The genetic modifications of cereals containing gluten over last 10,000 years for qualitative and quantitative reasons represent an error that is the evolutionary basis for the apparition of diseases that are dependent to the exposure to gluten.
Various forms of gluten intolerance affect a larger and larger number of people and is among the most common patholigies. Speaking only of Celiac disease, among the European population, approximately 1% of adults and children are affected.
Gluten reactivity is not limited to Celiac disease: when neither allergic nor autoimmune mechanisms are involved, the condition is refered to as gluten sensitivity or more correct Non Celiac Gluten Sensitivit(NCGS). NCGS is more common than other types of gluten reactivity. There are no typical markers for identifying gluten sensitivity.
Present in barley, wheat, rye and other grains, gluten is a protein aggregate whose chemical composition is represented by 45% glutenin and 55% gliadin. Not all grains contain gluten. Some examples of gluten-free grains are sorghum, millet, brown rice, buckwheat, wild rice, amaranth, quinoa, corn (polenta) and teff. Oats are also gluten-free, but can be contaminated during processing.
In a water solution gluten forms a visco-elastic reticular form that is elastic and porous, and represents the main structure for baking dough. The main characteristic of gluten is its visco-elasticity, which means that it is material that may alter its original shape.
The pratical applications of gluten include: Increasing the volume of baked goods, retaining starch during the baking process, and slowing the assimilation of starch during digestion.
Gliadins, shown in figure 1(40-50 different molecules) are monomeric proteins with few molecular interactions which gives dough extensibility.
Glutenins are polymeric multi-chain molecules with many molecular interactions which give dough elasticity and consist of both HMW-Hight molecular weight subunits (3-5 different molecules) and LMW-Low molecular weight subunits (16-25 different molecules).
Gliadin is a prolamin containing various amino acids including proline, glutamine, alanine and leucine. The greater the content of proline and glutamine, the greater the toxic effect for the patient with celiac disease or gluten sensitivity. Proline is considered difficult to digest due to the absence of digestive enzymes, namely prolyl endopeptidase (PEP), in the human gut.
Proline is an unessential amino acid synthetized by the human body. It can be synthesized from glutamic acid. The oxidation product, glutamate semialdehyde, cyclizes and can be converted to proline by further reduction.
One Gluten: Many different protiens
Proteins in a single wheat variety, even if they represent only 10-15% of the dry weight, are very heterogeneous with up to 100 different types of protein. Because of this polymorphism, every cultivated wheat variety can be identified based on its reserve protein content (fingerprint).
The allelic polymorphism for gliadins is highly elevated. For example, in some wheat varieties, 25 alleles for alpha gliadin were located on chromosome 6A, 22 alleles on chromosome 6B, and 19 alleles on chromosome 6D.
The toxic component of gluten derives from gliadins and was identified as a peptide of 33 amino acids from alpha gliadin corresponding to the 56-88 region with the following sequence:
- Gluten antigenic epitopes are located in regions rich in proline residue
- Epitopes rich in proline are resistant to gastric and pancreatic juice proteolysis, which causes the accumulation of these toxic peptides in the intestinal lumen (they remain intact throughout the digestive process and even act as potential antigens for T cell proliferation).
- These epitopes among others are the main substrate of tissue transglutaminase.
- Tissue transglutaminase (tTG) catalyzes a deamidation reaction of glutamine residues that results in glutamic acid residues which then results in increased affinity of these epitopes for HLA major histocompatibility complex.
Gliadin-derived peptides have different effects on various systems:
- LCQQQPFPPQQPY causes K538 cells agglutination, apoptosis, and induces morphological changes in celiac patient’s gut, but it is not immunogenic for T cells.
- QLQPFPQPQLPY is immunogenic for T cells but it does not induce changes in the gut
Celiac disease occurs in genetically predisposed subjects. The genes involved in celiac disease are located on the short arm of 6D chromosome and encode the major histocompatibility complex HLA (Human Leukocyte Antigen).
HLA system comprises class I and II genes that are essential for proper immune response. HLA class I molecules, comprising A, B, C genes, are located on the surface of all nucleated cells.
Their function is to bind infectious or tumor protein antigens or parts of them, after they phagocytized, and reduce them into smaller protein fragments, and present them to cytotoxic TCD8+ lymphocytes, which have cytotoxic activity.
Numerous studies have shown that celiac disease is frequently associated with the presence of certain genes in the HLA system encoding DQ2 and DQ8 heterodimers identifiable by DQA1*0501/DQB1*0201 or DQA1*0501/DQB1*0202 and DQB1*0302 alleles.
In celiac disease, the immune reaction is triggered by gliadin. At low a pH, as in the case in the digestive tract, transglutaminase enzyme ( tTG), gliadin is deamidated and converted into glutamic acid-rich peptides with high affinity for DQ2 heterodimer.
Gluten-specific T cells, which are separable in a celiac disease patient’s small intestine, activate and are capable of acting as helper cells for B lymphocytes and produce antibodies in response to gliadin and tTG. Anti-transglutaminase antibodies trigger a destructive immunological response to all tissues containing it. Intestinal mucosa is hardest hit, but damage can also affect other organs such as the nervous system, skin, teeth, liver and pancreas.
DQ2 haplotype was observed in about 95% of celiac patients whereas DQ8 haplotype is present only in about 5% of patients. There is a very small percentage, less than 2%, that show neither DQ2 nor DQ8, as there is about 25-30% of the population carrying predisposing haplotypes without ever developing the disease. Although the presence of these alleles is an important predisposing genetic factor, it cannot be considered the only cause of the disease as there are other genetic and environmental factors contributing to the pathogenesis.
CELIAC DISEASE IN THE PAST
- Characterized by diarrhea and malabsorption
- Triggered by the ingestion of certain grains
CELIAC DISEASE TODAY
- It can occur at any age, > 60% of those diagnosed are adults
- Frequently occurs without gastrointestinal symptoms
- Autoimmune disease with a strong genetic component
Clinical use of serum antibody Test and genetic tests
- Anti-transglutaminase antibodies, IgA and IgG
- Total IgA test
- Anti-endomysial antibodies test (in some cases)
- If positive → duodenal biopsy
- If negative – biopsy is only indicated if the estimated risk is high
- The genetic test may be useful if trying to exclude a diagnosis of celiac disease in case of doubt
Histology: Celiac disease histology
Marsh classification of histologic findings in celiac disease
Marsh classification of histologic findings in celiac disease 1
Marsh classification of histologic findings in celiac disease 2
Figure 2: Photomicrograph from: a) pretreatment duodenal biopsy showing partial villous atrophy and crypt hyperplasia [modified Marsh type 3a (H&E, x200)]; b) follow-up biopsy at 6 months with no histological improvement [modified Marsh type 3a (H&E, x200)]; c) pretreatment biopsy showing subtotal villous atrophy along with crypt hyperplasia [modified Marsh type 3b (H&E, x100)]; d) follow-up biopsy at 6 months with histological improvement [modified Marsh type 1 (H&E, x100)]; e) pretreatment duodenal biopsy showing lymphoid aggregates (H&E, x100) and f) submucosal inflammation (H&E, x200)
Histology is considered the Gold standard for diagnostics, however…
Requirements for a relevant biopsy
- Multiple samples (4-6 samples)
- Samples must taken on an empty stomach from different sites: distal duodenum (D3), intermediate duodenum (D2), proximal duodenum (D1) and duodenal bulb (B)
- Samples must be a appropriate size
- Samples must be stained with CD3 antibodies
- Samples must be evaluated by a pathology expert
A normal intestinal mucosa presents: intestinal villi with a ratio of villous height to crypt length of 3:1, normal height enterocytes of 29-34mm, and intraepithelial lymphocytes below 20 per 100 enterocytes (it is considered pathological above 40 lymphocytes per 100 enterocytes).
Description of the pathological intestinal mucosa is based on the Marsh classification for which Oberhuber proposed a new classification that divides lesion type (villous atrophy) into three subgroups a, b, c
Symptoms of celiac disease may include
- Short stature
- Weight loss
- Abdominal distension
- Mouth ulcers
- Dental enamel defects
- Folate deficiency
Osteoarticular and muscular
- Osteomalacia (softening of bones)
- Muscle cramps
- Peripheral neuropathy
- Dermatitis herpetiformis
Other conditions causing villous atrophy
- Whipple’s disease
- Eosinophilic gastroenteritis
- Autoimmune enteropathy
- Crohn’s disease
- Common immunodeficiency
NON celiac gluten sensitivity or GLUTEN SENSITIVITY
Gluten Sensitivity is characterized by a number of different symptoms, either intestinal or extra-intestinal, that are not attributed to either celiac disease or wheat allergy and disappear within a few hours or days after eliminating gluten from the diet but reappear if it’s the reintroduction of wheat.
So when neither allergenic or autoimmune mechanisms are involved, we have what we call today sensibility to gluten or gluten sensitivity. Although there is insufficient epidemiological data, it seems that this form of the disease is 6-7 times more common than celiac disease.
For example in Italy 1 in every 100 people has celiac disease. Currently 110,000 patients are already diagnosed and under observation, and it is believed that there are about half a million patients that are undiagnosed and thus exposed to the risk of complications.
Among these there are 2800 newborn cases, and 5000 cases of newly diagnosed adults.
Compared to these statistics it is estimated that the gluten-sensitive population is much higher. If certain case of reactivity to gluten understood as celiac disease or wheat allergy, it is well explained by allergic and autoimmune mechanisms, and therefore easy to diagnose. Even today there are no typical markers for identifiying gluten sensitivity.
The diagnostic algorithm for Gluten Sensitivity
In February 2011 the First Consensus Conference on Gluten Sensitivity, held in London for the first time, coded a diagnostic algorithm to clarify the different forms of gluten reactivity: allergy, celiac disease, and sensitivity.
Of utmost importance in diagnosing gluten sensitivity is patient history to determine whether or not the patient has symptoms associated with gluten sensitivity.
Since these symptoms are often overlap with those in the case celiac disease and wheat allergy, the following process should be followed:
Excluding gluten allergy
Classification includes wheat allergy respiratory allergy, prevalent in adults with asthma and rhinitis.
Food allergy by ingestion of wheat and derivatives (commonly found in children) with the following kinds of manifestations: gastrointestinal, urticaria, angio-edema, bronchial obstruction, and atopic dermatitis.
WDEIA (Wheat Dependent Exercise Induced Anaphylaxis) a wheat-dependent anaphylactic reaction mediated by omega 5 gliadin and 1B and encoded by the gene derived from Aegilops speltoides B genome of wheat.
exclusion celiac disease
Celiac disease is an autoimmune disease characterized by the presence of specific serological markers for anti-transglutaminase antibodies (anti-tTG) and anti-endomysial (EMA) antibodies of the immunoglobulin A (IgA). We can rule out celiac disease where results for markers are negative, after having checked the absence of IgA deficiency which can cause false negativity.
In the case of celiac disease, HLA DQ2/DQ8 gene research is indispensable, as the lack of heterodimers DQ2 / 8 consents practically excludes the disease when in doubt.
Here, our test
The absence of intestinal villous atrophy is a sign that we do not have celiac disease.
Another sign is the increase in the number of intraepithelial lymphocytes. These are the first and most sensitive indicator of the effect of gluten on the intestinal mucosa and the most important histological characteristic celiac disease in which the number of intraepithelial lymphocytes increases in the stomach and colon.
The possible presence of anti-gliadin antibodies AGA first generation
IgA antibodies are present in saliva, tears, mucous secretions. These antibodies represent a barrier on the mucous membranes opposed to an agent that threatens the body.
IgGs attack and eliminate external agents. These antibodies are not as relevant in celiac disease as tTG or EMA.
On the other hand, AGA IgG can be of great utility in sensitivity to gluten, however their absence does not eliminate the possibility of a positive diagnosis. It’s good to keep in mind that above normal markers can can occur in non-intolerant subjects such as children, people with gastrointestinal disorders manifest (e.g. diarrhea) and even other pathologies such as irritable colon.
Signs and symptoms of Gluten Sensitivity
The patient who exhibit sensitivity to gluten present:
- Severe abdominal pain (68% of cases)
- Eczema or other skin rash are present in (40% of cases)
- Hemicrania (35% of cases)
- Obfuscation (34% of cases)
- Chronic fatigue (34% of cases) , and as much shows diarrhea.
- Depression is present in 22% of subjects
- At points circulatory anemia in 20% of subjects
- Tingling and loss of sensation distal parts of the limbs, 20% of subjects
- Joint pain 11%
- Heartburn, nausea, vomiting 15% of cases
- Fainting (20% of cases)
- Inflamation of the limbs 10% of cases
- regression symptoms
Patients with gluten sensitivity who eliminate gluten from their diet experience relief all symptoms.
expert nutrigenetics, nutrigenomics and antiaging medicine.
Biogenonco Project coordinator, University of Medicine and Pharmacy Cluj Napoca