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, Gluten Proteases&
chymotrypsin) peptidases of gut
Peptides
Gliadin (one region on gluten)
(peptidase)
Membrane cells, enterocytes
Amino acids
villi
Disruption of tight junction bet. villi

(Only peptides as large 500 Allows larger than 3 a.a to enter circulation
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 cells-
plasma 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 intra-
epithelial 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 long-
standing 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 Kruskal-
Wallis 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) 0.13
0
Crypt depth (µm) 135.1 (124.8, 149.7) 150.9 (134.9, 160.6) 0.07
7
Vh/Cd 2.9 (2.5, 3.1) 2.2 (2.1, 2.9)3 0.01
9
IEL count (x100 22 (18, 24) 27 (23, 34)3 0.00
enterocytes) 2

1
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.
 Groups
Placebo(n=13) 10mg gluten 50mg P
Table 2: (n=13) gluten(n=13)
Marsh-Oberhuber grading of small bowel
histology
Baseline
0 8 8 7 0.263 (3)
1 4 1 5
2 1 4 1
After microchallenge
0 8 8 5 0.433 (3)
1 4 5 5
2 1 0 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)

IEL -4 (-22,42) 0(-14,47) 12 (-8,93) 0.514 (5)

1
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
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 (Kruskal-
Wallis 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.