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Name of candidate: Pooja GoswamiName of candidate: Pooja Goswami Date of enrolment- 15 February 2007Date of enrolment- 15 February 2007
Dept. of Gastroenterology and Human NutritionDept. of Gastroenterology and Human NutritionAll India Institute of Medical SciencesAll India Institute of Medical Sciences
New Delhi New Delhi
Name of candidate: Pooja GoswamiName of candidate: Pooja Goswami Date of enrolment- 15 February 2007Date of enrolment- 15 February 2007
Dept. of Gastroenterology and Human NutritionDept. of Gastroenterology and Human NutritionAll India Institute of Medical SciencesAll India Institute of Medical Sciences
New Delhi New Delhi
Characterization of Tight Junction Proteins in Patients with Crohn's Disease and Celiac
Disease
Background• Autoimmune DiseaseGenetic
predisposition(host factors)
Environmental trigger
(exogenous factors)
Immune response
(host factors)
How this antigen enter?SkinLungIntestineEyes
Th1
Th2
Amplified cycle of inflammation, allow further leakage of foreign Ag
Foreign Ag entry through barrier defect
T cell
APC
IFN-γTNF-α
Tissue damage
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
TJs regulate intestinal permeability
Normal TJ regulate normal IP
DiseaseNo disease
Disrupted TJ leads to increase IP
• 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
Why to study Tight Junction ?
PARA
CELLULA
R
LumenLumen
TRANSCELLU
LAR
Tight Tight JunctionJunction
BloodstreamBloodstream
Paracellular permeability between the epithelial cells (gate function )
Maintenance of apical basolateral cell polarity (fence function)
Tight Junction ProteinsName of Proteins Known Partner
Claudin family ZO-1
Occludin ZO-1, ZO-2,Vap33, Actin
JAM family ZO-1, MUPP1
ZO family ZO-1ZO-2ZO-3
Occludin, Claudin , ZO-2-3, Cingulin, Actin & ZONABZO-1, ActinZO-1, OccludinRas, Zo-1
Tricellulin
AF-6
DLg (Drosophila)
Scribble(Drosophila)
Cingulin Occludin, ZO-1-2, myosin
Symplekin
ASIP/Par3 PKC-αRab3b
Rab13 δ-PDERab8 G/C Kinase, exocyst
Sec6, Sec8
Tight Junction proteins
Claudin-2
Claudin-3
Claudin-4
Occludin
ZO-1
ZO-1
JAM
Scaffolding
Pore forming
Sealing
Cell migration
Cell polarity
Distribution of Tight Junctions
• 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
Villous
crypt
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)
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
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
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
Leads to ↑ IP, implicated in pathogenesis of CD and
celiac disease
Restoration of TJP & IP, may improve course of CD and Celiac disease
Differential expression of TJ proteins in CD and
celiac may be attributed toTJs disintegrity
Hypothesis
ObjectivesPrimary 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
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, 2006Best et al 1976Silverberg et al 2005
ESPGHAN, Arch Dis Child 1990Oberhuber 1999
Study criteriaDisease Inclusion criteria Exclusion criteria
Crohn’s disease 1. Age: 12 to 65 years2. Both genders3. Active disease
(Crohn’s disease activity index >150-450)
1. Severely sick patients2. Severe active disease (CDAI>450)3. Massive bleeding 4. Those on active NSAID use5. Patients with small intestinal resection6. Ulcerative colitis7. Indeterminate colitis8. Intestinal tuberculosis9. Patients with chronic or acute renal failure10. Patients with intestinal perforation11. Subject with other concomitants diseases12. Pregnant and lactating mothers13. Unwilling patients
Celiac disease 1. Age: 12 to 65 years2. Both genders3. Treatment Naïve
1. Patients who have received gluten free diet earlier
2. Those on active NSAID use3. Subject with other concomitants diseases4. Pregnant and lactating mothers5. Unwilling patients6. Patients with tropical sprue
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
Patient with Crohn’s disease, celiac disease and controls recruited from the Gastroenterology OPD of AIIMS
Biochemical test, IP test, UGI endoscopy followed by Bx in CD, celiac and Controls
Electron MicroscopyHistopathology
After 6 month follow up by clinician, we repeated all the test in patients with celiac and Crohn’s disease
Immunohistochemistry •ZO-1•Cld 2,cld-3 & cld-4 •Occludin,• JAM
Informed consent taken
Inclusion and exclusion criteria
Work Algorithm
Duodenal Biopsy
Diagnosis•ECCO guideline for Crohn’s disease•ESPGHAN criteria for celiac disease
TJ protein expression
Ultrastructural changes
Effect of treatment
Objectives
Screened (n=50)
Included (n=28)Active Crohn’s disease
Follow-Up
Post treatment (n=20)
Lost to follow up=8
Crohn’s disease
Patients Enrollment
Excluded (n= 22 ) •Over age (n=1)•Active bleeding (n=11)•Co morbidity (n=5)•Refused to participate (n=5)
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
Post treatment (n=21)
Lost to follow up=3
Celiac disease
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
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
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
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
MSICJCytoplasmNucleus
Intensity
DistributionNegative 0 V. Focal <20% surface area Focal >21%-50% surface area Diffuse >50% surface area
Negative- 0 Faint 1Moderate- strong 2Strong 3
Villous
Crypt
Mucosal Surface (MS)
Intracellular junction (ICJ)
Cytoplasm
Nucleus
Immunohistochemistry Methodology
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
Immunohistochemistry two day protocol
Villi Crypt
Claudin-2CryptVilli
CryptVilliCryptVilli
Claudin-4Occludin
Claudin-3
TJ protein expression in normal control biopsy villous vs. crypt
CryptVilli
CryptVilli
ZO-1
JAM
control
ZO-1 expression in celiac and Crohn’s disease
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
Control
Cld-2 expression in Celiac and Crohn’s disease
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
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
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
Claudin-4 expression in Celiac and Crohn’s disease
Treatment naïve celiac
Active Crohn’s disease
Control
Occludin expression in Celiac and Crohn’s disease
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
ControlJAM 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
JAM expression in Celiac and Crohn’s disease
Treatment naïve celiac
Active Crohn’s disease
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
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
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) ¢rifuge for 5 min at 3000 rpm
Analyzed the sample by ELSD-HPLC
Protocol for HPLC
Standard Urine sample
HPLC and ELSD components Standardized condition
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
Injector volume 10µl with needle wash
Standard chromatogram
Mannitol
Lactulose
Cellobiose
Mannitol Retention time 7.3 minuteLactulose Retention time 8.3 minuteCellobiose Retention time 9.6 minutesTotal Retention time 12 minutes
Urine Water
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
Calibration graph of urine and water
ConcentrationConcentration
Are
a
Are
a
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↑
Intestinal permeability in celiac and Crohn’s disease
Control Active Crohn’s disease
P
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
Data in mean ±SD
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
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)
Sample preparation for TEMSteps 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.
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
Continued
TJ
AJ
Desmosomes
Gap junction
Pentalaminar structure
X 120000
Pentalaminar structure of Tight Junction
25000 x
Tight junction Mitochondria
10000 x
5000 x 10000 x
Microvillous length Intervillous width
Morphometric measurement of TJ and other ultrastructures
Characteristics in Ultra structure
Controln=5(%)
Treatment naïve Celiac n=12(%)
P
Alternation of TJ 0 5(41.6) 0.6 Pentalaminar structure 0 3(25) 0.3ICJ 0 3(25) 0.3Desmosomes 0 4(33.3) 0.2Gap junction 0 0(0) 1.0Mitochondria dilation 0 6(50) 0.2Microvilli disarray 0 4(33.3) 0.2
Characteristics in Ultra structure
Controln=5(%)
Active Crohn’s disease n=10(%)
P
Alternation of TJ 0 6(60) 0.1 Pentalaminar structure 0 6(60) 0.1ICJ 0 2(20) 0.3Desmosomes 0 5(50) 0.0Gap junction 0 0(0) 1.0Mitochondria dilation 0 2(20) 0.4Microvilli disarray 0 3(30) 0.2
Ultrastructural changes in celiac and Crohn’s disease
Ultrastructural quantitative changes of celiac disease compare to control
TJ morphology Control(n=5)
Treatment naïve celiac(n=12)
P
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
Tight junction Mitochondria Microvillous length Desmosome
Treatment naïve celiac
Control
Ultrastructural quantitative changes of Crohn’s disease compare to controls
RESULTS
TJ morphology Control(n=5)
Active CD (n=10)
P
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
Tight junction Mitochondria Microvillous length Desmosome
Active Crohn’s disease
Control
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.
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%)
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 /Arthritis9(37.5%) 2(9.09%)
0.01
Clinical response in Crohn’s disease
Variable Pre treatment (n=28)
Post-treatment (n=20)
P
Diarrhoea 28(100%) 8(40%) <.01Abdominal pain 24(85.7%) 11(55%) 0.003Anaemia 23(82.1%) 12(60%) 0.02Malabsorption 20(71.4%) 12(60%) 0.04Joint pain 17(60.7%) 4(20%) 0.004Blood in stool 10(35.7%) 2(10%) 0.005Tensemus 14(50%) 2(10%) 0.005Intestinal Colic 20(71.4%) 5(25%) 0.005
Intestinal obstruction 13(46.4%) 2(10%) 0.005Perianal Pain 7(25%) 2(10%) <.01Fever 15(53.5%) 3(15%) <.01Oral Ulcer 5(17.8%) 0(0%) 0.002Fatigue 26(92.8%) 4(20%) <.01Wt loss 17(60.7%) 3(15%) 0.005
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
Histological changes in celiac disease 6 month 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%)
Disease activity and Histological change in Crohn’s disease after treatment
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
ZO-1 in celiac pretreatment
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. 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
ZO-1 expression in villous and crypt Control vs Celiac
(P)
Celiac pre vs post
(P)
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
Cld -2 Celiac disease pre-treatment
Cld-2 Celiac disease post treatment
Cld -2 Crohn’s disease pre-treatment
Cld-2 Crohn’s disease post treatment
a b c
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
Cld-2 expression in villous and crypt Control vs Celiac
(P)
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 -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
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
Cld-4expression in villous and crypt Control vs Celiac
(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
Occludin Celiac disease pre-treatment
Occludin Celiac disease post treatment
Occludin Crohn’s disease pre-treatment
Occludin Crohn’s disease post treatment
Occ expression in villous and crypt Control vs Celiac
(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
JAM Celiac disease pre-treatment
JAM Celiac disease post treatment
JAM Crohn’s disease pre-treatment
JAM Crohn’s disease post treatment
JAM expression in villous and crypt Control vs Celiac
(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
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↓
Effect of treatment on IP in celiac and Crohn’s disease
Active CD(n=28)
CD Post-treatment (n=20)
P
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
Data expressed in mean± SD
Ultrastructural changes in celiac disease 6 month after GFD
TJ morphology Treatment naïve celiac
(n=12)
Celiac after GFD
(n=12)
P
Diameter of TJ(nm) 31.14±21.6 23.96±8.4 0.14
Inter villous width(nm) 156.77±95.2 164.28±124.2 0.87
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
Pre-treatment10000x
Post-treatment10000x
Pre-treatment8000x
Post-treatment8000x
Pre-treatment16000x
Post-treatment16000x
Pre-treatment4000x
Post-treatment4000x
Celiac disease post GFD
Treatment naïve celiac
Tight junction Intervillous widthDesmosome Mitochondria
There was no correlation of the ultrastructural changes with changes in IP (LMR)
Effect of treatment on ultrastructural changes in Crohn’s disease
TJ morphology Active CD (n=10)
CD Post-treatment(n=10)
P
Diameter of TJ(nm) 45.10±32.1 20.37±7.2 0.04
Inter villous width(nm) 140.35±77.5 142.97±73.4 0.94
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
Pre-treatment10000x
Post treatment10000x
Pre-treatment3200x
Post treatment3200x
Pre-treatment8000x
Post treatment8000x
Pre-treatment4000x
Post treatment4000x
Crohn’s disease post-treatment
Active Crohn’s disease
Tight junction Intervillous widthDesmosome Mitochondria
Except from the inter-villous width, with lactulose excretion (p-0.01 & r-0.72) ,there was no correlation of the ultrastructural changes
with IP changes( LMR)
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
Celiac disease vs. controls Celiac disease pre vs. post
Summary: Celiac disease
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
Crohn’s disease vs. controls Crohn’s disease pre vs. post
Summary: Crohn’s disease
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
Celiac vs. Crohn’s disease pre Rx Celiac vs. Crohn’s disease post Rx
Summary: Crohn’s disease & celiac disease
Conclusion
Pretreatment TJ proteins
pore forming protein: ↑cld-2
Pore sealing protein : ↓ cld-3
Scaffold protein : ↓ ZO-1 Ultrastructure TJ dilated IP Increase
Celiac diseasePost-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-1Ultrastructure TJ dilatedIP 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
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
CHIEF GUIDEDr Govind K. Makharia
Additional ProfessorDept. of Gastroenterology & HNU
AIIMS, New Delhi
Dr. Vineet AhujaAdditional Professor
Dept. of Gastroenterology & HNUAIIMS, New Delhi
Dr S.K. AcharyaProfessor & Head
Dept. of Gastroenterology & HNUAIIMS, New Delhi
Dr Subroto SinhaProfessor & Head
Department of Biochemistry AIIMS, New Delhi
Dr. S. k. PandaProfessor & Head
Dept. of PathologyAIIMS, New Delhi
Dr. H.K. PrasadProfessor
Dept. of Biotechnology AIIMS, New Delhi
Dr Y.K. JoshiProfessor
Dept. of Gastroenterology & HNUAIIMS, New Delhi
Dr. V. SreenivasAssociate ProfessorDept. of Biostatistics
AIIMS, New Delhi
Shyam PrakashScientist
Dept. of Gastroenterology & HNUAIIMS, New Delhi
DC member CO-Guide
Dr. Siddhartha Datta GuptaProfessor
Dept. of PathologyAIIMS, New Delhi
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
PublicationsJournal 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.
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.