Wheat Proteins Cause Inflammation Gut

 

A large number of studies have focused on the effect gluten has on the digestive system within the human body, as well as how it reacts in the body. In reality, wheat products contain more than just gluten, which is why a new research study has turned their focus towards the effects of a different protein that is found in certain types of wheat products. EurekAlert! Science News explains that a group of scientists presented their findings of this study at the UEG Week 2016 event, which concluded that a different type of protein, known as amylase-trypsin inhibitors or ATIs, may have a larger negative impact on the digestive tract, as well as trigger inflammation that is known to cause numerous types of chronic disease to become worse.
The Impact Of Amylase-Trypsin Inhibitors
The press release associated with this new finding reports that approximately 4% of wheat proteins consist of ATIs. While this may seem like a considerably low mount of this specific type of protein, they report that, even in such small amounts, it can cause a powerful reaction in the immune system which starts within the gut, but quickly travels throughout the entire body. The research team was led by Detlef Schuppan, a professor at the Johannes Gutenberg University in Germany. He explained that their findings provided evidence that ATIs can contribute towards inflammation within the bowels, but may also have a potent impact on other parts of the body. One of the most important findings, other than the development of inflammation, is the fact that ATIs were found to contribute to non-coeliac gluten sensitivity developing among participants of their study.
Apart from these findings, the professor reported that they also discovered ATIs contribute towards the development of inflammation that causes certain chronic diseases to become worse, such as:

• Multiple sclerosis
• Lupus
• Rheumatoid arthritis
• Fatty liver disease (non-alcholic type)
• Asthma

With these new findings, the professor reports that further research now needs to be conducted in order to provide further details as to how much of an impact ATIs has on the body’s inflammatory response, as well as how much of an impact this protein family has on chronic diseases that worsens when inflammation in specific parts are triggered.

Avoiding ATIs In Your Daily Diet
With these new findings, many people are looking for ways to avoid this family of proteins in their daily diet. Due to the negative impact they have on numerous diseases, as well as on the digestive tract, compiling a diet that avoid ATIs may have potential benefits for people who are already suffering from inflammatory bowel conditions, problems with the digestive tract or any of the chronic diseases that were observed to become worse with the administration of ATIs.
Unfortunately, this new findings of ATIs are still relatively new, thus no further research has been conducted yet to determine the specific food types that contain them – reports do claim that not all wheat products contain ATIs. Thus, in order to be on the safer side, it is now highly recommended that patients who are suffering from any of the inflammatory conditions that may be worsened by ATIs to avoid wheat products. This is also often referred to as a gluten-free diet.
Celiac Disease Foundation reports that some of the most common foods that are often recommended to people who needs to follow a gluten-free diet includes vegetables, fruits, poultry, lean meat, dairy products such as milk and yogurt, beans, nuts, legumes, seafood and fish. By including more of these food choices in your daily diet, you will be able to avoid consuming a high amount of ATIs, thus also lowering the risk of triggering an inflammatory response within your body.
Mayo Clinic also reports that going wheat-free doesn’t mean you have to avoid using starches and grains. They explain that several options are available including arrowroot, flax, cornmeal, corn, buckwheat, amaranth, hominy, millet, rice, soy, teff, tapioca and sorghum. Flours that are gluten-free, such as soy, bean, potato, rice and corn flour can also be used as substitutes for baking. They also report that certain foods that usually contain wheat (gluten) often comes in gluten-free variants, such as beer, breads, gravies, croutons, cookies, pastas, salad dressings, soups, French fries and cereals. It is, however, important to consult the label of foods in order to determine if they are gluten-free – if these food choices are not specifically labelled “gluten-free”, then they most likely contain gluten and may also contain ATIs.
Conclusion
While many studies often look at how gluten affects the body, this new study rather focused on ATIs and found made some alarming conclusions that were overlooked in previous gluten-based studies. The study found that ATIs may worsen the effects of certain chronic diseases by trigger an inflammatory response in the body’s tissues. This is still a relatively new finding, which means additional research still needs to be completed in order to provide more insight into this new discovery.

New research reveals that a family of proteins in wheat may be responsible for activating inflammation in chronic health conditions such as multiple sclerosis, asthma, and rheumatoid arthritis. Scientists discovered that the proteins might also contribute to the development of non-celiac gluten sensitivity.

Amylase-trypsin inhibitors found in wheat may potentially lead to the development of inflammation in the lymph nodes, kidneys, spleen, and brain.

Findings were presented at UEG Week 2016 in Vienna in Vienna, Austria - a meeting organized by United European Gastroenterology for specialists to communicate the latest research in digestive and liver diseases.
Although wheat was only added to the human diet around 12,000 years ago, it has become a major dietary staple and is widely used in processed foods. One group of proteins found in wheat - amylase-trypsin inhibitors (ATIs) - has been shown to trigger an immune response in the gut that can spread to other tissues in the body.
ATIs are plant-derived proteins that inhibit enzymes of common parasites - such as mealworms and mealybugs - in wheat. ATIs also have an important role in metabolic processes that occur during seed development.
Many previous studies have focused on the impact of gluten on digestive health. However, lead researcher Prof. Detlef Schuppan, from the Johannes Gutenberg University in Germany, and team aimed to highlight the role that ATIs play in digestive health and beyond.
ATIs only make up a small amount of wheat proteins - around 4 percent - yet the immune response they induce significantly affects the lymph nodes, kidneys, spleen, and brain in some people, causing inflammation. ATIs have also been suggested to exacerbate rheumatoid arthritis, multiple sclerosis (MS), asthma, lupus, and nonalcoholic fatty liver disease, as well as inflammatory bowel disease.

"As well as contributing to the development of bowel-related inflammatory conditions, we believe that ATIs can promote inflammation of other immune-related chronic conditions outside of the bowel. The type of gut inflammation seen in non-celiac gluten sensitivity differs from that caused by celiac disease, and we do not believe that this is triggered by gluten proteins."

Prof. Detlef Schuppan

"Instead, we demonstrated that ATIs from wheat, that are also contaminating commercial gluten, activate specific types of immune cells in the gut and other tissues, thereby potentially worsening the symptoms of pre-existing inflammatory illnesses," Prof. Schuppan adds.

Wheat protein-free diet may help treat immunological disorders

Some individuals experience stomach symptoms when eating foods with ingredients containing gluten - such as wheat, barley, and rye - even if they do not have celiac disease. ATIs may contribute to this non-celiac gluten sensitivity (NCGS). This area of research is relatively new, and more research needs to be conducted to understand NCGS and who is at risk.
There are currently no biomarkers for NCGS to monitor a patient's status, and based on current understanding, no intestinal damage has been indicated in people with NCGS after exposure to gluten. Healthcare providers, as a result, rely solely on symptom resolution to observe whether intervention improves the condition.
While gluten is not believed to cause NCGS, people with the condition have been reported to benefit from a gluten-free diet. Some of their symptoms - such as abdominal pain and irregular bowel movements, headaches, joint pain, and eczema - rapidly improve when eating foods devoid of gluten.
Prof. Schuppan notes that the team's research could help redefine the condition to a more appropriate term: "Rather than non-celiac gluten sensitivity, which implies that gluten solitarily causes the inflammation, a more precise name for the disease should be considered."
Researchers are currently preparing studies to investigate further the effect of ATIs on chronic health conditions. "We are hoping that this research can lead us toward being able to recommend an ATI-free diet to help treat a variety of potentially serious immunological disorders." Prof. Schuppan concludes.
Read about how gluten-free diet is gaining popularity, despite no rise in celiac disease.

Written by Hannah Nichols

 “Gluten” is basically a buzzword at this point, but even if you’re avoiding it, do you really know what it is? And did you know that there’s other stuff in wheat that’s also worth avoiding: wheat is bad news for reasons that have nothing to do with gluten. Here’s a look at 11 reasons why.

The Basics

First of all, a refresher: wheat is a grain. The calories in wheat come mostly from carbohydrates, but wheat also contains a few problem proteins.

• Gluten
• Wheat Germ Agglutinin
• Amylase Trypsin Inhibitors

Problems caused by these proteins are not the same thing as blood sugar problems caused by the carbohydrates in wheat. It’s true that getting a majority of calories from wheat (especially refined wheat) can cause metabolic problems like blood sugar swings. But these problems would be caused by any high-carb diet, and they’re only relevant for people eating a large amount of wheat: something like a spoonful of soy sauce wouldn’t be a problem.
This post is not about metabolic issues like blood sugar and carbohydrates. It’s about a totally different list of problems caused specifically by wheat and the proteins it contains. These problems are relevant even for people eating a small amount of wheat, and even for people who do fine eating carbs.
So what’s so bad about wheat?

1. Wheat Problems Aren’t Restricted to People with Celiac Disease

The most famous problem with wheat is celiac disease, an autoimmune reaction provoked by gluten and treatable with a gluten-free diet. 30-40% of people have the genetic background to potentially develop celiac disease, but only about 1-3% of people actually do – it’s not clear why but it may have something to do with the gut microbiome.
Most people know that celiac disease requires absolutely strict avoidance of all gluten. But a lot of people also think that if you don’t have celiac disease, you’re completely in the clear.
That’s not true. Recently there’s been an increased amount of interest in non-celiac gluten sensitivity (NCGS). Plenty of people have documented sensitivities to gluten that aren’t actually celiac disease (as you’ll read below, there’s a different immune reaction involved). There’s also the overlapping problem of other proteins in wheat – wheat germ agglutinin and amylase trypsin inhibitors are not the same thing as gluten and you can be sensitive to them regardless of how your body handles gluten.
Wheat isn’t just a problem for people with celiac disease, and there’s more to wheat than gluten.

 2. Gut Inflammation

Inflammation is the natural response of your immune system to injury. You can see it in action whenever you get a cut or splinter and the surrounding area gets all red and tender. The proteins in wheat are gut irritants: they’re like that papercut or splinter digging into the lining of your gut, causing an inflammatory response.
The most famous case is the inflammation caused by gluten in people with celiac disease or non-celiac gluten sensitivity. But inflammation from wheat is also a problem even for people who aren’t sensitive to gluten specifically. Amylase trypsin inhibitors (ATIs for short) that can provoke an inflammatory immune response in the GI tract by stimulating immune cells. This occurs in people regardless of whether they have celiac disease or not – it’s a completely different problem from gluten and it can cause trouble for you regardless of whether or not you’re sensitive to gluten in particular.
That inflammation is dangerous because…

3. Increased Intestinal Permeability

Inflammation in the gut contributes to a problem called intestinal permeability. The gut has a very complex system of “border control” that lets digested food into your bloodstream (this is how you get nutrients from it) while keeping everything else out. Every day, you swallow millions of random viruses, bacteria, indigestible molecules like dust, and other stuff that needs to go out the other end, not into your bloodstream.
Inflammation in the gut messes up that system of border control. It loosens the junctions between cells in the gut wall so too much stuff can pass through. This is often described as making the gut “leaky” (hence the popular name of “leaky gut”).
On top of inflammation leading to increased permeability, gluten accelerates this process by stimulating the release of a protein called zonulin. Zonulin independently contributes to loosening the junctions between cells in the gut. Add together the inflammation and the zonulin, and wheat has a powerful effect on gut permeability, which is really a problem.
Intestinal permeability is a big problem – most notably because it’s an essential factor in the development of autoimmune diseases.

4. Double Trouble: Wheat Germ Agglutinin

Another one for the non-Celiac crowd: wheat germ agglutinin is an inflammatory, immune-disrupting protein found in wheat and despite the similar name it isn’t the same thing as gluten. Wheat germ agglutinin can provoke an inflammatory response in gut cells and disturb the natural immune barrier in the gut, making the gut more permeable to things that don’t belong in your blood.
Again, this is totally separate from the problem of gluten. Obviously, gluten and WGA usually come as a package deal, because they’re both found in wheat, but you can have trouble with WGA even if you had no reaction to a gluten elimination challenge.

5. Increased Vulnerability to Gut Autoimmunity

Items #1-4 on this list discussed how wheat makes the gut more permeable, so all kinds of stuff can get into the bloodstream even though it shouldn’t be there. Included in that stuff is…gluten! Specifically, gliadin, which is a component of gluten. Once it’s inside your bloodstream, gliadin runs into your immune system, and that’s where the problems really start, in the form of molecular mimicry.
Molecular mimicry works like this: some foreign thing gets into the bloodstream. The immune system forms antibodies against it. So far, so good: that’s how the immune system is supposed to work. But if that foreign thing looks enough like your own body’s tissue, then the antibodies formed to fight it might start attacking your own body as well.
Molecular mimicry may be the reason why people with celiac disease mount an attack on their own gut cells: to your immune system, gliadin looks a lot like the cells lining the gut. But it’s not just celiac disease! Gluten-related inflammation may also be a factor in the development of Crohn’s Disease, another autoimmune gut disease. In this study of patients with inflammatory bowel disease (Crohn’s Disease and ulcerative colitis), a gluten-free diet helped a majority of people who tried it.
And gut cells aren’t the only cells affected by gluten-related autoimmunity…

6. Increased Vulnerability to non-Celiac Autoimmune Diseases

If you go digging into the research on celiac disease and gluten, you’ll find a bunch of studies linking it to all kinds of other autoimmune diseases, including autoimmune thyroid disorders, type 1 diabetes, fibromyalgia (for both celiac disease and non-celiac gluten sensitivity!), rheumatoid arthritis, autoimmune liver disease, and a couple different autoimmune skin diseases.
The common factor here might be the gluten. Wheat gluten is a major potential trigger of Type 1 Diabetes (that’s the autoimmune type, not the diet-and-lifestyle type). In this study, feeding mice a gluten-free diet reduced the rate of Type 1 diabetes in their children. There’s also evidence that breastfeeding human children reduces the rate of type 1 diabetes, which would make sense if gluten is the problem because breastfeeding delays the introduction of gluten to the baby.
Hey, by the way, guess what other common health problems have an autoimmune component? Obesity and Type 2 Diabetes.

7. Autoimmune Reactions in People Without Celiac Disease.

Point #6 above gave a lot of reasons why celiac disease is associated with other autoimmune diseases, but it’s not limited to people with celiac disease. If you thought non-celiac gluten sensitivity was unrelated to autoimmune disease, you thought wrong! This study found that a lot of people with non-celiac gluten sensitivity have autoimmune markers in their blood, suggesting that the wheat exposure might be causing autoimmune issues even without celiac disease.
One interesting aspect of this is that patients with non-celiac gluten sensitivity may have a different type of autoimmune reaction, which just underlines that celiac disease and non-celiac gluten sensitivity are two different things. But the point is that both involve potentially serious autoimmune responses.

8. Damage to the Gut Biome

Not the all-important gut biome! The gut biome, aka the gut microbiome, aka the gut flora, is the collection of friendly bacteria that live in your gut. They help regulate your immune system, control intestinal permeability, digest your food, synthesize nutrients like vitamin K2, send hunger/fullness signals to your brain, and do all kinds of other stuff.
But they really don’t like gluten, and gluten really doesn’t like them. People with celiac disease often have very bad problems with the gut flora, but those problems are significantly reduced when the person eliminates gluten. Once again, it’s not limited to celiac disease: non-celiac gluten sensitivity also involves disturbances in the gut flora.
Even in people who aren’t sensitive to gluten at all, inflammation caused by other components of wheat can also rebound on the gut biome. And independently of any of that, wheat is also high in FODMAPs, which may be an issue for people with sensitivities to that.

9. Gastrointestinal Symptoms (Even for People who Don’t have Celiac Disease)

All this stuff about gut bacteria and intestinal permeability might seem totally abstract and disconnected from the real world, so let’s bring it back down to earth: this stuff has actual, noticeable consequences. Most of the direct damage involves the gut, so it makes sense to start there:

• In people with celiac disease, gluten causes immediate and severe symptoms (diarrhea and/or constipation, heartburn, pain, bloating, gas, stools that smell awful, sometimes vomiting…).

• In people with non-celiac gluten sensitivity, symptoms are typically similar to celiac disease.

• Even in people who aren’t sensitive to gluten specifically, the inflammatory action of other components of wheat (wheat germ agglutinin and amylase trypsin inhibitors) contributes to chronic, relapsing gut problems.

Of course, there are non-wheat-related reasons why a person might have GI problems (stress is a biggie, and stress is certifiably gluten-free). But gluten can contribute to the problem, even if it’s “only” a low-level inflammatory response that you’ve gotten used to. Sure, constipation and feeling bloated after meals might be your “normal,” but what if it didn’t have to be?

10. Brain Symptoms

Think of gluten or wheat issues, and you probably think of the gut first. The typical symptoms are all gut-related. But actually, there’s another important organ at stake: your brain.
Brain fog and fatigue are symptoms of both celiac disease and non-celiac gluten sensitivity. On a more serious note, the gut inflammation and microbiome disturbances involved in the immune-inflammatory response to gluten may increase vulnerability to dementia and Alzheimer’s disease. Autoimmunity in general (whether it’s celiac disease or some other gluten-related autoimmunity) may be involved in depression.
This doesn’t mean that gluten is the cause of all mental health problems or that eliminating gluten will cure them. Nobody is saying that. Mental health is complicated and there are all kinds of factors to consider. The point is that in some people, gluten may be one of them.

11. Skin Symptoms

The most famous cause of gluten-related skin problems is celiac disease, which can cause a skin disease called dermatitis herpetiformis. Symptoms of dermatitis herpetiformis include an itchy, red rash with raised blisters. Symptoms typically show up in a person’s 20’s.
And once again, this isn’t limited to celiac disease. This study describes the way non-celiac gluten sensitivity can show up as skin problems: “very itchy…similar to eczema, psoriasis, or dermatitis herpetiformis.” The itchy skin showed up most often on the arms and legs.
The upshot: wheat is pretty bad news even for people who don’t have celiac disease. And the symptoms don’t necessarily show up as dramatic episodes of vomiting and diarrhea. Why not try giving it up for a few weeks just to see how your body reacts – you might be surprised!

Wheat is one of the most consumed cereal grains worldwide and makes up a substantial part of the human diet. Although government-supported dietary guidelines in Europe and the U.S.A advise individuals to eat adequate amounts of (whole) grain products per day, cereal grains contain “anti-nutrients,” such as wheat gluten and wheat lectin, that in humans can elicit dysfunction and disease. In this review we discuss evidence from in vitro, in vivo and human intervention studies that describe how the consumption of wheat, but also other cereal grains, can contribute to the manifestation of chronic inflammation and autoimmune diseases by increasing intestinal permeability and initiating a pro-inflammatory immune response.

Keywords: cereal grains, celiac disease, gluten, gliadin, inflammation, intestinal permeability, lectins, wheat, wheat germ agglutinin

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1. Introduction

Inflammation is the response of the innate immune system triggered by noxious stimuli, microbial pathogens and injury. When a trigger remains, or when immune cells are continuously activated, an inflammatory response may become self-sustainable and chronic. Chronic inflammation has been associated with many medical and psychiatric disorders, including cardiovascular disease, metabolic syndrome, cancer, autoimmune diseases, schizophrenia and depression [1,2,3]. Furthermore, it is usually associated with elevated levels of pro-inflammatory cytokines and acute phase proteins, such as interferons (IFNs), interleukin (Il)-1, Il-6, tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP). While clear peripheral sources for this chronic inflammation are apparent in some conditions (i.e., fat production of cytokines in the metabolic syndrome), in other disorders, such as major depression, the inflammatory source is not completely understood. Genetic vulnerability, psychological stress and poor dietary patterns have all been repeatedly implicated as being of significant importance in the development of an inflammatory phenotype [3,4,5]. Dietary factors associated with inflammation include a shift towards a higher n-6:n-3 fatty acid ratio [5] and a high intake of simple sugars [6]. Other substances in our daily food, like those found in wheat and other cereal grains, are also capable of activating pro-inflammatory pathways.

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2. Wheat Grain, Gluten and Disease

2.1. Wheat Allergy and Intolerance

The ingestion of wheat products has been reported to be responsible for IgE-mediated allergic reactions. Wheat-dependent exercise-induced anaphylaxis is a syndrome in which the ingestion of a product containing wheat followed by physical exercise can result in an anaphylactic response. Several proteins present in wheat, most notably gluten proteins have been shown to react with IgE in patients [7]. Other allergic responses that appear to be related to a range of wheat proteins include baker’s asthma, rhinitis and contact urticaria [7,8].

More common than wheat allergies are conditions involving wheat intolerance, including celiac disease (CD), which is estimated to affect 1% of the population of Western Europe, and dermatitis herpetiformis, which has an incidence between about 2-fold and 5-fold lower than CD [9]. The close association between type 1 diabetes and CD [10] and the observation that autoimmune diseases seem to be more prevalent in celiac patients and their relatives [11] thus links the intake of wheat with several other conditions.

2.2. Wheat Grain and Gluten

Gluten is the main structural protein complex of wheat consisting of glutenins and gliadins. When wheat flour is mixed with water to form dough, the gluten proteins form a continuous network which provides the cohesiveness and viscoelasticity that allows dough to be processed into bread, noodles and other foods. The protein contents of wheat varies between 7% and 22% with gluten constituting about 80% of the total protein of the seed [9]. Glutenins are the fraction of wheat proteins that are soluble in dilute acids and are polymers of individual proteins. Prolamins are the alcohol-soluble proteins of cereal grains and are specifically named gliadins in wheat. Gliadins are monomeric proteins and are classified into three groups: α/β-gliadins, γ-gliadins, and ω-gliadins [7].

2.3. Gluten, Gliadin and CD

Gliadin epitopes from wheat gluten and related prolamins from other gluten-containing cereal grains, including rye and barley, can trigger CD in genetically susceptible people. The symptoms of this disease are mucosal inflammation, small intestine villous atrophy, increased intestinal permeability and malabsorption of macro- and micronutrients. CD, a chronic inflammatory disorder mediated by T-cells, is preceded by changes in intestinal permeability and pro-inflammatory activity of the innate immune system. Gliadin immunomodulatory peptides can be recognized by specific T-cells, a process that can be enhanced by the deamidation of gliadin epitopes by tissue transglutaminases that convert particular glutamine residues into glutamic acid resulting in a higher affinity for HLA-DQ2 or DQ8 expressed on antigen-presenting cells (APC) [10]. Serum antibodies, among which are antibodies against tissue transglutaminases, are also found in CD. The HLA-DQ2 or HLA-DQ8 is expressed in 99.4% of the patients suffering from CD [10], however, interestingly enough, there is a group of HLA-DQ2/DQ8-negative patients suffering from gastrointestinal symptoms that respond well to a gluten-free diet. This group of “gluten-sensitive” patients does not have the CD serology and histopathology, but does present the same symptoms and shows improvements when following a gluten-free diet [12,13].

2.4. Gliadin and Immunity

There are at least 50 gliadin epitopes that exert immunomodulatory, cytotoxic and gut-permeating activities that can be partially traced back to different domains of α-gliadin. Where some immunomodulatory gliadin peptides activate specific T-cells, others are able to induce a pro-inflammatory innate immune response [10]. Stimulation of immune cells by gliadin is not only restricted to CD patients; the incubation of peripheral blood mononuclear cells (PBMC) from healthy HLA-DQ2-positive controls and CD patients with gliadin peptides stimulated the production of IL-23, IL-1β and TNF-α in all donors tested. Nevertheless, the production of cytokines was significantly higher in PBMC derived from CD patients [14]. Similar results were obtained by Lammers et al. [15], who demonstrated that gliadin induced an inflammatory immune response in both CD patients and healthy controls, though IL-6, Il-13 and IFN-γ were expressed at significantly higher levels in CD patients. IL-8 production was only expressed in a subset of healthy and CD individuals after stimulation with a specific gliadin peptide and appeared to dependent on the CXCR3 chemokine receptor only in CD patients. Sapone et al. [16] showed that in a subset of CD patients, but not in gluten-sensitive patients (with 36% of the studied individuals in this group being HLA-DQ2/DQ8-positive), there is an increased IL-17 mRNA expression in the small-intestinal mucosa compared to healthy controls. The same group showed that in a subset of gluten-sensitive patients (with about 50% of the studied individuals being HLA-DQ2/DQ8-positive) there is a prevailing stimulation of the innate immune system, while in CD, both the innate and adaptive immune system are involved [13].

2.5. Gliadin and Intestinal Permeability

In order for gliadin to interact with cells of the immune system, it has to overcome the intestinal barrier. Gliadin peptides cross the epithelial layer by transcytosis or paracellular transport. Paracellular transport occurs when intestinal permeability is increased, a feature that is characteristic for CD [17]. It is indicated by several studies that increased intestinal permeability precedes the onset of CD and is not just a consequence of chronic intestinal inflammation [18,19]. Gliadin has been demonstrated to increase permeability in human Caco-2 intestinal epithelial cells by reorganizing actin filaments and altering expression of junctional complex proteins [20]. Several studies by Fasano et al. show that the binding of gliadin to the chemokine receptor CXCR3 on epithelial IEC-6 and Caco2 cells releases zonulin, a protein that directly compromises the integrity of the junctional complex [21,22]. Although zonulin levels were more up-regulated in CD patients, zonulin was activated by gliadin in intestinal biopsies from both CD and non-CD patients [21,22], suggesting that gliadin can increase intestinal permeability also in non-CD patients, yet increased intestinal permeability was not observed in a group of gluten-sensitive patients [13].

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3. Increased Intestinal Permeability

3.1. Increased Intestinal Permeability is Associated with Disease

Chronically increased intestinal permeability (or leaky gut syndrome) allows for the increased translocation of both microbial and dietary antigens to the periphery which can then interact with cells of the immune system. Shared amino acid motifs among exogenous peptides (HLA-derived peptides and self-tissue) may produce cross-reactivity through immunological mimicry, thereby disturbing immune tolerance in genetically susceptible individuals [23]. Not surprisingly, increased intestinal permeability has been associated with autoimmune diseases, such as type 1 diabetes [24], rheumatoid arthritis, multiple sclerosis [18], but also with diseases related to chronic inflammation like inflammatory bowel disease [18,25], asthma [26], chronic fatigue syndrome and depression. The latter two conditions see patients with significantly greater values of serum IgA and IgM to LPS of gram-negative enterobacteria compared to controls, implying intestinal permeability is increased in these patients [27,28,29].

3.2. Intestinal Barrier Function and Inflammation

The intestinal barrier allows the uptake of nutrients and protects from damage of harmful substances from the gut lumen. Macromolecules that can be immunogenic like proteins, large peptides, but also bacteria and lectins, can be endocytosed or phagocytosed by enterocytes forming the epithelial layer of the gut. Absorbed proteins will generally enter the lysosomal route and will be degraded to small peptides. Normally, only small amounts of antigen pass the barrier by transcytosis and interact with the innate and adaptive immune system situated in the lamina propria. Highly specialized epithelial microfold (M) cells function as active transporters of dietary and microbial antigens from the gut lumen to the immune system, where either a pro-inflammatory or tolerogenic immune response can be generated. The paracellular route is regulated by the junctional complex that allows the passage of water, solutes and ions, but under normal conditions provides a barrier to larger peptides and protein-sized molecules. When the barrier function is disrupted, there is an increased passage of dietary and microbial antigens interacting with cells of the immune system [25,30] (Figure 1).

Figure 1

Increased intestinal permeability allows for the passage of microbial and dietary antigens across the epithelial layer into the lamina propria, where these antigens can be taken up by APC and presented to T-cells. JC, junctional complex.

3.3. The Role of Zonulin Signaling on Intestinal Permeability

Intestinal permeability is a measure of the barrier function of the gut which relates to the paracellular space surrounding the brush border surface of the enterocytes and the junctional complexes consisting of tight junctions, adherent junctions, desmosomes and gap junctions [31]. The junctional complexes are regulated in response to physiological and immunological stimuli, like stress, cytokines, dietary antigens and microbial products [31]. As mentioned before, zonulin, a protein identified as prehaptoglobulin-2 (the precursor of haptoglobin-2) is also a regulator of intestinal permeability. Haptoglobin-2, together with haptoglobin-1, is one of the two gene variants of the multifunctional protein haptoglobin and is associated with an increased risk for CD (homozygotes and heterozygotes) and severe malabsorption (homozygotes) [32,33]. The haptoglobulin-2/zonulin allele has a frequency of about 0.6 in Europe and the U.S.A, but varies throughout the world depending on racial origin [34].

3.4. High Zonulin Levels are Observed in Auto-Immune and Inflammatory Diseases

Zonulin signaling is proposed to cause rearrangements of actin filaments and induces the displacement of proteins from the junctional complex, thereby increasing permeability [18,32,35]. Gliadin peptides initiate intestinal permeability through the release of zonulin, thereby enabling paracellular translocation of gliadin and other dietary and microbial antigens, which by interacting with the immune system give rise to inflammation. In this manner, a vicious cycle is created in which, as a consequence of the persistent presence of pro-inflammatory mediators, intestinal permeability will increase even further. High zonulin levels (together with increased intestinal permeability) have been observed in autoimmune and inflammatory diseases like CD, multiple sclerosis, asthma and inflammatory bowel disease and the haptoglobin polymorphism is associated with rheumatoid arthritis, ankylosing spondylitis, schizophrenia and certain types of cancer [32].

The zonulin inhibitor Larozotide acetate was tested in an inpatient, double-blind randomized placebo-controlled trial. The group of CD patients in the placebo group that were exposed to gluten showed a 70% increase in intestinal permeability, while no changes were seen in the group exposed to Larazotide acetate. Also gastrointestinal symptoms were significantly more frequent in the placebo group [32]. These results suggest that in CD patients, when intestinal barrier function is restored, autoimmunity will disappear while the trigger (gluten) is still there. Besides gliadin from wheat gluten, the lectin wheat germ agglutinin (WGA) has also been shown to stimulate cells of the immune system and increase intestinal permeability, as we will now discuss further.

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4. Wheat Germ Agglutinin (WGA)

4.1. Dietary WGA

Lectins are present in a variety of plants, especially in seeds, where they serve as defense mechanisms against other plants and fungi. Because of their ability to bind to virtually all cell types and cause damage to several organs, lectins are widely recognized as anti-nutrients within food [36]. Most lectins are resistant to heat and the effects of digestive enzymes, and are able to bind to several tissues and organs in vitro and in vivo (reviewed by Freed 1991 [37]). The administration of the lectin WGA to experimental animals caused hyperplastic and hypertrophic growth of the small intestine, hypertrophic growth of the pancreas and thymus atrophy [36]. Lectin activity has been demonstrated in wheat, rye, barley, oats, corn and rice, however the best studied of the cereal grain lectins is WGA [38].

The highest WGA concentrations are found in wheat germ (up to 0.5 g/kg [39]). Although unprocessed wheat germ, like muesli, contains far higher amounts of active WGA than do processed wheat germ products, WGA activity is still apparent in several processed breakfast cereals as assessed by hemagglutination and bacterial agglutination assays [40,41]. A summary of the amount of active WGA in commonly consumed wheat derived products is listed in Table 1.

Table 1

Amount of active WGA in wheat-derived products.

4.2. WGA Binds to Cell Surface Glycoconjugates

WGA binds to N-glycolylneuraminic acid (Neu5Ac), the sialic acid predominantly found in humans [44], allowing it to adhere to cell surfaces like the epithelial layer of the gut. The surface of many prokaryotic and eukaryotic cells are covered with a dense coating of glycoconjugates, also named glycocalyx. Sialic acids are a wide family of nine-carbon sugars that are typically found at the terminal positions of many surface-exposed glycoconjugates and function for self recognition in the vertebrate immune system, but they can also be used as a binding target for pathogenic extrinsic receptors and molecular toxins [45,46,47]. WGA binding to Neu5Ac of the glycocalyx of human cells (and pathogens expressing Neu5Ac) allows for cell entry and could disturb immune tolerance by evoking a pro-inflammatory immune response (discussed below).

4.3. WGA and Immunity

WGA induces inflammatory responses by immune cells. For example, WGA has been shown to trigger histamine secretion and granule extrusion from non-stimulated rat peritoneal mast cells [48], induce NADP-oxidase activity in human neutrophils [49] and stimulate the release of the cytokines IL-4 and IL-13 from human basophils [50]. In human PBMC, WGA induced the production of IL-2, while simultaneously inhibiting the proliferation of activated lymphocytes [51]. WGA stimulated the secretion of IL-12, in a T- and B-cell-independent manner in murine spleen cells. IL-12, in turn, activated the secretion of IFN-γ by T or natural killer cells [52]. In murine peritoneal macrophages WGA induced the production of the pro-inflammatory cytokines TNF-α, IL-1β, IL-12 and IFN-γ [53]. Similar results have been observed in isolated human PBMC, given that nanomolar concentrations of WGA stimulated the release of several pro-inflammatory cytokines. In the same study a significant increase in the intracellular accumulation of IL-1β was measured in monocytes after WGA exposure [54]. These results indicate that, when delivered in vitro, WGA is capable of directly stimulating monocytes and macrophages, cells that have the ability to initiate and maintain inflammatory responses. Monocytic cells have been shown to engulf WGA via receptor-mediated endocytosis or by binding to non-receptor glycoproteins [55].

Human data showing the influence of WGA intake on inflammatory markers are lacking, however, antibodies to WGA have been detected in the serum of healthy individuals [56]. Significantly higher antibody levels to WGA were measured in patients with CD compared to patients with other intestinal disorders. These antibodies did not cross-react with gluten antigens and could therefore play an important role in the pathogenesis of this disease [57].

4.4. WGA and Intestinal Permeability

After ingestion, WGA is capable of crossing the intestinal barrier. In animal models, WGA has been shown to reach the basolateral membrane and walls of the small blood vessels in the subepithelium of the small intestine [36]. WGA can be phagocytosed by binding to membrane non-receptor glycoproteins, a process that has been observed in Caco-2 cells [58]. WGA can also be endocytosed by antigen sampling M-cells [59,60] or by enterocytes via binding to epidermal growth factor receptors [61]. Another possible route for lectin entry into the periphery is by paracellular transport, a process that can be further aggravated by the binding of gliadin to the chemokine receptor CXR3 on enterocytes.

WGA itself has been found to affect enterocyte permeability. Investigations by Dalla Pellegrina et al. [54] showed, in vitro, that exposure to micromolar concentrations of WGA impairs the integrity of the intestinal epithelial layer, allowing passage of small molecules, like lectins. At the basolateral side of the epithelium, WGA concentrations in the nanomolar range induced the secretion of pro-inflammatory cytokines by immune cells [54]. This may further affect the integrity of the epithelial layer, heightening the potential for a positive feedback loop between WGA, epithelial cells and immune cells. When combined, these mechanisms are likely able to significantly increase the percentage of consumed WGA that can cross the epithelial layer compared to the low percentage of WGA crossing by means of transcytosis (0.1%) alone [54]. This suggests that, together with gliadin, WGA can increase intestinal permeability, resulting in an increase of translocating microbial and dietary antigens interacting with cells of the immune system.

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5. Animal Data on Cereal Grain Intake

There are two rodent models of spontaneous type 1 diabetes: the non-obese-diabetic (NOD) mouse and the diabetes-prone BioBreeding (BBdp) rat. In these animals, a cereal-based diet containing wheat induced the development of type 1 diabetes, while animals fed a hypoallergenic diet (gluten free) or a hypoallergenic diet supplemented with casein showed a decreased incidence and a delayed onset of this disease. BBdp rats fed a cereal-based diet showed increased intestinal permeability and a significant increase in the percentage of IFN-γ-producing Th1 lymphocytes in the mesenteric lymph nodes in the gut [30]. Compared to animals fed a hypoallergenic diet, NOD mice fed a wheat-based diet expressed higher mRNA levels of the pro-inflammatory cytokines IFN-γ and TNF-α and the inflammatory marker inducible NO synthase in the small intestine. While these diet-induced changes in gut-wall inflammatory activity did not translate to increased cytokine mRNA in Peyers patches, structures that contribute to immune regulation to exogenous antigens, it is possible that the gut-signal may promote systemic inflammation via other mechanisms, such as activating intraepithelial lymphocytes and mesenteric lymph node cells [62]. These in vivo results show that, in two rodent models of spontaneous type 1 diabetes, a cereal-containing diet induces the (early) onset of disease and increases markers of inflammation. In addition, Chignola et al. [63] have shown in rats that a WGA-depleted diet was associated with reduced responsiveness of lymphocytes from primary and secondary lymphoid organs after in vitro stimulation and attenuated spontaneous proliferation when compared to lymphocytes from rats fed a WGA-containing diet, indicating the stimulatory effect of WGA on cells of the immune system.

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6. Human Studies on Cereal Grain Intake and Inflammation

6.1. Human Epidemiological Data on Cereal Grain Intake and Inflammation

Observational prospective and cross-sectional studies show that the intake of whole grain products is associated with reduced risks for developing type 2 diabetes, cardiovascular diseases, obesity and some types of cancer [64]. Inflammation is associated with these conditions and some studies have shown that associations between the intake of whole grains and decreased inflammatory markers (CRP, Il-6) are found [65]. Intervention studies, however, do not demonstrate a clear effect of the intake of whole grains on inflammation [66,67,68,69,70,71] and it could therefore be that other components in the diet modulate the immune response.

It has been shown that the intake of whole grains is associated with healthier dietary factors and a healthier lifestyle in general. In a Scandinavian cross-sectional study, the intake of whole grains was directly associated with the length of education, the intake of vegetables, fruits, dairy products, fish, shellfish, coffee, tea and margarine and inversely associated with smoking, BMI and the intake of red meat, white bread, alcohol, cakes and biscuits [72]. Good quality epidemiological studies attempt to control these confounding factors, but with the consequence that associations are attenuated or become insignificant.

6.2. Human Intervention Trials on Cereal Grain Intake and Inflammation

To accurately estimate the causal relationship of cereal grain intake and inflammation, intervention trials provide us with better evidence. Wolever et al. [71] showed that a diet with a low glycemic index (containing whole grains) compared to high (containing refined grain products), resulted in sustained reductions in postprandial glucose and CRP levels on the long-term in patients with type 2 diabetes treated with diet alone. A refined grain is a whole grain that has been stripped of its outer shell (fiber) and its germ, leaving only the endosperm, resulting in lower levels of macro- and micronutrients and a higher dietary glycemic index for refined grains compared to whole grains. Refined wheat products contain less WGA, but still contain a substantial amount of gluten. It should be noted that whole grains contain phytochemicals, like polyphenols, that can exert anti-inflammatory effects which could possibly offset any potentially pro-inflammatory effects of gluten and lectins [73].

The substitution of whole grain (mainly based on milled wheat) for refined grains products in the daily diet of healthy moderately overweight adults for six weeks did not affect insulin sensitivity or markers of lipid peroxidation and inflammation [66]. Consistent with these finding are the results of Brownlee et al. [67], who showed that infrequent whole-grain consumers, when increasing whole grain consumption (including whole wheat products), responded with no improvements of the studied biomarkers of cardiovascular health, including insulin sensitivity, plasma lipid profile and markers of inflammation. The substitution of refined cereal grains and white bread with three portions of whole wheat food or one portion of whole wheat food combined with two servings of oats significantly decreased the systolic blood pressure and pulse pressure in middle-aged, healthy, overweight men and women, yet none of the interventions significantly affected systemic markers of inflammation [70]. In obese adults suffering from metabolic syndrome, there were significantly greater decreases in CRP and the percentage of body fat in the abdominal region in participants consuming whole grains compared to those consuming refined grains. It must be noted that both diets were hypocaloric (reduced by 500 kcal/d) [69]. Most of the intervention studies mentioned above attempted to increase whole grain intake and were using refined grain diets as controls, thereby making it very difficult to draw any conclusions on the independent role of cereal grains in disease and inflammation.

6.3. Health Effects of the Paleolithic Diet

There are few studies that investigate the influence of a paleolithic type diet comprising lean meat, fruits, vegetables and nuts, and excluding food types, such as dairy, legumes and cereal grains, on health. In domestic pigs, the paleolithic diet conferred higher insulin sensitivity, lower CRP and lower blood pressure when compared to a cereal-based diet [74]. In healthy sedentary humans, the short-term consumption of a paleolithic type diet improved blood pressure and glucose tolerance, decreased insulin secretion, increased insulin sensitivity and improved lipid profiles [75]. Glucose tolerance also improved in patients suffering from a combination of ischemic heart disease and either glucose intolerance or type 2 diabetes and who had been advised to follow a paleolithic diet. Control subjects who were advised to follow a Mediterranean-like diet based on whole grains, low-fat dairy products, fish, fruits and vegetables did not significantly improve their glucose tolerance despite decreases in weight and waist circumference [76]. Similar positive results on glycemic control were obtained in diabetic patients when the paleolithic diet was compared with the diabetes diet. Participants were on each diet for three months, whereby the paleolithic diet resulted in a lower BMI, weight and waist circumference, higher mean HDL, lower mean levels of hemoglobin A1c, triacylglycerol and diastolic blood pressure, though levels of CRP were not significantly different [77]. Although the paleolithic diet studies are small, these results suggest that, together with other dietary changes, the withdrawal of cereal grains from the diet has a positive effect on health. Nevertheless, because these studies are confounded by the presence or absence of other dietary substances and by differences in energy and macronutrient intake, factors that could all affect markers of inflammation, it is difficult to make a concise statement on the impact of cereal grains on these health outcomes.

6.4. Rechallenge Trial of Effects of Dietary Gluten

One human intervention study specifically focused on the effects of dietary gluten on inflammation. Biesiekierski et al. [12] undertook a double-blind randomized, placebo-controlled rechallenge trial to investigate the influence of gluten in individuals with irritable bowel syndrome but without clinical features of CD, who reached satisfactory levels of symptom control with a gluten-free diet. After screening the participants, about 50% of the individuals in both the gluten and placebo group were HLA-DQ2 and/or HLA-DQ8 positive. Participants received either gluten or placebo together with a gluten-free diet for six weeks. Endpoints in the study were symptom assessments and biomarkers of inflammation and intestinal permeability. The patients receiving gluten reported significantly more symptoms compared to the placebo group. The most striking outcome of this study was that for all the endpoints measured, there were no differences in individuals with or without HLA-DQ2/DQ8, indicating that the intake of gluten can cause symptoms also in individuals without this specific HLA-profile. No differences in biomarkers for inflammation and intestinal permeability were found between both groups, however, inflammatory mediators have been implicated in the development of symptoms in patients with irritable bowel syndrome [78]. It could therefore be inferred that the markers used to measure inflammation and intestinal permeability were not sensitive enough to detect subtle changes on the tissue level.