23 jan final slides copy (dr pooja-vaio's conflicted copy 2013-12-14)

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

Methodology

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



Cld-2 expression in villous and crypt Control vs treatment naïve celiac (P)


(P)


(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



Control

Cld-3expression in villous and crypt Control vs treatment naïve celiac (P)


(P)


(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


Control

Cld-4expression in villous and crypt Control vs treatment naïve celiac (P)


(P)


(P)

Distribution in villous ICJ 0.08 0.29 0.04↑ in CD

Intensity in villous ICJ 0.18 0.44 0.32



Claudin-4 expression in Celiac and Crohn’s disease



Control

Occludin expression in Celiac and Crohn’s disease



Occ expression in villous and

crypt

Control vs treatment naïve celiac (P)


(P)


(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)


(P)


(P)

Distribution in villous ICJ 0.21 1.0 0.05

Intensity in villous ICJ 0.39 0.07 0.30



JAM expression in Celiac and 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

N2 Cylinder

Binary Pump

Detector

Complete HPLC assembly with ELSD detector

Injector

Column

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) &centrifuge 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

•

200000x 1300x

Tight junction structure

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


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


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


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)


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)


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)









Cld-3expression in villous and crypt Control vs Celiac

(P)

Celiac pre vs post

(P)





Cld-3expression in villous and crypt Control vs CD

(P)

CD pre vs post

(P)









Cld-4expression in villous and crypt Control vs Celiac

(P)

Celiac pre vs post

(P)





Cld-4expression in villous and crypt Control vs. CD

(P)

CD pre vs post

(P)





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)





JAM expression in villous and crypt Control vs. CD

(P)

CD pre vs post

(P)





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


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


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


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


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


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


Dr. V. SreenivasAssociate ProfessorDept. of Biostatistics

AIIMS, New Delhi

Shyam PrakashScientist


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.

THANK YOU

23 jan final slides copy (dr pooja-vaio's conflicted copy 2013-12-14)

Health & Medicine

celiac disease zo

ip permeability

increased ip sapone

increased ip drago

increased ip pizzuti

mupp1zo family zo

crohns disease of occludin

intrinsic barrier