Celiac disease yields surprises

Celiac disease is linked to diet and can be fatal. The study of the disease revealed a possible link to many autoimmune diseases

by Alessio Fesano

The most important scientific revolution of all time, in my opinion, took place 10,000 years ago in the Middle East, when people first noticed that new plants sprouted from seeds dropped from other plants. This insight led to the birth of agriculture. Before that, the human diet was based on fruits, nuts, roots and occasionally meat. People wandered after their food, at the mercy of chance, and permanent settlement was impossible.

After humans discovered the secrets of seeds, they soon learned how to grow crops and eventually hybridize different grasses and create important grains such as wheat, rye and barley, which were nutritious, versatile, easy to store and of commercial value. For the first time, humans could abandon the nomadic life and build cities. It is no coincidence that the first agricultural areas were also the "cradle of culture".

But this introduction came at a high price: the appearance of a disease known today as celiac disease, caused by eating gluten, a protein found in wheat, or by eating similar proteins found in rye and barley. Before that, gluten and the like were absent from the human diet. But from the moment grains began to sustain stable communities, these proteins killed people (often children) whose bodies did not respond properly to the proteins. Eating these proteins regularly prevented people who are sensitive to them from being able to effectively absorb nutrients from their food. The victims also suffered from repeated stomach pains and diarrhea, their bodies were ashen and their stomachs were swollen, as happens to starving people. Poor nutrition and a variety of other complications made their lives short and miserable.

The cause of these deaths, even if they were noticed in those days, remained unknown. But in the last 20 years, scientists have been able to achieve a detailed understanding of celiac disease. Today they know that it is an autoimmune disease, in which the immune system attacks the body's own tissues. They also know that the disease is not caused only by exposure to gluten and the like, but by a combination of several factors, including genes that increase susceptibility to disease and defects in the structure of the small intestine.

Furthermore, celiac disease provides an eye-opening example of how such a trinity: an environmental factor, high-risk genes, and intestinal defects, may play a role in many autoimmune diseases. Research on celiac disease therefore points to new types of treatment not only for this disease but also for a variety of other autoimmune diseases, such as juvenile diabetes, multiple sclerosis and rheumatoid arthritis.

Early insights
Thousands of years passed from the advent of agriculture until cases of children who ate properly but suffered from malnutrition were recorded. Celiac disease got its name in the first century AD, when the Greek physician Arteus of Cappadocia gave the first scientific description and called it koiliakos, a term derived from the Greek word koelia which means belly [hence also the Hebrew name belly, the use of which is not common - the editors]. The British doctor Samuel Gee is considered the modern father of celiac disease. In a lecture he gave in 1887, he described it as "a type of chronic digestive problem that exists in people of all ages, but tends to affect children between the ages of one and five." He even correctly surmised that "mistakes in diet may be a cause." Although he was undoubtedly wise, the true nature of the disease eluded him as well, as evidenced by the nutritional recommendations he gave: he suggested feeding the sick children with thin slices of bread that had been toasted on both sides.
The identification of gluten as the cause of the disease occurred after World War II, when the Dutch pediatrician Willem-Carl Dicke noticed that due to the lack of bread in the Netherlands after the war, there was a significant decrease in the mortality of children with celiac disease, from 35% to more than zero. He also reported that once wheat was available again the death rate rose to its previous value. Following Dicka's observation, other scientists examined the various components of wheat, and discovered that the main protein in this grain, the gluten, is the cause of the disease.
When examining the biological effects of gluten, the researchers realized that constant exposure of celiac patients to gluten causes chronic inflammation and damage to the vasculature, finger-like structures in the small intestine, and therefore they cannot perform their function: to break down food and transfer nutrients through the intestinal wall into the bloodstream (and from there to the rest of the body) . Fortunately, if the disease is diagnosed at an early stage and patients adhere to a gluten-free diet, the structure of the small intestine almost always returns to its normal state or close to it, and the gastrointestinal symptoms disappear.

In a person prone to illness, gluten causes inflammation and damage to the intestine by activating various cells of the immune system. These cells damage the healthy tissue in an attempt to destroy what they see as causing disease.

A diagnostic discovery
Fuller details about the many mechanisms by which gluten affects immune system activity are still being studied, but one insight has already proven clinically useful: the key sign of a poor immune response to gluten is the production of antibodies against an enzyme called tissue transglutaminase. This enzyme leaks from the damaged cells in the inflamed areas of the small intestine and tries to help and restore the tissues around it.

The discovery that these antibodies are very common in celiac patients added a new tool for diagnosing the disease and allowed my research group and other researchers to examine the prevalence of the disease in a new way, namely by reviewing the presence of the antibody in people's blood. Before that, doctors only had indirect tests, and the most reliable way to diagnose the disease was to examine the patient's symptoms, confirm that there is intestinal inflammation with the help of a biopsy, and see if a gluten-free diet causes the symptoms to disappear. (A screening test for antibodies is not conclusive, because they can also be detected in non-celiac patients.)

For years, celiac disease was considered a rare disease outside of Europe. In North America, for example, classic symptoms of the disease were observed in less than one out of every 10,000 people. In 2003 we published the results of our study in which we conducted the largest survey of celiac patients in North America. In this study, 13,000 people were tested. To our amazement, we found that one out of every 133 apparently healthy people was sick. In other words, the disease is 100 times more common than previously thought. The work of other researchers revealed that in other countries the incidence is similar, and this without sparing any continent.

How is it that 99% of the patients were not diagnosed for so long? The classic external signs, upset stomach and chronic diarrhea, appear only when large and vital parts of the intestine are destroyed. If a small segment of the intestine is not functioning, or if the inflammation is relatively mild, the symptoms may be less dramatic or atypical.
Today it is also clear that celiac disease manifests itself in a variety of symptoms that were not sufficiently appreciated before and are caused by a local disturbance in the absorption of nutrients through the intestine. Impaired iron absorption, for example, can cause anemia, and poor absorption of folic acid can bring about a variety of neurological problems. The disease robs the body of certain components, so it can cause symptoms such as osteoporosis, joint pain, chronic fatigue, short stature, skin problems, epilepsy, delirium, schizophrenia and convulsions.
Because celiac disease often manifests in an atypical way, many cases remain undiagnosed. The new ability to identify the disease in all its forms at an early stage makes it possible to eliminate gluten from the diet before serious complications develop.

From gluten to an immune disorder
Celiac disease provides a valuable model for understanding autoimmune problems because it is the only example where the addition or omission of a simple environmental component, gluten, can start and stop the disease process. (Although environmental factors are also suspected of causing other autoimmune diseases, no cause has been identified with certainty.)

To understand how gluten can be so harmful to some people, you have to see how the body reacts to it in the majority of the population. In people who do not have celiac disease, the body simply does not react. The normal immune system mobilizes into action only when it detects significant amounts of foreign proteins in the body, and it reacts violently because these foreigners may signal the presence of disease-causing microorganisms, such as bacteria and viruses.

One of the main ways of introducing foreign proteins and other substances into the body is eating. The soldiers of the immune system are waiting under the epithelial cells lining the intestine (enterocytes), ready to attack and call for help. One of the reasons that our immune system is usually not activated by protein invasion, which occurs several times a day, is that usually even before our defense system encounters something that might interfere with it, the digestive system breaks down most of the proteins we have ingested into normal amino acids that are used as building blocks for all proteins.

But gluten has a strange structure. It is particularly rich in the amino acids glutamine and proline, a feature that protects part of the molecule from protein cutting mechanisms. Short segments of the protein, or peptides, therefore remain intact. Even so, in healthy people most of these peptides remain in the digestive system and are simply excreted before the immune system notices them. And if gluten sneaks through the intestinal wall, its quantity is often too small to provoke a noticeable response from a normal immune system.

Celiac patients, on the other hand, have inherited a mixture of genes that contribute to increased sensitivity of the immune system to gluten. For example, some genes encode proteins called human tissue antigens (HLA). 95% of celiac patients carry the gene encoding the HLA-DQ2 protein or the HLA-DQ8 protein, compared to only 30% to 40% of the general population who carry one of these two variants. This finding and others indicate that it is almost impossible to develop the disease in the absence of HLA-DQ2 and HLA-DQ8, although they are not the only cause of immune overactivity. The reason for the centrality of these genes becomes self-evident following studies on the activity of the proteins that the genes encode.

The proteins HLA-DQ2 and HLA-DQ8 are produced by antigen-presenting cells. These sentinels of the immune system ingest foreign organisms and proteins, chop them up, load selected protein fragments into recesses on HLA molecules, and display the resulting attachments on the surface of the cell for the perusal of other cells of the immune system called helper T lymphocytes. T cells, Able to identify the attachments, attach to them and call for reinforcements.

In celiac patients, transglutaminase, secreted by intestinal epithelial cells, attaches to the undigested gluten and modifies the peptides in a way that allows them to bind tightly to the DQ2 and DQ8 proteins. When antigen-presenting cells, located behind epithelial cells in the intestine, ingest the transglutaminase and gluten conjugates, they attach the gluten to the HLA proteins and launch it to the surface, where it activates T cells and causes them to release cytokines and chemokines (chemicals that stimulate even greater immune activity). These chemicals and the strengthening of the immune defense could have been useful against a bacterial attack, but in this case they are not useful and damage the intestinal cells responsible for absorbing nutrients.

Celiac patients have additional genetic tendencies, such as the tendency to overproduce the IL-15 protein that stimulates the immune system and contain hyperactive immune cells that stimulate the immune system to attack the intestine in response to gluten.

circumstantial evidence
What role do the transglutaminase antibodies play in this pathological reaction to gluten? The answer is still partial, but scientists have some information about what is going on. When intestinal epithelial cells release transglutaminase, B cells of the immune system engulf it, alone or in conjunction with gluten. In response, they release antibodies against the enzyme. If the antibodies target transglutaminase found on or near intestinal epithelial cells, they may destroy the cells directly, or activate another destructive process. No one knows if they actually cause such damage.

In the last nine years, my colleague and I have discovered that abnormal permeability of the intestine is also, apparently, a cause of celiac disease and other autoimmune diseases. Indeed, a growing number of evidence points to the same trio of factors that underlies most, and perhaps all, autoimmune diseases: a substance found in the environment and the body is exposed to it, a genetic tendency of the immune system to overreact to the substance and a particularly permeable intestine.

Locating the leak
It would be fair to say that the claim that leaky gut contributes to celiac disease and autoimmune diseases in general was met with great skepticism at first, partly due to scientists' perception of the gut. In the 70s, when I was studying medicine, the small intestine was described as a tube consisting of a single layer of cells, like tiles connected with the help of "mortar" known as tight junctions. The Immune System in the Tissues Around the Tubes This simplistic model of the occlusive junction as an inert, opaque filler did not encourage legions of researchers to study their structure, and I was not enthusiastic either.

It was an unexpected twist of fate and one of the most disappointing moments of my career that made me investigate blocking junctions. In the late 80s I tried to develop a vaccine against cholera. In those days, the explanation was that the cholera toxin was the only cause of the terrible diarrhea that characterized the infection. To test this assumption, my research group disabled the activity of the gene encoding the toxin in the Vibrio cholera bacterium. According to the prevailing opinion at the time, a bacterium neutralized in this way was supposed to serve as a perfect immune component, because the proteins remaining on the wall of the living bacterium would cause a strong immune response that would protect against diarrhea.
We gave the weakened bacteria to the volunteers, but it caused diarrhea that prevented its continued use.

The results blew my hands. Years of hard work were flushed down the toilet, literally, and we were faced with two unattractive options: to give up and move on to another topic, or to persevere and try to understand what went wrong. An intuitive feeling that there was something to discover here motivated us to choose the second way. This is how we discovered a new toxin that causes diarrhea in a previously undescribed mechanism. The toxin changed the permeability of the small intestine by breaking down the blocking junctions that were considered inactive, so fluids were able to flow from the tissues into the intestine. The "mortar" was interesting despite everything.

And indeed, at about the same time, a series of important discoveries clarified that it is a complex array of proteins that creates the blocking junctions, but there was almost no information about how these structures are controlled. Therefore, our toxin, which we called the toxin zonula occludens or Zot (zonula occludens is the Latin name for occluding nodes), was a valuable tool for studying the control process. We found that this substance, Zot, was sufficient to break up the tight structure of the blocking junctions. We also realized that the control system that enabled this degradation was too complex, and unlikely to have evolved to cause biological harm to the host. The cholera bacterium causes diarrhea, obviously, by exploiting an existing pathway in the host, which regulates intestinal permeability.

Five years after formulating this hypothesis, we discovered the protein zonulin, found in humans and other evolved animals, which increases intestinal permeability using the same mechanism used by the bacterial toxin Zot. It is still not clear how the body uses zonulin for its benefit, but it is likely that this substance, which is secreted by the epithelial cell tissue of the intestine and also secreted by cells in other organs (obstructive junctions have important roles in tissues throughout the body), performs several actions, including regulating the movement of fluids, large molecules and Immune system cells between different parts of the body.
The discovery of zonulin prompted us to search the medical literature for human diseases characterized by increased intestinal permeability. So I learned for the first time, much to my surprise, that poor intestinal permeability is a common denominator of many autoimmune diseases, including celiac disease, juvenile diabetes, multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease. In many of these diseases the increased permeability is caused by too high levels of zonulin. In celiac disease, it is now clear that the gluten itself causes an excessive secretion of zonulin (perhaps due to the genetic makeup of the patients).

This discovery made us think that the increased permeability of the intestine in celiac patients is what allows gluten, the environmental factor, to leak out of the intestine and react freely with the components of the immune system whose sensitivity is increased due to genetic reasons. This means that removing one of the three causes of the autoimmune disease - the environmental factor, hypersensitivity of the immune system or intestinal permeability - is enough to stop the disease process.

Methods to disrupt the trio
As I mentioned here, and as can be expected according to the theory, eliminating gluten from the diet results in recovery from the damage caused to the intestine. Unfortunately, it is difficult to stick to a gluten-free diet for life. Gluten is very common and in many countries its presence is not indicated on food product packaging. Another difficulty is that gluten-free products are not available everywhere and are more expensive than products containing it. It is also extremely difficult to strictly adhere to a medical diet for years. For these reasons, the nutritional solution is only a partial solution.
Therefore, several medical alternatives are being considered that focus on one or more of the components of the three-step process. The Alvin pharmaceutical company from San Carlos, California has developed a protein-enzymatic treatment, designed for ingestion and completely breaking down the peptides that are resistant to digestion. The product is in clinical trial stages. Other researchers are looking at ways to inhibit the enzyme transglutaminase so that it does not change the chemical structure of the gluten segments that allows them to bind very efficiently to the HLA-DQ2 and HLA-DQ8 proteins.

No one has yet developed a safe and ethical way to change the genes that increase a person's vulnerability to disease, but researchers are working on developing treatments that may moderate some of the genetic factors that contribute to the hypersensitivity of the immune system. For example, the Australian company Nexpep is developing an ingredient that will expose the immune system to small amounts of gluten of the type that causes a strong immune response. The hope is that repeated small exposures will eventually cause the immune system to tolerate the presence of gluten.

Since I wanted to correct the defect that causes intestinal permeability, I was one of the founders of the company Alba-Therapeutics, whose goal is to test an inhibitor of zonulin known as Larazotide. (Today I am Alba's scientific advisor and have options on its shares, but I no longer participate in the company's decision-making.) So far Larzotide has been tested in two human trials examining safety, tolerability and signs of efficacy among celiac patients who ate gluten. The experiments were conducted according to the standard: there was a control using a dummy drug (placebo), the subjects were randomly divided and the experiment was conducted double blind, meaning that both the patients and the therapists did not know until the end of the experiment who received real treatment and who received a placebo.

Both trials showed that taking Larzotide does not cause side effects that are more serious than those of the placebo. More importantly, the first, smaller trial showed that the substance reduced both the damage to intestinal activity due to exposure to gluten and the generation of the inflammatory molecules and symptoms in celiac patients. The second and larger trial, the results of which were reported at a conference in April 2009, showed that celiac patients who received the placebo produced antibodies against transglutaminase, unlike the treatment group. As far as I know, this is the first time that a drug stops an autoimmune process by inhibiting the immune response against a certain molecule produced by the body. Other drugs that suppress immune activity do not work in such a specific way. Alba recently received approval from the US Food and Drug Administration (FDA) to expand research on laresotide to other autoimmune diseases, including juvenile diabetes and Crohn's disease.

These new treatment options do not mean that celiac patients will not have dietary restrictions in the near future. But it is possible to use nutrition in a new way. My research group at the University of Maryland, under the direction of Carlo Catasi, has begun a long-term clinical trial to test whether the onset of celiac disease can be delayed, and possibly even prevented, in high-risk infants who avoid gluten in their first year of life. These babies carry genes that cause vulnerability and the disease is common in their immediate family.

We believe that this approach will be successful because the immune system matures considerably in the first 12 months of life and because research on at-risk babies has shown that avoiding gluten in the first year may train the developing immune system to tolerate gluten, as it does in healthy people. So far, more than 700 babies with genetic risk have been enrolled in the study, and preliminary findings show that delaying exposure to gluten reduces the risk of getting sick fourfold. But it will be decades before we know for sure if this strategy can prevent the disease completely.

In light of the discovery of a seemingly common mechanism for autoimmune diseases in general, scientists who study these diseases are eager to find out if some of the strategies for the treatment of celiac disease can also relieve other autoimmune diseases that do not yet have a successful treatment. And since there are several different approaches to treating celiac disease in the pipeline, we can hope that this disease, which has accompanied humanity since the dawn of civilization, is facing its last century on earth.

in short

• About XNUMX percent of the world's population has celiac disease, but most patients are unaware of it.
• More than two million people in the United States have the disease.
• Some of the common symptoms among babies and children are abdominal pain, bloating, constipation, diarrhea, weight loss and vomiting.
• About half of the sick adults do not suffer from diarrhea at the time of diagnosis.
• Other signs that may appear in adults are anemia, rheumatism, bone loss, depression, fatigue, infertility, joint pain, convulsions, and numbness in the hands and feet.

A reason for the late onset of the disease
Celiac patients are born with a genetic predisposition to the disease. Why then are there people who do not show signs of illness until old age? In the past I would have said that the disease process probably occurred at a young age, but the disease was mild and asymptomatic. But now it seems that the more appropriate answer has to do with the bacteria living in the digestive tract.

These bacteria, generally called the microbiome, may vary from person to person, from population to population and even in the same person during life. Apparently, these bacteria can also bring about the activation of genes of the host at any given time. That is, a person whose immune system has been able to tolerate gluten for many years can suddenly lose tolerance if the microbiome changes and causes genes that confer susceptibility to the disease, which were dormant, to act. If this theory is correct, one day it will be possible to prevent celiac disease or treat it by ingesting beneficial bacteria or "probiotics".

Why is it hard to find wheat substitutes?
Gluten is the main reason for the lightness and airiness of wheat pastries. During baking, gluten fibers capture water and carbon dioxide (from the yeast and other fermenting factors) and swell. To prepare gluten-free products, bakers usually use several types of flour (as well as starches and additives), because there is no single type that mimics the properties of wheat flour. This requirement raises the price of the product considerably. She also explains why gluten-free foods fail to compete in taste with gluten-containing products.

Ideas for treatment
Today, celiac patients have only one treatment option: to avoid food containing gluten. Because adhering to a restrictive diet can be difficult, researchers are now exploring other treatment options for these patients, such as those listed here. These are the first steps of the process. No drug in the table has yet reached the advanced stage of clinical trials, which are necessary to receive approval for marketing.

About the author
Alessio Fasano is a professor of medicine, pediatrics, and physiology and director of the Mucosal Biology Research Center and Celiac Research Center at the University of Maryland School of Medicine. Much of his basic clinical research focuses on the role played by intestinal permeability in the development of celiac disease and other autoimmune diseases.

More on:

Mechanisms of Disease: The Role of Intestinal Barrier Function in the Pathogenesis of Gastrointestinal Autoimmune Diseases. Alessio Fasano and Terez Shea-Donohue in Nature Clinical Practice Gastroenterology & Hepatology, Vol. 2, no. 9, pages 416-422; September 2005.

Diagnosis and Treatment of Celiac Disease. LM Sollid and KEA Lundin in Mucosal Immunology, Vol. 2, no. 1, pages 3-7; January 2009.