Pooja

1. Characterization of Tight Junction Proteins in
Patients with Crohn's Disease and Celiac
Disease
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2. Background
Genetic • Autoimmune Disease
predisposition
(host factors)
Environmental
trigger
(exogenous
factors)
Immune
response
(host factors)
How this antigen enter?
Skin
Lung
Intestine
Eyes
Tissue damage
Amplified cycle of inflammation,
allow further leakage of foreign Ag
Th1
Th2
Foreign Ag entry
through barrier defect
APC
T cell
TNF-α
IFN-γ

3. Intestinal mucosal barrier system
• The extrinsic barrier consisting i.e. mucus, bicarbonate, hormones,
cytokines prostaglandins
• The intrinsic barrier is composed of the epithelial cells lining i.e.
Junctions and channels
• Paracellular pathway (junction)
• Transcellular pathway (channel)
Paracellular pathway regulated by 4 specific junction
• Tight Junction
• Adherens junction
• Desmosomes
• Gap junction

4. TJs regulate intestinal permeability
• Permeability is a process where molecules are allowed to
pass through the epithelial lining by non mediated diffusion
• Barrier properties of the intestinal epithelium are regulated by
TJs. It is generally believed that disease related, increase in
IP is caused by defects in TJ structure
No disease Disease
Normal TJ
regulate normal IP
Disrupted TJ
leads to increase IP

5. Why to study Tight Junction ?
PARACELLULAR
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TRANSCELLULAR
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Paracellular permeability between the epithelial
cells (gate function )
Maintenance of apical basolateral cell polarity
(fence function)

6. Tight Junction Proteins
Name of Proteins Known Partner
Claudin family ZO-1
Occludin ZO-1, ZO-2,Vap33, Actin
JAM family ZO-1, MUPP1
ZO family
ZO-1
ZO-2
ZO-3
Occludin, Claudin , ZO-2-3, Cingulin, Actin & ZONAB
ZO-1, Actin
ZO-1, Occludin
Ras, Zo-1
Tricellulin
AF-6
DLg (Drosophila)
Scribble(Drosophila)
Cingulin Occludin, ZO-1-2, myosin
Symplekin
ASIP/Par3 PKC-a
Rab3b
Rab13 d-PDE
Rab8 G/C Kinase, exocyst
Sec6, Sec8

7. Tight Junction proteins
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
Scaffolding
Pore forming
Sealing
Cell migration
Cell polarity

8. Distribution of Tight Junctions
Villous
crypt
• At the tip of the villi the TJs are smaller in size but larger in number and
more accessible
• At the crypt the TJs are larger in size but less in number and less accessible
• Small molecules (mannitol , 6.7 Ao) = Villous
• Larger molecules (lactulose, 9.5 Ao) = Crypt

9. TJ proteins in celiac disease
↓ ZO-1 in celiac patients (n=10) which normalized 10 months
after treatment (Montalto 2002)
↓ ZO-1 in celiac patients (n=20) which reverse after
treatment along with increased IP (Pizzuti 2002)
↓ZO-1 and occludin , on exposure to gliadin in vivo and vitro,
zonulin signaling gets activated, leading to increased IP
(Drago 2006)
↓ZO-1 and occludin both in in vivo and vitro (Ciccocioppo
2006)
↑ of Claudin- 2 & claudin-3 in naïve celiac patients (n=33)
responsible for increased IP (Sapone 2011)
Alteration in TJ proteins leads to increased IP, which improves after
GFD( gluten free diet)

10. TJ proteins in Crohn’s disease
• ↓ of Occludin in colonic epithelial cells of active IBD( UC and
CD) play a role in enhanced paracellular permeability
(Kucharzik 2001)
• ↑ of Claudin- 2 and ↓ claudin 3 &4, which have different
effect of cytokines and leads to increased IP (Shyam Prasad
2005)
• ↑ of Claudin- 2 and ↓ claudin 5 &8 leads altered TJ structure
leads barrier dysfunction (Zeissig 2007)
• ↑ claudin-1 and claudin-2 expression may be involved at
early stages of transformation in IBD-associated
neoplasia( Weber 2008)
Alteration in TJ proteins along with increased IP

11. IP in celiac and Crohn’s disease
 Increased IP has been implicated in the pathogenesis of Crohn’s
disease, celiac disease
 Increased IP in celiac patients is 60-80%
 Studies for increased IP in case of Crohn’s disease is 36-40%, while
our study is also reported as 36% abnormal IP in Crohn’s disease
patients
 Abnormal IP in patients with celiac disease is because of increase
in mannitol, while high lactulose in those with Crohn’s disease
Vogelsang H, Oberhuber G, Wyatt J 1996, Benjamin J, Govind K Makharia 2008

12. Lacunae
 Lack of literature on the differences in tight junction
proteins in patients with Crohn’s disease and celiac
disease
 Lack of knowledge on whether there is a difference in
the expression of tight junction proteins in those with or
without increase in intestinal permeability
 Lack of literature on the effect of treatment in the tight
junction proteins expression in Crohn’s disease and
celiac disease

13. Differential expression of
TJ proteins in CD and
celiac may be attributed to
TJs disintegrity
Leads to ↑ IP, implicated in
pathogenesis of CD and
celiac disease
Restoration of TJP & IP,
may improve course of
CD and Celiac disease
Hypothesis

14. Objectives
Primary objective
 To study the expression of tight junction proteins (Zo-1, Claudin 2, 3
& 4, Occludin and JAM) using Immunohistochemistry in patients
with Crohn's disease, celiac disease and controls
 To study the ultrastructural changes of tight junction and to note the
changes of other junctions of a paracellular pathway by electron
microscopy
Secondary objective
 To study the effect of treatment on the expression pattern of tight
junction proteins examined, as well as to see if any ultrastructural
improvement has taken place post-treatment, in those with celiac
disease and Crohn's disease

15. Methodology

16. Patients
Crohn’s disease
• Diagnosis
ECCO guidelines
• Activity
CDAI score
• Location & Behavior
Montreal classification
Celiac disease
• Diagnosis
ESPGHAN criteria
• Pathologic changes (Activity)
Modified Marsh classification
ECCO, Gut, 2006
Best et al 1976
Silverberg et al 2005
ESPGHAN, Arch Dis Child 1990
Oberhuber 1999

17. Study criteria
Disease Inclusion criteria Exclusion criteria
Crohn’s disease 1. Age: 12 to 65 years
2. Both genders
3. Active disease
(Crohn’s disease
activity index >150-450)
1. Severely sick patients
2. Severe active disease (CDAI>450)
3. Massive bleeding
4. Those on active NSAID use
5. Patients with small intestinal resection
6. Ulcerative colitis
7. Indeterminate colitis
8. Intestinal tuberculosis
9. Patients with chronic or acute renal failure
10. Patients with intestinal perforation
11. Subject with other concomitants diseases
12. Pregnant and lactating mothers
13. Unwilling patients
Celiac disease 1. Age: 12 to 65 years
2. Both genders
3. Treatment Naïve
1. Patients who have received gluten free diet
earlier
2. Those on active NSAID use
3. Subject with other concomitants diseases
4. Pregnant and lactating mothers
5. Unwilling patients
6. Patients with tropical sprue

18. Selection of controls
Patients with functional dyspepsia, age (12-65)
of both gender served as controls, with no
alarming signal
 Diarrhea
 Anemia
 Normal liver function test
Use of NSAID 15 days prior to study
Negative for celiac serology
Normal UGI endoscopy and normal histology

19. Work Algorithm
Patient with Crohn’s disease, celiac disease and controls recruited from the
Gastroenterology OPD of AIIMS
Inclusion and exclusion criteria
Informed consent taken
Diagnosis
•ECCO guideline for Crohn’s disease
•ESPGHAN criteria for celiac disease
Biochemical test, IP test, UGI endoscopy followed by Bx in CD, celiac and Controls
Duodenal Biopsy
Immunohistochemistry
•ZO-1
•Cld 2,cld-3 & cld-4
•Occludin,
• JAM
Histopathology Electron Microscopy
After 6 month follow up by clinician, we repeated all the
test in patients with celiac and Crohn’s disease
TJ protein
expression
Ultrastructural
changes
Effect of treatment
Objectives

20. Patients Enrollment
Crohn’s disease
Screened (n=50)
Excluded (n= 22 )
•Over age (n=1)
•Active bleeding (n=11)
•Co morbidity (n=5)
•Refused to participate (n=5)
Included (n=28)
Active Crohn’s disease
Follow-Up
Lost to follow up=8
Post treatment (n=20)
Celiac disease
Screened (n=80)
Excluded (n= 56 )
• Already on GFD (n=20)
• Co morbidity (n=31)
• Refused to participate (n=5)
Included (n=24)
Treatment naïve celiac
Follow-Up
Lost to follow up=3
Post treatment (n=21)

21. Analysis TJ structure and TJ protein
• TJ protein expression
– Immunohistochemistry, RT PCR, Western blotting
• Functional analysis of TJ
– IP by High performance liquid chromatography (HPLC), Ussing
chamber
• Ultrastructural of TJ
– Transmission electron microscopy (TEM), freeze fracture
Celiac, Crohn’s disease and
controls
TJ protein expression
Immunohistochemistry
(IHC)
Functional analysis of TJ
High performance liquid
chromatography(HPLC)
Ultrastructure of TJ by
Transmission electron
microscopy (TEM)
After 6 month test only in Celiac, Crohn’s disease

22. Statistical test
 STATA 11.0 (College Station Texas USA) software used
 Qualitative expression of TJ protein done by Fisher Exacts Test
 Quantitative parameters of TEM and HPLC, by one way ANOVA
followed by Boneforroni for multiple regression
 The effect of treatment assessed by using Mc Nemar’s test
 Pairwise correlation coefficient for correlation
 p value of <0.05 was considered statistically significant

23. Primary objective
To study the expression of tight junction proteins
(Zo-1, Claudin 2, 3 & 4, Occludin and JAM)
using Immunohistochemistry in patients with
Crohn's disease, celiac disease and controls

24. Immunohistochemistry Methodology
ZO-1 1:20
Claudin-2 1:50
Claudin-3 1:40
Claudin-4 1:400
JAM-A 1:40
occludin 1:20
Villous
Crypt
MS
ICJ
Cytoplasm
Nucleus
Distribution
Intensity
Negative 0
V. Focal <20% surface area
Focal >21%-50% surface area
Diffuse >50% surface area
Negative- 0
Faint 1
Moderate- strong 2
Strong 3
Villous
Crypt
Mucosal Surface (MS)
Intracellular junction
(ICJ)
Cytoplasm
Nucleus

25. Immunohistochemistry two day protocol
Steps Methods
Tissue Paraffinized section of duodenal biopsy
Deparaffinization Deparaffinized the slide in xylene and followed by acetone
& ethanol washing with running tap water
Blocking Blocking in 0.3% H2O2 for 30 minute washing in x PBS 3
times
Retrieval Prewarm then retrieval treatment
Primary Ab( Zymed, San
Francisco, CA) binding
Primary antibody binding for overnight incubation at 40 C
Secondary Ab (DAKO, Envision
real system) binding
After 3 washing x PBS incubate with secondary for 1
hour.
Colour development Di- aminobenizidine tetra hydrochloride (DAB)
Washing To stop the reaction, we washed the slides in water
Counterstaining By using hematoxylin stain
Mounting DPX Mount
Detection Examined using light microscope

26. TJ protein expression in normal control biopsy
villous vs. crypt
Villi Crypt
Claudin-2
Villi Crypt
Claudin-3
Villi Crypt
ZO-1
Villi Crypt
JAM
Villi Crypt Villi Crypt
Occludin Claudin-4

27. ZO-1 expression in celiac and Crohn’s disease
control
Treatment
naïve celiac
Active Crohn’s
disease
ZO-1 expression in villous and
crypt
Control vs
treatment naïve
celiac (P)
Control vs. Active
Crohn’s disease
(P)
Celiac vs. Crohn’s
(P)
Distribution in villous mucosal
surface
0.02 0.03 0.29
Intensity in villous mucosal surface 0.003 0.03 0.13
Distribution in crypt mucosal surface 0.05 0.008 0.84
Intensity in crypt mucosal surface 0.02 0.02 1.0

28. Cld-2 expression in Celiac and Crohn’s disease
Control
Treatment
naïve celiac
Active Crohn’s
disease
Cld-2 expression in villous and crypt Control vs
treatment naïve
celiac (P)
Control vs. Active
Crohn’s disease
(P)
Celiac vs. Crohn’s
(P)
Distribution in villous ICJ 0.002 0.001 0.27
Intensity in villous ICJ 0.02 0.03 0.03↑ in CD
Distribution in crypt ICJ 0.001 0.007 0.70
Intensity in crypt ICJ 0.01 0.001 0.73

29. Claudin-3 expression in Celiac and Crohn’s disease
Treatment
naïve celiac
Active Crohn’s
disease
Control
Cld-3expression in villous and crypt Control vs
treatment naïve
celiac (P)
Control vs. Active
Crohn’s disease
(P)
Celiac vs. Crohn’s
(P)
Distribution in villous ICJ 0.003 0.78 0.008 in CD
Intensity in villous ICJ 0.17 0.64 0.03 in CD
Distribution in crypt ICJ 0.01 0.43 0.009 in CD
Intensity in crypt ICJ 0.26 0.33 0.17

30. Claudin-4 expression in Celiac and Crohn’s disease
Control
Cld-4expression in villous and crypt Control vs
treatment naïve
celiac (P)
Control vs. Active
Crohn’s disease
(P)
Celiac vs. Crohn’s
(P)
Distribution in villous ICJ 0.08 0.29 0.04↑ in CD
Intensity in villous ICJ 0.18 0.44 0.32
Distribution in crypt ICJ 0.10 0.18 0.07
Intensity in crypt ICJ 0.24 0.20 0.28
Treatment
naïve celiac
Active Crohn’s
disease

31. Occludin expression in Celiac and Crohn’s disease
Control
Treatment
naïve celiac
Active Crohn’s
disease
Occ expression in villous and
crypt
Control vs treatment
naïve celiac (P)
Control vs. Active
Crohn’s disease
(P)
Celiac vs. Crohn’s
(P)
Distribution in villous ICJ <0.01 <0.01 0.66
Intensity in villous ICJ <0.01 <0.01 0.06
Distribution in crypt ICJ <0.01 <0.01 0.15
Intensity in crypt ICJ <0.01 <0.01 0.74

32. JAM expression in Celiac and Crohn’s disease
Control
JAM expression in villous and crypt Control vs
treatment naïve
celiac (P)
Control vs. Active
Crohn’s disease
(P)
Celiac vs. Crohn’s
(P)
Distribution in villous ICJ 0.21 1.0 0.05
Intensity in villous ICJ 0.39 0.07 0.30
Distribution in crypt ICJ 0.76 0.58 0.53
Intensity in crypt ICJ 0.15 1.0 1.0
Treatment
naïve celiac
Active Crohn’s
disease

33. Functional analysis of TJ
 Assessment of IP by dual sugar
permeation test(LM ratio)
 Lactulose mannitol ratio estimated
in urine sample by HPLC
 Pumping unit
 Sample-injection unit(injector)
 Separation unit ( Column)
 Detection unit (ELSD)
 Data-processing unit

34. Complete HPLC assembly with ELSD detector
N2 Cylinder
Column
Binary Pump
Injector
Detector

35. ELSD (Evaporating light scattering detector)
• Nebulisation of the effluent: Transformation of solvent from the
HPLC column into fine droplets
• Evaporation of the effluent: Droplets are carried by the gas flow
into the heated area
• Detection: The sample particles pass through a flow cell where
they get detect

36. Protocol for HPLC
Standard Urine sample
 Serial dilution of standard i.e. lactulose,
Mannitol & cellobiose (75μg-5μg)
 Conditioning by Maxi-Clean C18 600-mg
cartridges with 5 ml of methanol followed
by 5 ml of water
 Passed the sugars in conditioned C-18
cartridge, residual volume was collected
and diluted 1:1 with water (cellobiose and
amberlite IRA-400) vortex for 10 s and
centrifuged for 2 min at 3000 rpm
 Filter the supernatant in syringe filters
(Nylon 66, 0.2 mm) & centrifuge for 5 min
at 3000 rpm
 Analyzed the standard by ELSD-HPLC
 Centrifuge the urine (30%TCA) sample at
3000 rpm for 10 min
 Conditioning by Maxi-Clean C18 600-mg
cartridges with 5 ml of methanol followed by
5 ml of water
 Passed the urine from C-18 cartridge,
residual volume was collected and diluted
1:1 with water (cellobiose and amberlite
IRA-400) vortex for 10 s and centrifuged for
2 min at 3000 rpm
 Filter the supernatant in syringe filters
(Nylon 66, 0.2 mm) &centrifuge for 5 min at
3000 rpm
 Analyzed the sample by ELSD-HPLC

37. Standard chromatogram
HPLC and ELSD components Standardized condition
Mannitol
Column 250 ´ 4.6 mm column
Mobile phase Water & Acetonitrile (ACN)
Mobile phase flow rate 1.0 ml/min
ELSD detector temperature 650C
ELSD flow rate 2 litre/min
Column temperature 400C
Lactulose
Cellobiose
Mannitol Retention time 7.3 minute
Lactulose Retention time 8.3 minute
Cellobiose Retention time 9.6 minutes
Total Retention time 12 minutes
Injector volume 10μl with needle wash

38. Calibration graph of urine and water
Urine Water
Concentration Concentration
Recovery = Concentration of (L or M) in Urine ×100
Concentration of (L or M) in water
Recovery was (97%-100%)
Lower detection limit for lactulose is 6.25mg/L and 3.25mg/L for mannitol
Area
Area

39. Intestinal permeability in celiac and
Crohn’s disease
Control Treatment naïve
celiac
P
Lactulose % 0.002±.001 0.004±.003 0.013↑
Mannitol % 0.04±.02 0.024±.013 0.037↓
LMR 0.07±.04 0.19±0.19 0.006↑
Control Active Crohn’s
disease
P
Data in mean ±SD
Lactulose % 0.002±.001 0.005±.002 0.0005↑
Mannitol % 0.040±.02 0.024±.019 0.038↓
LMR 0.07±.04 0.22±.16 <.01↑
Data in mean ±SD

40. Primary Objective
To study the ultrastructural changes of tight
junction and to note the changes of other
junctions of a paracellular pathway by electron
microscopy

41. Transmission Electron Microscope
 To increase resolution of the
object many-folds over
conventional light microscope
Image analyses were performed
to measure
 TJ diameter,
 Microvilli (MV) length
 Inter-MV width
 Mitochondrial diameters (MD)

42. Sample preparation for TEM
Steps Description
Primary fixation Very small 1-2mm2 thick duodenal fragments were directly fixed in 2.5%
gluteraldehyde, made in 0.1M sodium phosphate buffer (pH=7.4).
Washing After fixation, repeated washing was made in 0.1M sodium phosphate
buffer (pH=7.4).
Post fixation OsO4(1% solution) was used as the secondary fixative, and it preserves
the lipid and again washed in 0.1M sodium phosphate buffer (pH=7.4).
Dehydration As the tissue dehydrated by passing through a series of ascending
concentration of ethanol
Clearing Epoxy propane was used for removing the traces of water in case of
incomplete dehydration.
Embedding Embedding was done in embedding capsule using gelatine or beam
capsule.
Polymerization To polymerize tissue, kept at 400 c - 500 c for overnight. After
polymerization capsules were washed in water at 700 c.

43. Continued
Steps Description
Staining for LM The semithin sections (thick 0.5 -2μm) was then stained with toludine
blue. After cleaning the stained section they were observed under light
microscope to mark the area of interest and went for ultra thin section
cutting
Staining for EM with
Uranyl acetate
Staining of ultrathin section ( thick 70-80 nm) was done with 50μl of
uranyl acetate
Staining with
Lead acetate
After washing of grid in double distilled water and dried carefully on a
filter paper. And stain with Lead acetate and lead citrate inside a
petridish.
Washing The grids were then washed in 0.02 M NaOH, followed by twice in
double distilled water. The grids were then dried carefully.
Acquiring of images The stained sections were then observed under a Morgagni 268D
TEM at an operating voltage 80 kV. Images were digitally acquired by
using a CCD camera (Megan view III, Fei Company) attached to the
microscope

44. •
Tight junction structure
1300x 200000x

45. Pentalaminar structure of Tight Junction
TJ
AJ
Desmosomes
Gap junction
Pentalaminar structure
X 120000

46. Morphometric measurement of TJ and other
ultrastructures
25000 x
Tight junction Mitochondria
10000 x
5000 x 10000 x
Microvillous length Intervillous width

47. Ultrastructural changes in celiac and Crohn’s
disease
Characteristics in Ultra
structure
Control
n=5(%)
Treatment naïve
Celiac n=12(%)
P
Alternation of TJ 0 5(41.6) 0.6
Pentalaminar structure 0 3(25) 0.3
ICJ 0 3(25) 0.3
Desmosomes 0 4(33.3) 0.2
Gap junction 0 0(0) 1.0
Mitochondria dilation 0 6(50) 0.2
Microvilli disarray 0 4(33.3) 0.2
Characteristics in Ultra
Control
Active Crohn’s
structure
n=5(%)
disease n=10(%)
P
Alternation of TJ 0 6(60) 0.1
Pentalaminar structure 0 6(60) 0.1
ICJ 0 2(20) 0.3
Desmosomes 0 5(50) 0.0
Gap junction 0 0(0) 1.0
Mitochondria dilation 0 2(20) 0.4
Microvilli disarray 0 3(30) 0.2

48. Ultrastructural quantitative changes of celiac
disease compare to control
TJ morphology Control
(n=5)
Treatment naïve
celiac(n=12)
P
Control
Diameter of TJ(nm) 21.67±20.6 31.14±21.6 0.5
Inter villous width(nm) 94.47±27.0 156.77±95.2 0.4
Length of microvilli(nm) 1250.31±343.9 1075±321.5 0.9
Diameter of Mitochondria(nm) 741.60±238.7 784.86±200.3 0.7
Treatment naïve
celiac
Tight junction Mitochondria Microvillous length Desmosome

49. Ultrastructural quantitative changes of Crohn’s
disease compare to controls
RESULTS
TJ morphology Control
(n=5)
Active CD
(n=10)
P
Control
Diameter of TJ(nm) 21.67±20.6 45.10±32.1 0.8
Inter villous width(nm) 94.47±27.0 140.35±77.5 0.7
Length of microvilli(nm) 1250.31±343.9 1110±436.2 0.7
Diameter of Mitochondria (nm) 741.60±238.7 826.77±236.4 0.3
Active Crohn’s disease
Tight junction Mitochondria Microvillous length Desmosome

50. Secondary objective
To study the effect of treatment on the
expression pattern of tight junction proteins
examined, as well as to see if any ultrastructural
improvement has taken place post-treatment, in
those with celiac disease and Crohn's disease.

51. Standard Treatment
Medication Crohn’s Disease
(n=28)
Celiac disease
(n=24)
Azathiaprine 17(60.7%)
Gluten free Diet
(GFD)
Mesalamine 17(60.7%)
Glucocorticoid 25(89.2%)

52. Clinical response in celiac disease
Variables Pre treatment
(n=24)
Post-treatment
(n=21)
P
Diarrhoea 16(66.6%) 2(9.09%) 0.0001
Pain abdomen 17(70.83%) 5(22.7%) 0.001
Anemia 23(95.83%) 12(54.5%) 0.01
Malabsorption 16(66.6%) 6(28.57%) 0.002
Arthralgia /Arthritis 9(37.5%) 2(9.09%)
0.01

53. Clinical response in Crohn’s disease
Variable Pre treatment
(n=28)
Post-treatment
(n=20)
P
Diarrhoea 28(100%) 8(40%) <.01
Abdominal pain 24(85.7%) 11(55%) 0.003
Anaemia 23(82.1%) 12(60%) 0.02
Malabsorption 20(71.4%) 12(60%) 0.04
Joint pain 17(60.7%) 4(20%) 0.004
Blood in stool 10(35.7%) 2(10%) 0.005
Tensemus 14(50%) 2(10%) 0.005
Intestinal Colic 20(71.4%) 5(25%) 0.005
Intestinal obstruction 13(46.4%) 2(10%) 0.005
Perianal Pain 7(25%) 2(10%) <.01
Fever 15(53.5%) 3(15%) <.01
Oral Ulcer 5(17.8%) 0(0%) 0.002
Fatigue 26(92.8%) 4(20%) <.01
Wt loss 17(60.7%) 3(15%) 0.005

54. Histological changes in celiac disease 6 month
after treatment
Post treatment
Pre Rx 0(N) 1(N) 2(3a) 3(3b) 4(3c) Total P
0 0 0 0 0 0 0
1 0 0 0 0 0 0
2(3a) 1 0 0 0 0 2 0.009
3(3b) 0 0 2 4 0 6
4(3c) 0 3 2 3 6 14
Total 1 4 4 7 6 22

55. Disease activity and Histological change in Crohn’s
disease after treatment
Variable Pre treatment
(n=28)
Post-treatment
(n=20)
P
CDAI Score 250.95±65.07 140.24±81.05
Remission (£150) 0(0%) 11(56.54%) 0.0005
Mild (151-220) 13(43.33%) 6(21.73%)
Moderate (221-400) 17(56.67%) 3(21.73%)
Severe (>400) 0(0%) 0(0%)
Post treatment
Pre Treatment 0(N) 2(3a) 3(3b) 4(3c) Total P
0 (N) 9 0 1 0 10
0.31
2(3a) 2 1 0 2 5
3(3b) 2 0 1 0 3
4(3c) 1 0 1 0 2
Total 14 1 3 2 20

56. Celiac pre vs post
(P)
ZO-1 in celiac pretreatment
Distribution in villous mucosal surface 0.02 0.02
Intensity in villous mucosal surface 0.003 0.03
Distribution in crypt mucosal surface 0.05 0.34
Intensity in crypt mucosal surface 0.02 0.75
ZO-1 in Celiac post-GFD
ZO-1 in CD pre-treatment
ZO-1 in CD post-treatment
ZO-1 expression in villous and crypt Control vs Celiac
(P)
ZO-1 expression in villous and crypt Control vs. Crohn’s
(P)
Crohn’s disease pre vs post
(P)
Distribution in villous mucosal surface 0.3 0.04
Intensity in villous mucosal surface 0.03 0.13
Distribution in crypt mucosal surface 0.008 0.14
Intensity in crypt mucosal surface 0.02 0.12

57. Cld -2 Celiac disease pre-treatment
Cld-2 expression in villous and crypt Control vs Celiac
Celiac pre vs post
(P)
Distribution in villous ICJ 0.002 0.12
Intensity in villous ICJ 0.02 0.14
Distribution in crypt ICJ 0.001 0.71
Intensity in crypt ICJ 0.01 0.55
Cld-2 Celiac disease post treatment
Cld -2 Crohn’s disease pre-treatment
Cld-2 Crohn’s disease post treatment
a b c
(P)
Cld-2 expression in villous and crypt Control vs. CD
(P)
CD pre vs post
(P)
Distribution in villous ICJ 0.001 1.0
Intensity in villous ICJ 0.03 0.25
Distribution in crypt ICJ 0.007 0.25
Intensity in crypt ICJ 0.001 0.37

58. Cld -3 Celiac disease pre-treatment
Cld-3 Celiac disease post treatment
Cld -3 Crohn’s disease pre-treatment
Cld-3 Crohn’s disease post treatment
Cld-3expression in villous and crypt Control vs Celiac
(P)
Celiac pre vs post
(P)
Distribution in villous ICJ 0.003 0.59
Intensity in villous ICJ 0.17 0.19
Distribution in crypt ICJ 0.01 0.45
Intensity in crypt ICJ 0.26 1.0
Cld-3expression in villous and crypt Control vs CD
(P)
CD pre vs post
(P)
Distribution in villous ICJ 0.78 1.0
Intensity in villous ICJ 0.64 1.0
Distribution in crypt ICJ 0.43 1.0
Intensity in crypt ICJ 0.33 1.0

59. Cld-4expression in villous and crypt Control vs Celiac
Cld -4 Celiac disease pre-treatment
Cld-4 Celiac disease post treatment
Cld -4 Crohn’s disease pre-treatment
Cld-4 Crohn’s disease post treatment
(P)
Celiac pre vs post
(P)
Distribution in villous ICJ 0.08 0.57
Intensity in villous ICJ 0.18 0.3
Distribution in crypt ICJ 0.10 0.7
Intensity in crypt ICJ 0.24 0.23
Cld-4expression in villous and crypt Control vs. CD
(P)
CD pre vs post
(P)
Distribution in villous ICJ 0.29 0.88
Intensity in villous ICJ 0.44 0.57
Distribution in crypt ICJ 0.18 0.59
Intensity in crypt ICJ 0.20 0.43

60. Occludin Celiac disease pre-treatment
Occ expression in villous and crypt Control vs Celiac
Occludin Celiac disease post treatment
Occludin Crohn’s disease pre-treatment
Occludin Crohn’s disease post treatment
(P)
Celiac pre vs post
(P)
Distribution in villous ICJ <0.01 1.0
Intensity in villous ICJ <0.01 1.0
Distribution in crypt ICJ <0.01 0.5
Intensity in crypt ICJ <0.01 1.0
Occ expression in villous and crypt Control vs. CD
(P)
CD pre vs post
(P)
Distribution in villous ICJ <0.01 0.6
Intensity in villous ICJ <0.01 0.3
Distribution in crypt ICJ <0.01 0.25
Intensity in crypt ICJ <0.01 0.05

61. JAM Celiac disease pre-treatment
JAM expression in villous and crypt Control vs Celiac
JAM Celiac disease post treatment
JAM Crohn’s disease pre-treatment
JAM Crohn’s disease post treatment
(P)
Celiac pre vs post
(P)
Distribution in villous ICJ 0.2 1.0
Intensity in villous ICJ 0.39 1.0
Distribution in crypt ICJ 0.76 1.0
Intensity in crypt ICJ 0.15 1.0
JAM expression in villous and crypt Control vs. CD
(P)
CD pre vs post
(P)
Distribution in villous ICJ 1.0 1.0
Intensity in villous ICJ 0.07 0.21
Distribution in crypt ICJ 0.58 0.5
Intensity in crypt ICJ 1.0 0.25

62. Effect of treatment on IP in celiac and Crohn’s
disease
Treatment naïve celiac
(n=24)
Celiac after GFD
(n=21)
P
Lactulose % 0.004±.003 0.002±0.001 0.01↓
Mannitol % 0.024±.013 0.020±0.007 0.18
LMR 0.19±0.19 0.12±0.06 0.04↓
Active CD
(n=28)
CD Post-treatment
(n=20)
P
Data expressed in mean± SD
Lactulose % 0.005±.002 0.002±0.001 0.002↓
Mannitol % 0.024±.019 0.020±0.019 0.48
LMR 0.22±.16 0.14±0.08 0.01↓
Data expressed in mean± SD

63. Ultrastructural changes in celiac disease 6
month after GFD
TJ morphology Treatment
Tight junction Desmosome Mitochondria Intervillous width
naïve celiac
(n=12)
Celiac after
GFD
(n=12)
P
Diameter of TJ(nm) 31.14±21.6 23.96±8.4 0.14
Pre-treatment
10000x
Pre-treatment
8000x
Pre-treatment
16000x
Pre-treatment
4000x
There was no correlation of the ultrastructural changes
Treatment naïve celiac
Inter villous width(nm) 156.77±95.2 164.28±124.2 0.87
Post-treatment
10000x
with changes in IP (LMR)
Post-treatment
8000x
Post-treatment
16000x
Post-treatment
4000x
Length of microvilli(nm) 1075±321.5 1110.89 ± 337.6
0.97
Diameter of Mitochondria (nm) 784.86±200.3 892.11± 173.3 0.85
Celiac disease post GFD

64. Effect of treatment on ultrastructural changes in
Crohn’s disease
TJ morphology Active CD
Tight junction Desmosome Mitochondria Intervillous width
(n=10)
CD Post-treatment(
n=10)
P
Diameter of TJ(nm) 45.10±32.1 20.37±7.2 0.04
Pre-treatment
10000x
Pre-treatment
3200x
Except from the inter-villous width, Pre-with treatment
lactulose excretion Pre-treatment
(p-0.01
& Inter r-0.72) villous ,there width(nm) was no correlation 140.35±77.5 8000x
of the ultrastructural 142.97±73.4 4000x
changes
0.94
Active Crohn’s disease
Post treatment
10000x
with IP changes( LMR)
Post treatment
3200x
Post treatment
8000x
Post treatment
4000x
Length of microvilli(nm) 1110±436.2 1506.84±1439
0.46
Diameter of Mitochondria(nm) 826.77±236.4 715.45±210.1 0.04
Crohn’s disease post-treatment

65. Summary: Celiac disease
Celiac disease vs. controls Celiac disease pre vs. post
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
IP Increased
Dilated TJ
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
IP improved
Dilated TJ

66. Summary: Crohn’s disease
Crohn’s disease vs. controls Crohn’s disease pre vs. post
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
IP Increased
Dilated TJ
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
IP improved
TJ improved

67. Summary: Crohn’s disease & celiac disease
Celiac vs. Crohn’s disease pre Rx Celiac vs. Crohn’s disease post Rx
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
IP , No change
Dilated TJ,
No change
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
IP, No change
Dilated TJ,
No change

68. Conclusion
Pretreatment
 TJ proteins
Celiac disease
pore forming protein: ↑cld-2
Pore sealing protein : ↓ cld-3
Scaffold protein : ↓ ZO-1
 Ultrastructure TJ dilated
 IP Increase
Post-treatment
 TJ proteins
pore forming protein : ↓ cld-2
Scaffold protein : ↑ ZO-1
 Ultrastructure TJ dilated
 IP Reduced
Crohn’s disease
Pretreatment:
TJ proteins
pore forming protein : ↑cld-2
Scaffold protein : ↓ ZO-1
Ultrastructure TJ dilated
IP Increase
Post-treatment:
 TJ proteins
pore forming protein : ↓ cld-2
Scaffold protein : ↑ ZO-1
 Ultrastructure partially resolved
 IP Reduced
There was no difference in TJ protein expression, IP and TJ dilation in celiac
and Crohn’s disease at baseline as well as 6 month after treatment

69. Conclusion
Possibly the expression patterns of TJ proteins
- ZO-1
- pore forming protein claudin-2
- and pore sealing protein claudin-3
regulate the tight junction structure and intestinal
permeability

70. CHIEF GUIDE
Dr Govind K. Makharia
Additional Professor
Dept. of Gastroenterology & HNU
DC member CO-Guide
AIIMS, New Delhi
Dr. Vineet Ahuja
Additional Professor
Dept. of Gastroenterology & HNU
AIIMS, New Delhi
Dr S.K. Acharya
Professor & Head
Dept. of Gastroenterology & HNU
AIIMS, New Delhi
Dr Subroto Sinha
Professor & Head
Department of Biochemistry
AIIMS, New Delhi
Dr. S. k. Panda
Professor & Head
Dept. of Pathology
AIIMS, New Delhi
Dr. H.K. Prasad
Professor
Dept. of Biotechnology
AIIMS, New Delhi
Dr Y.K. Joshi
Professor
Dept. of Gastroenterology & HNU
AIIMS, New Delhi
Shyam Prakash
Scientist
Dept. of Gastroenterology & HNU
AIIMS, New Delhi
Dr. V. Sreenivas
Associate Professor
Dept. of Biostatistics
AIIMS, New Delhi
Dr. Siddhartha Datta Gupta
Professor
Dept. of Pathology
AIIMS, New Delhi

71. Awards
 ISG WIN Medicare Award for Best oral Paper presentation :
P. Goswami*1, P. Das2, S. P. Khanel3, V. Sreenivas3, S. Datta
Gupta2, G. K. Makharia. “Expression of tight junction protein (ZO-1,
Claudin-2, 4 and Occludin) in patients with celiac disease and active
Crohn’s disease and change in their expression 6 months after
treatment” in ISG 2009 held at Kolkata
 Prof K.C. Basumallick Award for best research paper : P Das,
Pooja Goswami, Siddharth Datta Gupta, Subrat K Panda & Govind
K Makharia, Tight Junction proteins (Zo-1, Claudin 2, 3 & 4,
Occludin and JAM) expression and Intestinal permeability in
patients with Celiac and Crohn's disease, and their association with
light microscopic and ultrastructural findings in APCON 2011 held at
Patiala

72. Publications Journal publication
 Prasenjit Das, Pooja Goswami, Tapash K Das, Tapas Nag, Vishnubhatla Sreenivas, Vineet
Ahuja,Subrat K Panda, Siddhartha Datta Gupta, Govind K Makharia.. Comparative Tight junction
protein expressions in colonic Crohn’s disease, ulcerative colitis and tuberculosis: a new
perspective: Virchows Archiv 2012
 Sood A, Midha V, Makharia GK, Ahuja V, Singal D, Goswami P, Tandon RK. . Probiotics for
ulcerative colitis ... Are the good bugs back? Gastroenterology, 2010 Sep; 139(3):1054-6. [Epub
ahead of print] PubMed PMID: 20667486.
 Makharia GK, Srivastava S, Das P, Goswami P, Singh U, Tripathi M, Deo V, Aggrawal A, Tiwari
RP, Sreenivas V, Gupta SD. Clinical, endoscopic, and histological differentiations between
Crohn's disease and intestinal tuberculosis. Am J Gastroenterology, 2010 Mar; 105(3):642-51.
Epub 2010
 G. K Makharia, A. Seth, S. K. Sharma, A. Sinha, P. Goswami, A. Aggrawal, K. Puri & V.
Sreenivas. Structural and functional abnormalities in lungs in patients with achalasia.
Neurogastroenterol Motility .2009 1365-2982.
 Sood A, Midha V, Makharia GK, Ahuja V, Singal D, Goswami P, Tandon RK. The Probiotic
Preparation, VSL#3, Induces Remission in Patients With Mild-to-Moderately Active Ulcerative
Colitis. Clin Gastroenterol Hepatol. 2009;(11):1202-9
 Das CJ, Makharia G, Kumar R, Chawla M, Goswami P, Sharma R, Malhotra A. PET-CT
enteroclysis: a new technique for evaluation of inflammatory diseases of the intestine. Eur J Nucl
Med Mol Imaging. 2007 ;(12):2106-14.
 Gupta AK, Chauhan DS, Srivastava K, Das R, Batra S, Mittal M, Goswami P, Singhal N, Sharma
VD, Venkatesan K, Hasnain SE, Katoch VM. Estimation of efflux mediated multi-drug resistance
and its correlation with expression levels of two major efflux pumps in mycobacteria. J Commun
Dis. 2006 Mar; 38(3):246-54.
Book-Chapter:
 Pooja Goswami, Chandan Jyoti, Govind K Makharia .Inflammatory bowel disease ; Update
2008.Non-invasive Diagnostic Tools for Inflammatory Bowel Disease: 32-35..Kolkata
Gastroenterology society on eve of Enterocon 2008.

73. Abstracts
 P. Goswami*1, P. Das2, TC Nag, TK Das V. Sreenivas3, S. Datta Gupta2, G. K. Makharia. Tight junction alteration in
Celiac disease and Crohn’s disease and effect of treatment on them. J. of Gastroenterology and Hepatology 2010.
(584small intestine)
 P. Goswami*1, Meenakshi Sharma, P. Das2, S. Datta Gupta2, G. K. Makharia. Difference in the features of celiac disease
in adolescents and adults. J. of Gastroenterology and Hepatology 2010.(582 small intestine)
 P. Das2, G. K. Makharia, P. Goswami*1, , TC Nag, TK Das V. Sreenivas3, S. Datta Gupta2. Expression of tight junction
proteins in Crohn’s disease , ulcerative colitis and intestinal tuberculosis. – 2009 (585 large intestine)
 P. Goswami*1, P. Das2, S. P. Khanel3, V. Sreenivas3, S. Datta Gupta2, G. K. Makharia. “Changes in the expression of
ZO-1, claudin-2, claudin-4 and Occludin in the duodenal mucosa of patients with celiac disease before and after treatment
with Gluten free diet”. J. of Gastroenterology and Hepatology 2009, P0079
 S. Srivastava*1, G. K. Makharia1, P. Das2, P. Goswami1, S. Datta Gupta2 J.Development of a therapeutic algorithm for
management of intestinal tuberculosis and Crohn’s disease in tuberculosis endemic countries. of Gastroenterology and
Hepatology 2009,OP412
 , U. Sharma1, R. R. Singh1, P. Goswami*2, V. Ahuja2, G. K. Makharia2, N. R. Jagannathan. Similarity in the metabolic
profile in macroscopically involved and uninvolved colonic mucosa in patients with inflammatory bowel disease: An in-vitro
proton MR. Spectroscopy study J. of Gastroenterology and Hepatology 2009, P1744
 P. Goswami*2, Das C.J.,V. Ahuja2, Rakesh Kumar G. K. Makharia2 PET-CT Colonography: A novel non-invasive
technique for assessment of extent and activity of the disease in patients with ulcerative colitis. Gastroenterology and
Hepatology 2009, P1059
 P. Goswami*1, P. Das2, S. P. Khanel3, V. Sreenivas3, S. Datta Gupta2, G. K. Makharia Expression of tight junction protein
(ZO-1, Claudin-2, 4 and Occludin) in patients with celiac disease and active Crohn’s disease and change in their expression
6 months after treatment Indian J.Gastroenterol.2009.
 Pooja Goswami, Namrata Singh, Rajesh Khadgawat,* Siddhartha Datta Gupta,** Govind K Makharia. Response to
treatment in adult patients with celiac disease. J. of Gastroenterology and Hepatology 2008:23 Suppl.5):A-93
 S. Srivastava, G.K. Makharia, U Singh, P Goswami, M. Tripathi, SD Gupta, YK Joshi. Development of therapeutic algorithm
of intestinal tuberculosis endemic area. J. of Gastroenterology and Hepatology 2008:23 Suppl.5): A-95.
 Chandan J Das, Govind Makharia*, Raju Sharma, Pooja Goswami*, Rakesh Kumar, AK Malhotra. PET-CT Colonography:
A Novel Noninvasive Technique for Assessment of disease extent in Ulcerative Colitis. J. of Gastroenterology and
Hepatology 2008:23 Suppl.5):A-109
 Chandan J Das, Govind Makharia*, Raju Sharma, Rakesh Kumar, Rajender Kumar *, R Reddy*, Pooja Goswami*, AK
Malhotra, AK Gupta. Assessment of terminal ileal Crohn’s disease activity by PET-CT enteroclysis and ileocolonoscopy. J.
of Gastroenterology and Hepatology 2008:23 Suppl.5): A-95.
 RR Singh, U Sharma, GK Makharia, P Goswami, V. Ahuja NR Jagannathan. Similarity in metabolically involved and
uninvolved colonic mucosa of patients with inflammatory bowel disease (IBD): a 1 H MR spectroscopic study. J. of
Gastroenterology and Hepatology 2008:23 Suppl.5): A-111.

74. THANK YOU