triplet found in TG2.

Arthritis Is Mediated by Autoantibodies to Peptidylarginine Deiminase and Citrullinated Proteins

Parallel to the celiac example, in some forms of arthritis, antibodies are produced against an enzyme that modifies proteins.  In arthritis, the enzyme involved, peptidylarginine deiminase (PAD) removes the terminal nitrogen from arginine (deimination) to produce citrullinated proteins.  Antibodies are produced to both PAD and citrullinated proteins.

PAD Also Has a Triplet of Basic Amino Acids for Internalization

I of course wondered if PAD had the same triplet of basic amino acids, e.g. RRK, that I had found on all other autoantigens and allergens.  Examining the sequence of human PAD in the NCBI sequence databases and comparing to other sequences, I found the basic triplet near the carboxy terminus.  The same or an alternative basic triplet was found in PADs from other mammals.

Autoantigens and Predicted Basic Triplets of Amino Acids Reveal the Cause of Arthritis

Arthritis is an inflammatory disease.  That means that without inflammation, arthritis cannot start and if inflammation is inhibited, arthritis cannot progress.  It is likely that arthritis is the result of chronic inflammation plus a precipitating event, such as joint injury or joint infection.  Alternatively, in a manner similar to Hashimoto’s thyroiditis, in which celiac produces anti-TG2 antibodies that attack the TG2 also produced in the thyroid gland, arthritis may be produced by autoantibodies stimulated in the inflammation of other tissues and spreading to the joints.  Celiac is also a risk factor for arthritis.  Trauma-based inflammation of a joint can also result in migration of Clamydia pneumonia (Cpn)-infected macrophages to the site of inflammation.  Cpn could contribute to joint inflammation and promote immunological presentation of autoantigens and autoantibody production.

reference:
Stenberg P, Roth B, Wollheim FA.  Peptidylarginine deiminases and the pathogenesis of rheumatoid arthritis: a reflection of the involvement of transglutaminase in coeliac disease.  Eur J Intern Med. 2009 Dec;20(8):749-55. Epub 2009 Sep 19.

Biofilm Transformation, Helicobacter, Klebsiella

Helicobacter pylori causes stomach cancer, but it feeds on hydrogen gas produced by Klebsiella pneumoniae in gut biofilms. DNA released by biofilm bacteria not only transfers antibiotic resistance, but it also provides protection against host antibacterial peptides, such a cathelicidins and defensins.

Exploding Labs

When I was working on host/pathogen interactions and plant disease resistance, I also became familiar with research on the formation of the plant equivalent of cancer, crown galls, and symbiotic bacterial nitrogen fixation. I mention this, because this also exposed me to the free-living bacterial nitrogen fixing system in Klebsiella and to the memorable urban legion of exploding labs. As the story goes, as bacteria convert atmospheric nitrogen gas into ammonia, nitrogen fixation, they use high energy electrons, e.g. from ferrodoxin, and lots of ATP, but they also produce hydrogen gas. In labs where they are researching nitrogen fixation, the excess hydrogen gas would accumulate on the ceiling until... boom! Now those labs are properly vented.

Helicobacter Uses Hydrogen as an Energy Source

Helicobacter pylori is considered the most common bacterial pathogen of humans and is the primary cause of ulcers and stomach cancer. H. pylori lives in the stomach by neutralizing stomach acid with ammonia. Another interesting ability of this bacterium is its ability to use hydrogen dissolved in circulating blood as an energy source. The high energy electrons from molecular hydrogen are transported to its electron transport chain, and the energy is used in membrane transport and ATP production. The circulating hydrogen is produced by gut bacteria.

Klebsiella Is not just a Soil Bacterium, Gut Gases

Klebsiella pneumonia is a lung pathogen and it also forms gut biofilms. Presence in the gut and the ability to produce hydrogen gas has some implications for hydrogen utilizing bacteria like H. pylori. Clearly, the stomach of someone with an abundant source of hydrogen fuel in their blood is a better target for H. pylori colonization. This explains why even at age 50, individuals who were exclusively breastfed have a lower incidence of H. pylori and stomach cancer, since even a single bottle of formula can shift an infant to adult, i.e. Klebsiella gut flora.

Klebsiella Needs Carbs to Produce Hydrogen

K. pneumoniae has been associated with Crohn’s Disease and Ankylosing Spondylitis. It grows in gut biofilms and produces pullulanase, an enzyme that can utilize the branched glucosides left over from the action of amylase on plant starch. So K.p. has an untapped food source and it needs lots of ATP to produce hydrogen gas. The nitrogenase needed for nitrogen fixation and hydrogen production is very sensitive to oxygen, so this means that K.p. needs a partially anaerobic environment and must get its energy from fermentation. Fermentation yields much less ATP than respiration using oxygen, which means that K.p. can only produce hydrogen with lots of glucose from starch.

Low Carb Diet Cures Crohn’s Disease

It turns out that the antigen causing Crohn’s disease is the pullulanse (with collagen mimetics.) As you should expect, it has a basic triplet. Eating a low carb diet reduces the flareups of Crohn’s disease, presumably by starving out the K.p.. It is interesting that nitrogenase is the antigen involved in Ankylosing Spondylitis.

Biofilms Promote Transformation and Antibiotic Resistance

Just as a footnote to the benefit of K.p. as a citizen of a biofilm community, H.p. should also live in those biofilms, since that is the source of the hydrogen it uses. Biofilms also stimulate the exchange of DNA, because the quorum sensing chemical signals trigger the release of DNA. The DNA is a component in the matrix that binds bacteria in the biofilm and can work in conjunction with bacterial acidic polysaccharides and host heparan sulfate. These acidic polymers tend to bind the basic antimicrobial peptides, e.g. defensins and cathecidins produced as a major non-adaptive defense against bacteria. Thus, the release of DNA triggered by quorum sensing, builds matrix, facilitates DNA transformation that is the foundation for the spread of antibiotic resistance in gut biofilms and provides resistance against antimicrobial peptides.

Cause of Autoimmunity and Allergies

Inflammation-Induced Presentation by Mannose Receptor

Speculation on how innocuous proteins become the targets of our immune systems, and result in allergies, asthma, celiac, arthritis, lupus, and other inflammatory autoimmune diseases.

Inflammation

It all starts with chronic inflammation. In most cases diet is the predominant source of inflammation, but infections (bacterial, viral, fungal) may also contribute. Inflammation sets the stage for faulty processing of proteins at the focal location where lymphocytes and antigen-presenting cells are congregating.

Antigen Presentation

At this point a major mistake occurs. Cells that have been alerted to danger by inflammatory cytokines, start to internalize and process proteins in the vicinity, so that peptide fragments of the “antigens” can be displayed on their surfaces in the clutches of major histocompatibility complex proteins. The problem arises when self proteins are internalized, processed and displayed as candidate peptides.

Basic Triplets of All Allergens and Autoantigens

I have looked at dozens of allergens and autoantigens, and they all have one peptide sequence in common, a triplet of basic amino acids. The impetus for this article was finding this morning that one of the autoantigens for Hashimori’s thyroiditis is thyroglobulin, the serum carrier of thyroid hormones. I checked the sequence of thyroglobulin and it has two of the rare basic triplets.

Thyroiditis Autoantigen Is Thyroglobulin

I checked the Pubmed literature to see if thyroglobulin is naturally taken up by cells, since I have been trying to figure out the receptor for basic triplets. What I found was an article on the binding of thyroiditis autoantigens to mannose receptor. The idea of the paper was that the autoantigens in this case were heavily glycosylated and maybe the mannose receptor that is involved in antigen presentation of glycosylated antigens, would bind the selected autoantigens as well. I didn’t get much past the title of the paper.

Candidates for Allergen/Autoantigen Receptor

What I put together was the fact that antigen presenting cells use mannose receptor to internalize antigens. This places this receptor in the right place and the right time to be a receptor for basic triplets. But what would a basic triple binding protein domain look like and could mannose receptor be a candidate?

Structure of Thyroglobulin

I downloaded a structure for mannose receptor and began looking for a surface region that would bind carbohydrates, aromatics and basic amino acids. The critical amino acids in all of these cases are aromatic amino acids, phenylalanine, tyrosine and tryptophan. Tryptophan is at the heart of most carbohydrate binding proteins. Since the mannose receptor is a member of the carbohydrate-binding lectins, I expected to find tryptophans on the surface of the mannose receptor. I just looked for the tryptophan that binds the carbohydrates. It was exactly as I predicted. The mannose receptor should be able to form very stable tryptophan/arginine-like ladders with basic triplets.

I made a figure of the mannose receptor bound to a carbohydrate (red and grey). One of the exposed tryptophans (yellow) is bound to the carbohydrate. Several other tryptophans could be exposed and oriented toward a basic amino acid spread over the surface of the tryptophan in place of the carbohydrate. A similar kind of structure is used by importin to transport proteins with nuclear localization sites (NLS, basic quartets) into the nucleus. Many proteins with NLSs are also autoantigens, e.g. lupus.

I think that the peculiar circumstances that lead to allergy and autoimmunity result in the binding of self-proteins or allergens to mannose receptor and result in antibody production.

Why the Mannose Receptor?

Why does the mannose receptor make mistakes? The exposed tryptophan may also bind numerous plant products. In fact, the phytochemicals, e.g. alkaloids, flavonoids and terpenoids, are an abundant and varied group of chemicals that would bind to the exposed tryptophan of the mannose receptor and compete for binding with basic triplets. Protection offered by plant “anti-oxidants” may be due in part to this activity.

Autoimmunity, Allergies and Basic Triplets

Basic Triplets Only in Primate Forms and Allergens

I have examined the proteins, autoantigens, that are the focus of a dozen autoimmune diseases and a similar number of allergens. All of these proteins have basic triplets that I previously associated with heparin-binding. I have had two recent revelations. First, these triplets appear to not be involved with heparan sulfate proteoglycans for internalization. In fact, HSPGs don’t appear to be involved, even though the process is inhibited by heparin. So this suggests a transport system, perhaps using the LDL receptor or the mannose-6-phosphate receptor, (or a protein with an acidic triplet or quartet.)

The second interesting observation is that the mouse, cat, dog, etc. versions of the human autoantigens lack the basic triplets. This suggests that these diseases cannot occur in non-primates by the same mechanisms. So what is the role of these basic triplets in humans? They didn’t evolve to cause problems under unusual conditions of chronic inflammation, so what is their adaptive advantage?

I think that the answers to these questions could yield the identification of a fundamental cellular transport process and associated cellular phenomena that could be worth a Nobel prize in medicine and it could be unravelled by a group of high school kids doing some straightforward bioinformatics.

Oh. I just remembered that IL-1 beta, the inflammatory cytokine, has a basic triplet. I just checked NCBI, and that basic triplet is found in all of the mammalian IL-1 betas. Oddly, the soluble receptor for IL-1 beta has an acidic quartet. I looked up the protein structure of the IL-1 beta bound to the IL-1 receptor and did a quick illustration using Chimera:

I put the surface on the IL-1 beta and left the IL-1R wrapping around it in as a ribbon. The basic triplet of IL-1 beta is in blue and the acidic quartet of the receptor is in red. One of the basic amino acids is stabilized by hydrophobic bonding over the face of a tyrosine, in yellow. Clearly, there is a simple ionic, plus-minus charge, bonding between the basic and acid amino acids. I don’t normally see this interaction between basic heparin-binding domains and other proteins. Other proteins that bind to heparin-binding domains use aromatic amino acids to make hydrophobic bonds with the hydrophobic arms of the basic amino acids, e.g. importin and nuclear localization signals or tryptophan/arginine ladders. The use of simple acid-base connections (with the projection of each stiffened by adjacent prolines) shows that this interaction is selected to be irreversible.

I don’t know all of the ramifications of basic triplets, but they are very important and are the basis for most modern allergic and autoimmune diseases.

Celiac Causes Allergies and Autoimmune Diseases

Anti-Tissue Transglutaminase Can Lead to Hashimoto’s Thyroiditis

Celiac, gluten intolerance, causes intestinal inflammation and immunological presentation of the common intestinal protein, tissue transglutaminase (tTG). The result is anti-tTG autoantibodies that stimulate an immune attack on intestines and other tissues.

Heparan-Binding Proteins Involved in Autoimmunity and Allergy

Those familiar with my blog know that I am obsessed with heparin-binding protein domains. The reason that I am focused on these parts of proteins, is because most cells rapidly sweep heparan sulfate polysaccharides across their surfaces from sites of secretion to sites of internalization. During inflammation, proteins with strong heparin-binding domains, consisting of triplets of basic amino acids, e.g. KRK (lysine-arginine-lysine), are internalized along with the heparan sulfate. The result is an aberrant presentation of these internalized proteins to the immune system and production of inappropriate antibodies, e.g. autoantibodies.

Basic Triplets in Hasimoto’s Autoantigens

One of my hobbies is checking for the unusual occurrence of basic triplets in autoantigens and allergens. I have found dozens of examples. The most recent is associated with Hashimoto’s Thyroiditis. I knew that attack on the thyroid was common in celiacs, because the celiac autoantigen tTG (it has a basic triplet) is also present in the thyroid and the celiac autoantibodies to tTG also cause an attack on the thyroid. But the autoantigen for Hashimoto’s Thyroiditis is thyroid peroxidase (TPO).

I was momentarily perplexed, but then examined the TPO amino acid sequence and immediately found a couple of basic triplets (KKR and KRK).

MRALAVLSVTLVMACTEAFFPFISRGKELLWGKPEESRV
SSVLEESKRLVDTAMYATMQRNLKKRGILSPAQLLSFSK
LPEPTSGVIARAAEIMETSIQAMKRKVNLKTQQSQHPTD
ALSEDLLSIIANMSGCLPYMLPPK...


Hashimoto’ Thyroiditis Linked to Celiac

Then, I did a PubMed search for “celiac and Hashimoto’s”. As expected, there is a recent paper (see below) that shows that celiac commonly leads to Hashimoto’s Thyroiditis.

An obvious explanation is that the initial attack on the thyroid by anti-tTG autoantibodies of celiac leads to thyroid inflammation and presentation of TPO, with a second round of autoantibodies produced to TPO resulting in Hashimoto’s Thyroiditis. Celiac may be the initial autoimmune trigger for many other autoimmune diseases and allergies.

Autism has been associated with maternal autoimmunity and placental abnormalities. Guess where tTG is found in high abundance?

reference:
Bardella MT, Elli L, Matteis SD, Floriani I, Torri V, Piodi L. Autoimmune disorders in patients affected by celiac sprue and inflammatory bowel disease. Ann Med. 2009;41(2):139-43.

An Autoantigen for Pancreatitis

Pancreatic Secretory Trypsin Inhibitor (PSTI) Has Internalization Basic Triplet

Pancreatitis is an inflammation of the pancreas resulting from lack of adequate inhibition of proteases. Autoantibodies against PSTI would explain some forms of pancreatitis.

I was researching the maintenance of baby gut flora by mother’s milk, when the reference discussed here was brought to my attention by my wife, who happens to be a lactation consultant. The paper showed that PSTI is present in colostrom, the first milk that a baby gets, before the true milk comes in. PSTI protects the new gut from digestion by its own pancreatic proteases, since PSTI is a protease inhibitor that sticks to the gut.

I naturally assumed that PSTI stuck to the gut by heparin-binding domains that would stick to the heparan sulfate proteoglycans on the gut surface. [Recall that it is via these HSPGs that viruses and bacteria infect the gut and the HSPGs in turn are protected during infections by the release of heparin from mast cells. The heparin in the guts of cattle and pigs are used to make commercial heparin to block blood clotting.] So I looked up the structure (above, with basic amino acids in blue and basic triplet on right) sequence of human PSTI at NCBI:

>gi|190694|gb|AAA36522.1| PSTI
MKVTGIFLLSALALLSLSGNTGADSLGREAKCYNELNGCTKIYD
PVCGTDGNTYPNECVLCFENRKRQTSILIQKSGPC

The basic triplet (RKR,arg-lys-arg), from my perspective, should result in presentation to the immune system during high levels of inflammation, and as a consequence result in autoantibodies against PSTI. The result would be the neutralization of the protease inhibitor and damaging production of active protease to attack the pancreas, i.e. pancreatitis.

It would be fairly easy to test this hypothesis by looking for the anti-PSTI antibodies in some people with pancreatitis. Other autoantibodies, e.g. against tissue transglutaminase, might also be checked, because the inflammation that produced one autoantibody may produce others and both PSTI and tTG are produced in the intestines. In fact, celiac may be the cause of some cases of autoimmune pancreatitis.

Note added in proof:

I just checked the literature on PubMed and found that PSTI is in fact an autoantigen in pancreatitis and produces antibodies against PSTI:
Raina A, Greer JB, Whitcomb DC. Serology in autoimmune pancreatitis. Minerva Gastroenterol Dietol. 2008 Dec;54(4):375-87.

and
I found that pancreatitis is often found associated with celiac (gluten intolerance):
Patel RS, Johlin FC Jr, Murray JA. Celiac disease and recurrent pancreatitis. Gastrointest Endosc. 1999 Dec;50(6):823-7.

ref:
Marchbank T, Weaver G, Nilsen-Hamilton M, Playford RJ. Pancreatic secretory trypsin inhibitor is a major motogenic and protective factor in human breast milk. Am J Physiol Gastrointest Liver Physiol. 2009 Apr;296(4):G697-703.

Insulin-like Growth Factor, Diabetes Autoantigen

IGF Binding to Heparin is Basis for Receptor Interaction, Internalization and Immunization

Examination of the protein sequence of insulin-like growth factors reveals strong heparin-binding domains (triplet of basic amino acids) that are also associated with internalization. Similar heparin internalization domains are also found on allergens and autoantigens. It was a small leap to expect that IGFs would also become autoantigens under inflammatory conditions that minimize heparan sulfate proteoglycan production.

Triplets of Basic Amino Acids Internalize Proteins

In several articles on this blog, I have discussed proteins that are internalized by their heparin binding domains. Heparin binding domains consistent only of a pair of basic amino acids, e.g. RK, flanked by one or more basic amino acids within a hydrophobic sequence of protein, are not sufficient to mediate internalization on heparan sulfate proteoglycans. A triplet of basic amino acids is usually required. Simple inspection of amino acid sequences is sufficient to identify these regions.

Internalization Triplet Identified in Insulin-like Growth Factor Binding Proteins

I noticed in a paper that insulin-like growth factors bind to epidermal growth factor receptors. I have previously written an article showing that EGF1 binds to its receptor via heparin, i.e. both the EGF and the receptor have heparin-binding domains. So I suspected that IGFs also had heparin binding domains. Inspection of the sequences readily identified simple heparin binding domains with pairs, but not triplets of basic amino acids. A search of the literature confirmed that heparin mediated IGF binding to receptors. A further search indicated that the heparin binding domains from proteins that bind and control the activity of IGFs could mediate internalization of proteins into cells and also into nuclei.

Internalization Triplets Are Associated with Allergens and Autoantigens

I have previously noted that all allergens and autoantigens have internalization triplets of basic amino acids. The presence of these triplets in IGF binding proteins suggested that IGF binding proteins might also be autoantigens. A quick check of the literature showed that antibodies against IGFs themselves frequently occur in type I diabetes. This suggests that the IGF-binding protein complexes are internalized and IGFs are immunologically presented during inflammation to produce anti-IGF antibodies. It is interesting that the other autoantigens for type I diabetes, e.g. transglutaminase, also have the expected internalization triplets.

references:
Maruyama T, Murayama H, Nagata A, Shimada A, Kasuga A, Saruta T.
Anti-insulin-like growth factor-1 autoantibodies in type 1 diabetes. Ann N Y Acad Sci. 2002 Apr;958:267-70.

Miao D, Yu L, Eisenbarth GS. Role of autoantibodies in type 1 diabetes. Front Biosci. 2007 Jan 1;12:1889-98.

Goda N, Tenno T, Inomata K, Shirakawa M, Tanaka T, Hiroaki H. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5. Exp Cell Res. 2008 Aug 1;314(13):2352-61. Epub 2008 May 29.

Stem Cells Using HSPG Uptake of Recombinant Transcription Factors

Stem Cells from Adult Cells using Transcription Factor Genes
Stem cells have been produced from adult cells using transformation with genes for transcription factors. The problem with this approach was that the embryonic transcription factors had a tendency to promote cancer-like proliferation. What was needed was a temporary push toward embryonic gene expression by temporarily introducing a dose of embryonic transcription factors to dominate gene expression long enough to convert adult, differentiated cells into pluripotent stem cells.

Transcription Factors Synthesized by Recombinant Bacteria
The technical solution was tested and successful results were announced in a prior to publication paper in the journal Cell Stem Cell. Four transcription factors successfully used in prior experiments to induce stem cell transformation were synthesized using recombinant bacteria. The problem was getting the proteins into skin cells that were already growing in cell culture.

Protein Uptake via Triplets of Basic Amino Acids (Heparin-Binding Domains, NLS)
Transcription factors bind to DNA via basic amino acids and many of those basic amino acids are parts of the nuclear localization signals (NLS, quartet or two neighboring pairs of basic amino acids) that bind to importin and transport transcription factors from the cytoplasm to the nucleus.

HSPG Circulation Should Take in Transcription Factors
By inspection, I have demonstrated that proteins observed to be taken up by cells, without specific receptors, e.g. HIV-TAT, lactoferrin, heparanase, allergens, autoantigens, have triplets (or neighboring pairs) or basic amino acids, and this uptake is inhibited by heparin. One would expect that transcription factors would be naturally taken into cells by HSPG circulation. Just adding recombinant transcription factors to cultured skin cells should transform them into stem cells. I don’t believe that this was tested. Instead, more powerful heparin-binding domains were added.

Poly Arginine was used for Uptake of Transcription Factors
The investigators ensured a high efficiency of uptake by adding potent poly arginine sequences to the ends of the transcription factors and synthesized them in recombinant bacteria. The recombinant, arg-tailed transcription factors were taken up by the cultured skin cells and changed the pattern of gene expression in the skin cells. The cultured cells reverted to embryonic patterns of gene expression of pluripotent stem cells. The recombinant proteins were eventually metabolized, but the stem cells had been stably transformed.

reference:
Zhou, H. et al., Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins, Cell Stem Cell (2009), ahead of publication 04.005

Enteroviruses, Autoimmunity, Diabetes

Insulin-producing cells of the pancreases of diabetics have been found to harbor viruses common to the gut. Antibodies to the virus coat proteins also bind to pancreas proteins.

A recent paper, referenced below, shows that in a collection of samples from the pancreases of individuals that had been diagnosed with type I diabetes less than one year prior to the sampling, insulin producing cells are also infected with enterovirus. The same association between enterovirus infection was found to a lesser extent in type II diabetics, but not in non-diabetic controls.

Enteroviruses have been repeated associated with diabetes over the last decade and antigenic determinants of the enterovirus protein coat also bind, i.e. cross react, with antigenic determinants of human cellular proteins.

I examined the enterovirus coat protein, VP1, and found the same three amino acid sequence (three basic amino acids, lysine [K] or arginine [R], highlighted) that I also found in all allergens (peanut, ragweed, dust mite, bee venom) and autoantigens of autoimmune diseases (lupus, MS), and is associated with heparan sulfate-based internalization and presentation of protein immunogens. This observation is consistent with my hypothesis that inflammation plus the presence of one of these proteins, results in production of B and T lymphocytes specific for antigenic determinants on the surface of the immunogen protein. Note that the antigenic determinants usually do not include the three basic amino acid sequence, e.g. RRK, that is involved in uptake and presentation of the protein.

VP1 [Human enterovirus B]
HVINYHTRSESSVENFMGRAACVYIAQYATEKVNDELDR
YTNWEITTRQVAQLRRKLEMFTYMRFDLEVTFVITSSQR
TSTTYASDSPPLTHQVM

reference:
Richardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG. 2009. The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia. Mar 6. [Epub ahead of print]