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Celiac disease Celiac disease (CD) is an immune – mediated enteropathy. It’s a chronic (long-term) digestive disease in which patients have inflammation or irritation in the small intestine, which causes difficulties with absorbing nutrients from the diet. Patients with CD often have other family members with the condition and are therefore susceptible to this disease. Inflammation in the bowel occurs when a patient with CD begins to eat food that contains gluten. Gluten is the name given to certain types of proteins found in wheat, barley, rye and related grains When food containing the gluten protein arrives in the small bowel, the immune system reacts against the gluten, causing an inflammatory reaction in the wall of the bowel.

Gluten Gluten is a composite of gliadin and glutenin. These exist in conjoined with starch, in the endosperm of some grass related grains notably wheat,rye and barley. Gluten as an additive Gluten is used as stabilizing agent in products like ice-cream and ketchup. Foods of these kind may present as a problem because the hidden gluten constitute a hazard for the people with celiac disease.

Hidden gluten and its significance Celiac patients need to learn how to recognize the hidden sources of gluten.

Food Products like malt syrups, salad dressing, soup mixes, bakers yeast, vegetable gums, seasonings, soy sauce, beer, cocoa, some food starches shows the presences of gluten.

Non-Foods Products like stamps and envelop, medicines, lipstick lip gloss,verbal and vitamin supplements.

Cross Contamination as in using the same knife contaminated with bread crumbs while butter it. Many overseas countries have different levels of acceptable gluten to label their products "gluten free". Australia's acceptable level is less than 0.003% (30 parts per million - which is the smallest quantity that current testing can identify) Europe is allowed less than 0.02% ( 200 parts per million) to label the products as gluten-free.

Pathogenesis of celiac disease 1)Genetics: There is strong evidence for an inherited predisposition CD. This inherited susceptibility is closely associated with HLA B8, HLA-DR3, HLA-DQ alleles. The reason 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. Therefore, these forms of the receptor are more likely to activate T lymphocytes and initiate the autoimmune process in CD.

2) Prolamines: Term prolamin given for the extracts of gliadin. Peptides such as 33-MER, cannot be broken down any further. In people with celiac disease, 33-MER stimulates T-cells to produce antibodies. The antibodies, in turn, attack the villi in the small intestine, reducing their ability to absorb nutrients.

Digestion of proteins Normal conditions

gluten sensitive gut

Proteins (pepsin, trypsin, chymotrypsin)

Peptides (peptidase) villi

Amino acids

Gluten

Proteases& peptidases of gut

Gliadin (one region on gluten) Membrane cells, enterocytes Disruption of tight junction bet. villi

Allows larger than 3 a.a to enter circulation (Only peptides as large 500 Daltons(4 a.a residue length can cross.) Damage to the intestinal mucosa Decrease digestive &absorptive functioning

3) Tissue tranglutaminase Persons in whom C. D is suspected are screened using serological tests, which include the presences of antiendomysial antibodies (AEAs), (IgA), IgG-AGA antibodies or IgA tissue transglutmainase. Tissue transglutaminase modifies gluten peptides into a form that may stimulate the immune system more effectively. Anti-transglutaminase antibodies to the enzyme tissue transglutaminase (tTG) are found in an overwhelming majority of cases. .

4) villous atrophy and malabsorption: The inflammatory process, mediated by T cells, leads to disruption of the structure and function of the small bowel's mucosal lining.This leads to malabsorption of nutrients. There is a loss of villous height,elongated crypts resulting in reduced thickness of mucosa.Within lamina propria there is a marked infiltration of inflammatory cellsplasma cells and Lymphocytes.

Pathophysiology of celiac disease

Symptoms of Celiac Disease The symptoms or signs of disease will depend on how much and how badly the intestine is inflamed. The most common symptoms are: Bloating and gas ,Abdominal pains ,Diarrhea ,Stools that may float or smell very bad, Weight loss , Poor growth or weight loss in children, Anemia Other symptoms are: Feeling weak, Tiredness Low vitamin levels - especially iron, calcium and folate, Bone and joint pains , Osteoporosis , A skin rash that lasts(dermatitis herpetiformis)

Diagnosis in celiac disease Blood test Serological blood tests are the first-line investigation required to make a diagnosis of coeliac disease Serum antibodies to gliadin(IgA isotype),IgA antibodies to reticulin and IgA endomysial antibodies ,anti gliadin anti have high specificity and sensitivity for the disease. Serum IgG class AGA and IgAclass anti t-TG are measured by standard enzyme-linked immunosorbent assay methods(ELISA)

Endoscopy Establishing a firm diagnosis of CD requires taking biopsy samples of the small bowel using endoscopy. An upper endoscopy with biopsy of the duodenum or jejunum is performed. Until the 1970s, biopsies were obtained using metal capsules attached to a suction device. The capsule was swallowed and allowed to pass into the small intestine. After X-ray verification of its position, suction was applied to collect part of the intestinal wall inside the capsule. This method has now been largely replaced by fibre-optic endoscopy, which carries a higher sensitivity and a lower frequency of errors. Small –bowel biopsy remains a golden standard for measuring prevalence of celiac disease.

Pathology The classic pathology changes of coeliac disease in the small bowel are categorized by the "Marsh classification” Marsh stage 0: normal mucosa Marsh stage 1: increased number of intraepithelial lymphocytes, usually exceeding 20 per 100 enterocytes Marsh stage 2: proliferation of the crypts of Lieberkuhn Marsh stage 3: partial or complete villous atrophy Marsh stage 4: hypoplasia of the small bowel architecture. The changes classically improve or reverse after gluten is removed from the diet.

Treatment for celiac disease. It doesn’t require any surgery treatment.. Avoiding all foods that contain gluten treats celiac disease. Nutritional supplements may be necessary at the start of treatment. because of mucosal damage and loss of absorption of nutrients. Intestinal biopsy must be obtained to check for histological recovery. Hidden gluten sources can often be found by reading labells carefully, enquiring the food which is been ordered, or by consulting skilled dietician.

Bacterial Endopeptidase A new treatment for celiac disease is been formulated. Recently scientist identified a 3 a.a peptide fraction from 266a.a gliadin that triggers the destructive inflammatory response in celiac disease(Shan et al 2002). It appears that this peptide fraction is resistant to digestion by normal human digestive enzyme,but it can be degraded by bacterial endopeptidases. In preliminary studies, destruction of peptide fragment by addition of endopeptidase enzyme prevented the typical immunological response seen in CD. The hope is that oral endopeptidase enzyme can be used as as oral supplement to digest and destroy the specific fragment of gliadin in foods that contains gluten. Thus allowing their consumption.

Prevalence in India The prevalence of celiac disease (CD) in India is not known as large epidemiological studies are not available, but a study from northern India suggests prevalence similar to Western countries. Study were conducted among school children from north India It showed frequency of 1 in 310.(journal of gastroenterolgy & hepatology 2006) Therefore it clearly shows the CD is not rare in wheat-eating areas of North India. C D has world wide distribution , but there are significant variations in prevalence, ranging from 1:300 in Ireland to 1:3500 Finland. It has extremely rare in blacks, Chinese & Japanese. Peak prevalence in women occurs bet. 35 – 44 yrs of ages.

A prospective, double-blind, placebo-controlled trial to establish a safe gluten threshold for patients with celiac disease (Carlo Catassi, Elisabetta Fabiani, Giuseppe Iacono, Cinzia D’Agate, Ruggiero Francavilla, Federico Biagi, Umberto Volta, Salvatore Accomando, Antonio Picarelli, Italo De Vitis, Giovanna Pianelli, Rosaria Gesuita, Flavia Carle, Alessandra Mandolesi, Italo Bearzi and Alessio Fasano)

Back ground: treatment of celiac disease (CD) is based on the avoidance of gluten-containing food. However, it is not known whether traces amounts of gluten are harmful to treated patients.

Objective: The objective was to establish the safety threshold of prolonged exposure to trace amounts of gluten(contaminating gluten).

Study design •This was a prospective, multicentre, placebo-controlled, double-blind, randomized trial performed between the years 2001 and 2004. •The patients were 49 adults with biopsy proven CD who had consumed a GFD for 2 yrs. •Patients were excluded if they 1)were younger than 18y 2)were poorly compliant with the GFD 3)were positive for anti-tissue transglutaminase(tTG)antibodies or had villous height /crypt depth(Vd/Cd)<1.5 at baseline(t0) 4)associated conditions such as selective immunoglobulin A(IgA)deficiency or other autoimmune diseases. • Patients qualifying for the study were interviewed.

• Controlled subjects were adults who were negative for serologic CD markers and for Helicobacter pylori(urea breath test) and were undergoing upper endoscopy for diagnostic purpose. •Patients qualifying for trial underwent a screening and a dietary interview(t-1).They were asked to maintain strict GFD during the study period. The only cereal based food they were allowed to eat was the special GFD products on the market in Italy, which Italian law established as having a gluten contamination of <20ppm(20ppm=20mg/kg product. • After a month the subjects returned for a baseline evaluation which involved: clinical examination, a dietary interview, blood collection for serum anti-tTGantibody, antigliadin antibody measurements, an endoscopy and small intestinal biopsy.

• While still adhering to a strict GFD, the patients were randomly assigned to ingest daily 90d ; a capsule containing either 10mg purified gluten, 50 mg purified gluten, or 50 mg corn starch as a placebo. • After completing 3 month microchallange (t1),patients repeated the same clinical, serologic, and histologic tests as at t0. • If any patient had symptoms suggestive of CD relapse during the microchallange, the protocol was stopped and the patient was asked to perform the t1 evaluation before out of the study. •The study protocol was approved by the Ethical committee of the Universita Politecnica delle Marche, Ancona, Italy.

Methodology •Purified gluten was used for the preparation of the capsules. •Whole gluten was used rather than single gluten fractions because it has been shown that not only gliadins, but also glutenins contain toxic epitopes. •Gelatin capsules that would quickly dissolve in the stomach were prepared by the pharmacy of the coordinating center •On a dry weight basis, the capsules contained 10mg raw gluten, 50mg raw gluten, or 50 mg corn starch(placebo). •All the testing and biopsy specimens were analyzed at Universita Politechnica delle Marche. •Serum IgG class AGA and IgA class anti-tTG were measured by standard enzyme linked immunosorbent assay methods.

•Small-bowel biopsies were taken from the second part of duodenum. All biopsy specimens were oriented and fixed in 10% formalin, embedded in paraffin wax. •The sections were stained with hematoxylin and eosin and immunostained by using anti-human CD3 antibody to enhance diagnostic accuracy in counting intraepithelial lymphocytes(IEL) •The specimens were examined by 2 pathologists with longstanding experience in morphometric analysis,who were blinded to subject assignment. •The morphometric analysis of the sections was performed on 10 well oriented villi, arranged like fingers and 10 well oriented crypts, arranged in perpendicular to the muscolorais mucosae by a computerized image analyzer.

•The following variables were evaluated: Vh/Cd and IEL counts •The numbers of IEL was calculated by computing their mean value out of 1000 entercytes and expressed as a percentage of enterocytes in the area analyzed. •The overall architecture of the small-intestinal mucosa was also evaluated according to the March-Oberhuber classification.

Analysis of the background gluten intake •The consumption of gluten free products was measured in a different samples of 46 adults with CD who had been consuming a GFD long term(>2yrs). • These subjects were asked to report the type and the amount of all the special gluten free products consumed during 30 consecutive days in a diary. •A random sample of 42 gluten free products consumed by these subjects was collected and analyzed for gluten contamination by enzyme linked immunosorbent assay with a sensitivity limit of 3ppm of gluten.

Statistic analysis •Considering a 20% dropout rate, 49 patients were recruited. •The normality of the distribution of the examined variables was assessed by using the Shapiro-Wilk test. •Nonparametric methods were used for the statistic analysis of the data because the morphometric variables were not normally distributed. •A nonparametric analysis of variance(kruskal-Wallis test) was performed to evaluate the treatment groups at t0. •Wilcoxon’s test was used to compare pooled CD patients with control subjects at t0. •The correlation between Vh/Cd and IEL was analyzed by Spearman’s coefficient. •The comparisons between 3 groups were performed by the KruskalWallis test.

Result •49 subjects were enroled in the microchallenge study:37 women and 12 men with a median age of 30.5 y(19.8-55.4) •After t0, 7 of the 49 patients had to be excluded because Abnormal small intestinal histology Development of thyroid carcinoma Gastric polyposis confirmed by gastroduodenoscopy Subjects refusal on randomization •3 other cases did not complete the micro challenge because Change of residence, Poor adherence to protocol,Development of symptoms •39 patients completed study protocol.Of these 13 were challenged with placebo, 13 with 10mg gluten and 13 with 50 mg gluten. •The controlled subjects were 20 adults.

Clinical and serologic evaluation: •One patient challenged with 10mg gluten/d showed typical signs of relapse after 6-8 wk of microchallenge, but refused to repeat the t1 evaluation. •The comparison between baseline and post challenge findings in 39 patients completing microchallenge did not show any significant changes in the clinical outcome between 3 grps. •In all patients IgA anti TG and IgG antigliadin antibody values were normal range, both at baseline and after micro challenge. •No significant difference was detected between 3 groups at baseline(kruskal-wallis test; p=0.30 and p=0.45) •After the micro challenge the IgA anti TG did not show a significant change(p=0.12)while IgG AGA showed a significant decrease in the grp challenged with 50 mg of gluten in comparison with placebo.(p=0.04)

Results: Clinical serologic evaluation:

FIGURE 1. Median and interquartile concentrations of serum immunoglobulin A (IgA) class anti-tissue transglutaminase (tTG) and IgG class anti-gliadin (AGA) antibodies before ( ) and after ( ) the gluten challenge in the 3 study groups: placebo (n = 13), 10 mg gluten/d (n = 13), and 50 mg gluten/d (n = 13). No significant differences at baseline and no significant increase after the microchallenge were detected (Kruskal-Wallis test). The dashed line represents the normal cutoff. AU, arbitrary units.

Small- bowel biopsy results: •The small intestinal morphometric measures in the CD patients and control subjects at baseline showed:  Vh/Cd ratio was significantly lower in CD patients than in control subjects. IEL count was significantly higher in CD patients than in control subjects. •In the pooled CD group (n=39), the correlation between the Vh/Cd and the IEL count was weak and not statistically significant at either baseline (p=0.06) or after the microchallenge (p=0.07). •After the microchallenge, the Vh/Cd index improved in (fig.2) 11 of 13 subjects in placebo 6 of 13 subjects in 10mg group 2 of 13 subjects in 50 mg grps

•A significant improvement in the median percentage variation betweent1 and baseline of Vh/Cd index was observed in placebo group but not in the 10mg and 50 mg group. •Conversely at t1 the Vh/CD index showed a significant decrease in group challenged with 50mg gluten •The kruskal-Wallis test showed a significant differnce in the percentage in Vh/CD between placebo and 50 mg group(0.029 •An increase in IEL was observed in only 5 of 13 subjects in placebo 6 of 13 subjects in 10mg group 8 of 13 subjects in 50 mg group •However no significant difference in percentage change in IEL count were found between 3 groups.

TABLE 1 Intestinal morphometric evaluation of the subjects before and after the gluten microchallenge1 Control subjects (n CD patients at baseline (n = 20) = 39) P2 Villous height (µm)

372.7 (339.5, 385.4)

354.3 (318.0, 380.5)

Crypt depth (µm)

135.1 (124.8, 149.7)

150.9 (134.9, 160.6)

2.9 (2.5, 3.1)

2.2 (2.1, 2.9)3

22 (18, 24)

27 (23, 34)3

Vh/Cd IEL count (x100 enterocytes)

0.13 0 0.07 7 0.01 9 0.00 2

All values are medians; 95% CIs in parentheses. CD, celiac disease; Vh/Cd, villous height/crypt depth; IEL, intraepithelial lymphocyte. 2 The Wilcoxon test was used for the comparison between groups. 3 Significantly different from the control subjects, P < 0.05. 1

Groups Placebo(n=13)

10mg gluten (n=13)

50mg gluten(n=13)

P

0

8

8

7

0.263 (3)

1

4

1

5

2

1

4

1

0

8

8

5

1

4

5

5

2

1

0

3

Table 2: Marsh-Oberhuber grading of small bowel histology Baseline

After microchallenge 0.433 (3)

Intestinal morphometric results at baseline (4) Villous height (µm)

354 (315,437)

318 (306.366)

380 (266,396)

0.431 (5)

Crypt depth (µm)

150 (134,165)

161(124,187)

131(110,159)

0.197 (5)

Vh/Cd

2.2 (2.1,3)

2 (1.8,3.4)

2.4 (2.2,3.2)

0.273 (5)

IEL count( * 100 entrecytes)

26 (23,45)

29 (23,46)

27 (17,48)

0.968 (5)

Morphometric changes after the microchallenge (%) Villous height

5 (-5,13)

9(-9,19)

-17 (-30,23)

0.198 (5)

Crypt depth

-2 (-16,9)

3(-28,21)

3 (-17,24)

0.693 (5)

Vh/Cd

9 (3,15)

-1(-18,68)

-20 (-22, -13)

0.029 (5)

-4 (-22,42)

0(-14,47)

12 (-8,93)

0.514 (5)

IEL

n = 39. Vh/Cd, villous height/crypt depth; IEL, intraepithelial lymphocyte. 2 0 = normal, 1 = infiltrative lesions (increased IEL count), 2 = hyperplasic lesions (increased IEL count and increased crypt depth). 3 Fisher’s exact test. 4 All values are medians; 95% CIs in parentheses. 5 Kruskal-Wallis test. 6 Significantly different from placebo, P < 0.05 1

FIGURE 2. Individual results of the small-intestinal morphometric evaluation before and after the 3-mo gluten microchallenge (MC) in the 3 study groups: placebo (n = 13), 10 mg gluten/d (n = 13), and 50 mg gluten/d (n = 13). A significant difference in villous height/crypt depth (Vh/Cd) was observed between the placebo and the 50-mg groups (KruskalWallis test, P = 0.029). No significant differences in intraepithelial lymphocyte (IEL) ratio were detected between the 3 groups.

Analysis of background gluten intake •The daily (+/- SD)of commercially available gluten-free products was 332+/-98g(range:177-574g) in 46 adults being treated for CD. •The median gluten content of the 42 gluten free products analyzed was 5.2ppm(ppm = mg/kg)(range:0-50ppm). •On the basis of these values, we estimated that the background gluten intake from the GFD followed by our patients during the micro challenge study was <5mg/d.

Discussion •The evaluation of Vh/CD index and IEL count is reference method for quantitive assessment of gluten-induced damage in CD. •The IEL count is considered to be the most sensitive index of celiac disease. •In our study the analysis of the effects of gluten microchallenge indeed suggested that the Vh/Cc index reflected more closely the damaged induced by traces of gluten in the diet than did the IEL count. •Decreased median Vh/Cd and increased median IEL count showed evidence of histologic damage in adult CD patients. •These findings has been shown to be related to the ongoing ingestion of gluten, causing persistent inflammation in the small intestinal mucosa.

•This interpretation is supported by our results because we noticed a significant improvement in intestinal architecture in placebo after 3 months. •On the other hand it conformed that neither IgA anti TG nor IgG AGA showed a correlation with mucosal changes found in some patients, confirming that these serologic makers are not sensitive enough to detect the residual enterpathy that cant be found in apparently healthy CD patients receiving treatment with a GFD. •The gluten microchallenge disclosed large interpatient variability in the sensitivity to gluten traces. Some CD patients showed a damaged intestinal architecture after ingesting only 10mg gluten/d, whereas others had an apparent improvement in mucosal histology after the 3 month challenge with 50 mg gluten/d. •Despite this wide individual variability,which must be taken into account when implementing threshold that is safe for all patients, we showed that 50 mg gluten/d if introduced for 3 months was sufficient to cause a significant decrease in Vh/Cd index.

•The effects of a low gluten intake in CD patients have been investigated in a limited number of studies. •Ciclitra et al analyzed the gliadin toxicity in single patient.They concluded that 10mg produces no change,100mg a very slight measurable change,500mg a moderate change,and 1 g extensive damage to small intestine morphology •Ejderhamn et al showed that a daily intake of 4-14 mg gliadin did not affect the morphology of the small bowel mucosa in CD patient. •Recent Finnish studies indicate that an intake of 20-36mg gluten/d has no detectable effect on mucosal histology. •Finally, a higher gluten intake(1-5g/d),still lower than the normal gluten intake for the non CD population in western countries,caused relapse of diseases at a clinical, laboratory. •On the basis of the evidence from the current study it appears that 50mg gluten/d id the minimum dose required to produce measurable damaged to the small intestinal mucosa in CD patients.

•A more prudent value of 20ppm has been adopted in north American and southern European countries. •The decision about what threshold is depends,however not only on the minimum toxic dose but also on the amount of gluten-free product consumed. •Our result indicate that 200ppm is not a safe threshold because the harmful gluten intake of 50mg/d could be reached even with a moderate consumption(250g/d) of nominally gluten free products. •The threshold of 20ppm keeps the intake of gluten well below the amount of 50 mg/d,which allows a safety margin for the variable gluten sensitivity and dietary habits of patients.

Limitations •Because of the limited number of patients,we were not able to reach firm conclusions about the potential toxicity of 10mg gluten/d. •For ethical reasons duration of the microchallenge was limited to 2 months

Conclusion •This study confirmed that as abnormal small-bowel morphology persisted in a significant proportion of CD patients being treated with a GFD, most likely because of the persistent ingestion of trace amounts of gluten •The protracted intake of 50mg gluten/d produces significant damage in the architecture of the small intestine. •Hence it is necessary to set a safe gluten threshold. •These findings should be confirmed by further studies in larger numbers of CD patients.