Development of Screening for Coeliac Disease

Results:

In this practical anti-endomysial antibodies were detected using an indirect immunofluorescence method. The kit used was a NOVA Lite monkey oesophagus IFA kit. Monkey oesophagus slides were used as a substrate for the screening of anti-endomysial antibodies, which can be used as an aid in the diagnosis of coeliac disease.

The procedure principle of the kit used is an indirect immunofluorescence technique where patient samples and appropriate controls are incubated with the money oesophagus slides. The unreacted antibodies (non-endomysial antibodies) are washed off and then the appropriate fluorescein labelled conjugates (IgA FITC conjugate) are added. Unbound conjugate is removed off as before. Slides are visualised with a fluorescence microscope and positive samples (anti-endomysial antibodies present) produce apple-green fluorescence which corresponds to areas of the section where autoantibody has bound.

Serum samples from two patients were tested. The patients were called patient one and two.

The controls used were:

Positive control which was an endomysial (coeliac) positive control (Inova Diagnostics)

Negative control which was IFA system negative control (Inova Diagnostics)

A known positive sample was also used which was obtained from the hospital.

Table 1: Indirect immunofluorescence results for detection of anti-endomysial antibodies

Fig.1: Endomysial antibodies present producing bright apple-green staining pattern “reticulin-like fibres” in the connective tissue around smooth muscle in the monkey oesophagus slide

Discussion:

In this practical, indirect immunofluorescence method was used for the detection of anti-endomysial antibodies. Two patient samples were tested along with a known positive sample and controls.

The control results were the first results to be analysed to ensure that the method and reagents used gave the correct results and therefore ensuring that the test was valid.

Two controls were used, a positive and a negative control. The positive control contained serum derived from human serum which gives a reticulin like endomysial pattern on monkey oesophagus sections. The negative control contained serum that was universally negative for all autoantibodies.

The results of the two controls in table 1, were as follows. The positive control produced expected results where a bright apple-green staining pattern in the connective tissue around smooth muscle which was reticulin-like fibres for the endomysial control. The negative control produced expected results also where dull green staining was observed in the tissue, with no discernible fluorescence. Since the controls met expected results, the test was valid and the patient results could then be read.

A known positive sample was also included in this practical. This was included as an extra positive control. The result of this sample again met expected results where bright-apple green staining pattern was observed in the connective tissue around the smooth muscle.

For the patient results in table 1, patient one had no fluorescence present in the well when viewed under the fluorescent microscope. This meant that no anti-endomysial antibodies were detected and therefore the patient more than likely does not have coeliac disease.

Patient two had immunofluorescence present, where a bright apple green pattern was produced like the one in figure 1. This was interpreted that endomysial antibodies were present. This is diagnostic of the patient having coeliac disease.

Endomysium antibodies were first described by Chorzelski in 1983. Endomysium is a connective tissue found between microfibrils. Endomysium antibody detection in serum is one of the tests carried out for diagnosing coeliac disease.

Coeliac disease is a lifelong autoimmune disorder that primarily affects the small intestine. Typical symptoms include gastrointestinal problems such as chronic diarrhoea, abdominal distension, malabsorption, loss of appetite and among children failure to grow normally. Coeliac disease was first discovered in childhood, however, it may develop at any age. It is connected with other autoimmune diseases, such as diabetes mellitus type 1 and thyroiditis, among others (Fasano, 2005).

Coeliac disease is the result of a reaction to gluten, which are various proteins found in wheat, barley and rye. Moderate quantities of oats free from contamination with other gluten containing grains, are usually tolerated. When gluten is ingested, an abnormal immune response may lead to the production of several different autoantibodies that can affect a number of different organs. In the small intestine, this causes an inflammatory reaction and may produce shortening of the villi lining the small intestine (villous atrophy). This affects the absorption of nutrients, often leading to anaemia.

Diagnosis is usually made by a combination of blood antibody tests and intestinal biopsies, helped by specific genetic testing. The diagnosis of coeliac disease is not always straightforward. Often, the autoantibodies in the blood are negative and many people have only minor intestinal changes with normal villi (Lebwohl, Ludvigsson and Green, 2015).

The only effective treatment is a strict lifelong gluten-free diet, which leads to recovery of the intestinal mucosa, improves symptoms and reduces risk of developing complications in most people. If left untreated, it may result in cancers such as intestinal lymphoma.

There is a strong genetic component in coeliac disease. The vast majority of people (95%) with coeliac disease have one of two types of the HLA-DQ protein: HLA-DQ2 or less commonly HLA-DQ8. HLA-DQ is part of the MHC class II antigen-presenting receptor system and differentiates cells between self and non -self for the purpose of the immune system (Dieli-Crimi, Cénit and Núñez, 2015).

There are seven HLA-DQ variants (DQ2 and DQ4-DQ9). Greater than 95% of people with coeliac have the isoforms of DQ2 or DQ8, which is inherited in families. The reason why these genes produce an increase in risk of coeliac disease is that the receptors formed by these genes bind to gliadin peptides more tightly than other forms of the antigen-presenting receptor. So, these forms of the receptor are more likely to activate T lymphocytes and initiate the autoimmune process. Therefore this genetic association is important as it contributes to the pathophysiology of coeliac disease (van Heel, 2018).

The specific tissue changes and the pathology which is the result of a strong pro-inflammatory cytokines burst within the small intestine that occurs in coeliac disease can be depicted with the Marsh classification.

The Marsh classification was introduced in 1992 by M.N. Marsh as a means to characterise the spectrum of changes seen in the small intestinal architecture under a microscope. The original Marsh description ranged from type 0 to type 4 seen in figure two (Marsh and Heal, 2017).

Fig.2: Marsh classification of the classic pathology changes of coeliac disease in the small bowel.

This classification system has undergone some modifications since then but remains the foundation of diagnosing coeliac disease. Dr. Marsh was the first to understand that there is a spectrum of inflammatory changes that can occur in coeliac disease. Before this classification, it was thought that only the most advanced changes were the hallmark of coeliac disease.

Patients being evaluated for coeliac disease undergo an upper endoscopy to obtain tissue samples called biopsies from the first part of the small intestine known as the duodenum. The individual must be on a diet that contains gluten when these samples are being taken, as gluten is the trigger of intestinal inflammation in coeliac disease.

The architecture of a normal small intestine has finger-like projections of lining cells called epithelial cells. These projections are called villi and are responsible for normal absorption of nutrients including iron. Crevices between the villi are called crypts that contain regenerating epithelial cells. The normal villous length to crypt length ratio is between 3:1 and 5:1. Finally, there should be no more than 30 immune cells known as lymphocytes interspersed between the top layer of villous epithelial cells per 100 cells in a normal small intestine. The currently used Marsh classification spans from Marsh I to Marsh IV with Marsh III being subdivided into IIIa, IIIb and IIIc. The Marsh III category is where the majority of coeliac patients fall into.

The histology of Marsh I comprises normal villous architecture with an increase in the number of intraepithelial lymphocytes. This finding is not coeliac disease specific as it can be seen in other inflammatory and infectious diseases involving the small bowel or drug injury. The histology of Marsh II includes increased intraepithelial lymphocytes along with a finding known as crypt hypertrophy in which crypts appear enlarged.

Originally Marsh III was described as the “destructive” change because it characterised flattening of the villi. Marsh III was later sub-divided into IIIa, IIIb and IIIc to reflect the spectrum of villous atrophy along with crypt hypertrophy and increased intraepithelial lymphocytes. Marsh IIIa classification is partial villous atrophy in which the finger-like projections are partially shortened. Marsh IIIb also called subtotal villous atrophy, has finger-like projections that are visibly shortened but are still recognisable. Marsh IIIc called total villous atrophy is characterised by near total absence of villi. The histology of Marsh IV is called “Hyper plastic” or completely atrophic and describes histology at the extreme end of gluten sensitivity. Here is where lymphoma is more likely to occur (Kupfer, 2018).

Measuring tissue transglutaminase (tTG) antibodies is another test used for diagnosing coeliac disease. TTG is a major autoantigen recognised in coeliac disease. It is also the target for endomysial antibodies. TTG antibodies are an IgA type antibody that are measured using an Enzyme Linked Immunosorbent Assay (ELISA). The level of TTG antibodies correlates with the disease severity. They are also useful in monitoring adherence to a gluten free diet and how well the patient is responding to therapy.

TTG gene is expressed in a wide range of tissues, under complex but tight regulation. TTG is constitutively expressed in endothelial cells, in smooth muscle cells and in some other organ-restricted cells. TTG in coeliac disease has been detected in all layers of the small intestinal wall. TTG is most densely expressed in the submucosa and only 1% of the small intestinal tTG is located within the epithelium. tTG is not expressed in Crypt cells, but the intracellular expression increases as the cells maturate and migrate toward the small intestinal villi (Molberg, McAdam and Sollid, 2000).

The role of tTG in coeliac disease is that tTG is upregulated in intestinal inflamed sites and tTG may produce additional antigenic epitopes by cross linking gliadin peptides to itself and/or to other protein substrates. Complexes of TTG-gliadin bind to tTG-specific B cells, are endocytosed and then processed. Complexes of Gliadin-DQ2 are then presented by the tTG-specific B cells to gliadin-specific T cells which provide the necessary aid to produce anti-tTG antibodies. It is not proven the existence of tTG specific T cells in the intestinal mucosa of untreated patients, however, it is thought that the production of anti-tTG antibodies is driven completely by intestinal gliadin-specific T cells. It is observed that anti-tTG antibody titres fall and can become undetectable during a gluten free diet suggesting that B cell activity depends on persistent antigen presentation.

An explanation of how tTG generates neo-antigens in coeliac disease has been proposed is that tTG undergoes a large-scale structural rearrangement in its closed (GTP-bound) and open (Calcium activated) conformations. In a stress-free normal environment, most extracellular tTG is predominantly in a closed conformation despite relatively high extracellular calcium concentrations. Some signals from the innate immune system can trigger rapid activation of tTG into its catalitically active, open conformation that exposes self-epitopes that are ordinarily inaccessible to the immune system. These so called “neo-epitopes” trigger an autoantibody response (Caputo et al., 2008).

The sensitivity and specificity of measuring tTG antibodies and their importance in coeliac disease diagnosis was assessed in a study (Troncone et al., 1999), where the sensitivity and sensitivity values of IgA and IgG antibodies to tTG in the diagnosis of coeliac disease compared with endomysial antibodies were assessed. The method used was an ELISA.

It was found that the sensitivity of IgG anti-tTG was significantly lower (21%) than the sensitivity of IgA anti-tTG, the specificity was 98%. The concordance rate between IgA antiendomysium and IgA anti-tTG was very high (95%).

Both anti-tTG IgG and IgA were normal or significantly lower in patients receiving a gluten free diet. In patients treated with coeliac disease, IgA antibodies disappeared more rapidly than IgG after gluten was excluded from the diet.

In patients challenged with gluten, IgA anti-tTG levels began to rise as early as seven days after gluten was introduced, paralleling with a pattern already observed with antiendomysium antibodies. In a few cases this increment was accompanied by an increase of anti-tTG IgG. There was good correlation with antiendomysium.

The conclusion from this study was that tTG based ELISA is an effective method of diagnosing coeliac disease. However, the results of the tTG ELISA method were compared with immunofluorescence based assays, where it was found that those assays were more sensitive, especially in the gluten challenge (Troncone et al., 1999).

In conclusion to this practical where the detection of anti-endomysial antibodies was performed using indirect immunofluorescence, it was found that this was an effective diagnostic test for coeliac disease. From analysing the results from the study regarding the sensitivity and specificity of the tTG antibodies, it was also found that immunofluorescence was a more sensitive method than a tTG based ELIZA.

References:

• Caputo, I., Barone, M., Martucciello, S., Lepretti, M. and Esposito, C. (2008). Tissue transglutaminase in celiac disease: role of autoantibodies. Amino Acids, 36(4), pp.693-699.
• Dieli-Crimi, R., Cénit, M. and Núñez, C. (2015). The genetics of celiac disease: A comprehensive review of clinical implications. Journal of Autoimmunity, 64, pp.26-41.
• Fasano, A. (2005). Clinical presentation of celiac disease in the pediatric population. Gastroenterology, 128(4), pp.S68-S73.
• Kupfer, S. (2018). Making sense of Marsh. [online] Cureceliacdisease.org. Available at: https://www.cureceliacdisease.org/wp-content/uploads/0909CeliacCtr_News_v3final.pdf [Accessed 2 Dec. 2018].
• Lebwohl, B., Ludvigsson, J. and Green, P. (2015). Celiac disease and non-celiac gluten sensitivity. BMJ, pp.43-47.
• Marsh, M. and Heal, C. (2017). Evolutionary Developments in Interpreting the  Gluten‐Induced Mucosal Celiac Lesion: An  Archimedian Heuristic. Nutrients, 9(3), p.213.
• Molberg, Ø., McAdam, S. and Sollid, L. (2000). Role of Tissue Transglutaminase in Celiac Disease. Journal of Pediatric Gastroenterology and Nutrition, 30(3), pp.232-240.
• Troncone, R., Maurano, F., Rossi, M., Micillo, M., Greco, L., Auricchio, R., Salerno, G., Salvatore, F. and Sacchetti, L. (1999). IgA antibodies to tissue transglutaminase: An effective diagnostic test for celiac disease. The Journal of Pediatrics, 134(2), pp.166-171.
• van Heel, D. (2018). Recent advances in coeliac disease. Gut Review. 55 (7): 1037-46.