Poster No.

Department Presenting Author

Designation Abstract Title Authors

Phd-135 Community Medicine

Venkatesh Vinayak Narayan

PhD Student Estimates of influenza-associated mortality in India, 2010-13: A modelling study

Narayan Vinayak Venkatesh, Haldar Partha, Sreenivas Vishnubatla, Kant Shashi, Krishnan Anand

Phd-71A

RPC Ms. Renu Venugopal

PhD Student Expression of SPARC and MMP9 in Stevens - Johnson syndrome (SJS) chronic ocular sequelae undergoing cultivated oral mucosal epithelial transplantation (COMET).

Venugopal Renu, Sen Seema, Mohanty Sujata, Kashyap Seema, Agarwal Tushar, Sharma Anjana, Kaur Jasbir, Sharma Namrata.

Phd-71B

RPC Ms. Renu Venugopal

PhD Student Role of MMP9 in pathogenesis in chronic ocular Stevens Johnson syndrome

Venugopal Renu, Sen Seema, Mohanty Sujata, Kashyap Seema, Agarwal Tushar, Sharma Anjana, Kaur Jasbir, Sharma Namrata

Phd-72 RPC Shahzan Siddiqui

PhD Student Clinical Relevance of Notch 1 in Lacrimal Gland Adenoid Cystic Carcinoma

Anjum Shahzan, Sen Seema, Kashyap Seema, Chosdol Kunzang, Pushker Neelam, Bakhshi Sameer, Bajaj Singh Mandeep, Sharma Chand Maher, Meel Rachna

Phd-78 RPC (Ocular Pathology)

Jayanti Jha PhD Student Role of Ataxia Telangiectasia mutated in Uveal Melanoma and its prognostic significance

Jha Jayanti, Pushker Neelam, Singh Kumar Mithalesh, Singh Lata, Sen Seema, Kashyap Seema

Phd-129 RPC (Ocular Pharmacology)

Sukrit Bhattacharyya

PhD Student Identifying proteomic signatures in aqueous humor of diabetic rat

Bhattacharyya Sukrit, Halder Nabanita, Singh Basker S., Chawla Rohan, Velpandian Thirumurthy

Phd-133 RPC (Ocular Pharmacology)

Ujjalkumar S Das

PhD Student QSPR modeling of 8 congeneric antifungal and antiprotozoal agents in rabbits

Das U, Titiyal JS, Singh B, Sreenivas V, Jayaram B, Halder N, Velpandian T

Phd-140 RPC (Ocular pharmacology)

Gowtham.L PhD Student Aqueous humor metabolomics of glaucomatous patients

Gowtham. L, Nabanita Halder, Tanuj Dada, Rama jayasundar, Baskar singh, Velpandian T

Phd-71A

Title: Expression of SPARC and MMP9 in Stevens - Johnson syndrome (SJS) chronic

ocular sequelae undergoing cultivated oral mucosal epithelial transplantation (COMET)

Authors:Venugopal Renu1, Sen Seema1, Mohanty Sujata2, Kashyap Seema1,Agarwal

Tushar1, Sharma Anjana1, Kaur Jasbir1, Sharma Namrata1

Affiliation:

1- Dr.R.P.Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New

Delhi-110029 2- Stem Cell Facility, All India Institute of Medical Sciences, New Delhi-110029

Presenting Author

Name- Renu Venugopal

Email id- vrenu.aiims@gmail.com

Corresponding author

Name- Namrata Sharma

Email id – namrata.sharma@gmail.com

Abstract

Introduction: Molecular pathogenesis remains unknown in chronic ocular sequelae of

Stevens Johnson Syndrome (SJS). Persistent underlying conjunctival inflammation and tear

deficiency in SJS affects prognosis of visual rehabilitation surgeries like cultivated oral

mucosal epithelial transplantation (COMET). Secreted protein acidic and rich in cysteine

(SPARC), an extracellular matrix (ECM) protein is a key modulator of fibrosis and tissue

scarring. Matrix metalloproteinase (MMP) genes have been reported to get activated by

SPARC mediated wound healing mechanism. SPARC implicated in post glaucoma surgical

conjunctival scarring. There are no studies documenting SPARC in SJS chronic cases.

Aim: To investigate the role of SPARC and MMP9 in surgical prognosis of COMET in

chronic ocular SJS.

Materials & methods: Pannus tissues were excised from 45 SJS eyes undergoing COMET

and clinico-pathological parameters were examined. Preoperative clinical ocular severity

was graded by using a modified method. Immunohistochemistry (IHC) and Real Time PCR

were performed for SPARC, MMP2, MMP3, MMP9, MMP11 and TIMP-1 expression. IHC

positivity was assessed by staining intensity of >+2. Cadaveric cornea was used as a

control. Postoperative MMP9 levels were assessed in tears using Inflammadry MMP9 assay.

Statistical correlation was done with clinicopathological parameters and follow up.

Results: Mean age of patients was 25 ± 11.7 years. The primary causative agent in inciting

SJS was NSAIDS (55%) followed by sulpha drugs (15%).The logMAR BCVA improved post

COMET in 82.2% eyes and 55.5% eyes were removed from blindness category. Total

severity score improved in 88.8% eyes and 62.2% eyes were removed from Grade IV

(severe) to Grade III, II and I (less severe). Cytoplasmic MMP9 and SPARC overexpression

was seen in pannus epithelium of 48.9% and 44.4% in eyes while in stroma in 57.7% and

77.7% eyes.Epithelial MMP9 expression correlated with preoperative corneal

neovascularization (p=0.047) and keratinization (p=0.01) while stromal expression correlated

with conjunctival hyperaemia (p=0.14) and symblepharon (p=0.028). SPARC epithelial

expression correlated with conjunctivalization (p=0.018) while stromal expression correlated

with fibrosis (p=0.002). RNA expression of MMP9 and SPARC correlated with fibrosis

(p=0.04) and epithelial hyperplasia (p=0.05).On Univariate analysis, MMP9 and SPARC

epithelial immunoexpression and MMP9 RNA overexpression correlated with poor COMET

graft survival (p=0.02). MMP9 performed in SJS post COMET eyes showed 83.3% positivity.

There was no statistical significance found between MMP9 tear positivity and COMET

surgical outcomes.

Results: Ours is the first study to reveal the possible role of SPARC and MMP9 in promoting

tissue vasculature and remodelling in SJS chronic sequelae. Their over expression

correlated with poor visual acuity outcomes of COMET. MMP9 overexpression in tears was

suggestive of persistent secretion of MMP9 protein in SJS eyes postoperatively leading to

prolonged dryness. Our results may help in further novel therapeutics.

Phd-71B

Title: Role of MMP9 in pathogenesis in chronic ocular Stevens Johnson syndrome

Authors: Venugopal Renu1, Sen Seema1, Mohanty Sujata2, Kashyap Seema1, Agarwal

Tushar1, Sharma Anjana1, Kaur Jasbir1, Sharma Namrata1

Affiliation:

1- Dr.R.P.Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New

Delhi-110029

2- Stem Cell Facility, All India Institute of Medical Sciences, New Delhi-110029

Presenting Author

Name- Renu Venugopal

Email id- vrenu.aiims@gmail.com

Corresponding author

Name- Namrata Sharma

Email id – namrata.sharma@gmail.com

Abstract Introduction: Molecular pathogenesis remains unknown in chronic ocular sequelae of

Stevens Johnson Syndrome (SJS). Persistent underlying conjunctival inflammation and tear

deficiency in SJS affects prognosis of visual rehabilitation surgeries like cultivated oral

mucosal epithelial transplantation (COMET). Secreted protein acidic and rich in cysteine

(SPARC), an extracellular matrix (ECM) protein is a key modulator of fibrosis and tissue

scarring. Matrix metalloproteinase (MMP) genes have been reported to get activated by

SPARC mediated wound healing mechanism. SPARC implicated in post glaucoma surgical

conjunctival scarring. There are no studies documenting SPARC in SJS chronic cases.

Aim: To investigate the role of SPARC and MMP9 in surgical prognosis of COMET in chronic

ocular SJS.

Materials & methods: Pannus tissues were excised from 45 SJS eyes undergoing COMET

and clinico-pathological parameters were examined. Preoperative clinical ocular severity

was graded by using a modified method. Immunohistochemistry (IHC) and Real Time PCR

were performed for SPARC, MMP2, MMP3, MMP9, MMP11 and TIMP-1 expression. IHC

positivity was assessed by staining intensity of >+2. Cadaveric cornea was used as a

control. Postoperative MMP9 levels were assessed in tears using Inflammadry MMP9 assay.

Statistical correlation was done with clinicopathological parameters and follow up.

Results: Mean age of patients was 25 ± 11.7 years. The primary causative agent in inciting

SJS was NSAIDS (55%) followed by sulpha drugs (15%).The logMAR BCVA improved post

COMET in 82.2% eyes and 55.5% eyes were removed from blindness category. Total

severity score improved in 88.8% eyes and 62.2% eyes were removed from Grade IV

(severe) to Grade III, II and I (less severe). Cytoplasmic MMP9 and SPARC overexpression

was seen in pannus epithelium of 48.9% and 44.4% in eyes while in stroma in 57.7% and

77.7% eyes. Epithelial MMP9 expression correlated with preoperative corneal

neovascularization (p=0.047) and keratinization (p=0.01) while stromal expression correlated

with conjunctival hyperaemia (p=0.14) and symblepharon (p=0.028). SPARC epithelial

expression correlated with conjunctivalization (p=0.018) while stromal expression correlated

with fibrosis (p=0.002). RNA expression of MMP9 and SPARC correlated with fibrosis

(p=0.04) and epithelial hyperplasia (p=0.05). On Univariate analysis, MMP9 and SPARC

epithelial immunoexpression and MMP9 RNA overexpression correlated with poor COMET

graft survival (p=0.02). MMP9 performed in SJS post COMET eyes showed 83.3% positivity.

There was no statistical significance found between MMP9 tear positivity and COMET

surgical outcomes.

Results: Ours is the first study to reveal the possible role of SPARC and MMP9 in promoting

tissue vasculature and remodelling in SJS chronic sequelae. Their over expression

correlated with poor visual acuity outcomes of COMET. MMP9 overexpression in tears was

suggestive of persistent secretion of MMP9 protein in SJS eyes postoperatively leading to

prolonged dryness. Our results may help in further novel therapeutics.

Phd-72

Clinical Relevance of Notch 1 in Lacrimal Gland Adenoid Cystic Carcinoma

Anjum Shahzan1, Sen Seema

1, Kashyap Seema

1, Chosdol Kunzang

3, Pushker Neelam

2,

Bakhshi Sameer4, Bajaj Singh Mandeep

2, Sharma Chand Maher

5, Meel Rachna

2

Ocular Pathology1, Ophthalmology

2, Biochemistry

3, Medical Oncology

4, Pathology

5

All India Institute of Medical Sciences, New Delhi-110029, India

Presenting Author:

Name: Shahzan Anjum

Email: shahzansiddique@yahoo.com

Corresponding Author:

Name: Dr. Seema Sen

Email: ssenop@rediffmail.com

Abstract

Introduction

Adenoid Cystic Carcinoma (ACC) is the commonest epithelial malignant tumour of lacrimal

gland associated with high risk of local recurrence & distance metastasis. There is no targeted

treatment due to lack of well characterized molecular markers. Although Notch signaling is

well established in salivary gland ACC, role of Notch signaling in lacrimal gland ACC has

not been explored.

Aims & Objectives

The present study aims to evaluate the status of Notch1 in ACC & to correlate it with

clinicopathological high risk features.

Material & Methods

Nine prospective cases of histopathologocally proven lacrimal gland ACC & 5 normal lacrimal gland samples

were included in this study. Tumor staging was done by 7th

AJCC, 2007. Histologic classification was divided

into cribriform, tubular & solid patterns. 18 months follow up was available. Protein expression of Notch1 was

evaluated by immunohistochemistry (IHC) using monoclonal antibody (clone: D1E11). Gene expression

analysis of Notch1 gene was done by quantitative real time PCR. The relative mRNA expression of Notch1 in

tumour samples with respect to normal lacrimal gland was calculated by ΔΔCt method.

The results were correlated with clinicopathological high risk features and follow-up.

Results

Of the nine cases of lacrimal gland ACC, there were 8 females & one male with a mean age of 38 years (±25.8).

Right eye was involved in 67% (6/9) patients. Advance T stage (>T2) was found in 67% cases (6/9).

Cytoplasmic overexpression of Notch1 was detected in 88% (8/9) cases. The main histologic growth pattern was

cribriform (n=4), solid pattern in two case, tubular in two cases & mixed pattern observerd in one case.

Perineural invasion was found in 3 cases. Recurrence occurred in 33% cases (3/9), lymph node & systemic

metastasis in 2/9 cases (22%) & two patients with metastasis died. Gene expression analysis of all 9 cases

showed upregulation in 78% cases (7/9). Kaplan Meier survival analysis showed significant association of poor

disease free survival with perineural invasion (p = 0.045) & advance T stage (p= 0.045). No statistically

significant correlation was found between the protein expression of Notch1 & clinicopathological high risk

features.

Conclusion

Overexpression of Notch1 protein & gene was observed in 88% & 78% ACC cases respectively in this study. A

poor disease free survival was found significantly associated with perineural invasion (p=0.045) & advance T

stage (p=0.045). A larger patient cohort would give better insight into role of Notch 1 as a prognostic &

therapeutic target.

Phd-78

Title:

Role of Ataxia Telangiectasia mutated in Uveal Melanoma and its prognostic significance

Name of the authors: Jha Jayanti1, Pushker Neelam2, Singh Kumar Mithalesh 1, Singh Lata3,

Sen Seema1, Kashyap Seema1

Affiliations: 1Department of Ocular Pathology, Dr.R.P.Centre for Ophthalmic Sciences, All India

Institute of Medical Sciences; 2Department of Ophthalmology, Dr.R.P.Centre for Ophthalmic

Sciences, All India Institute of Medical Sciences; 3Department of Biosciences, JMI, New Delhi,

INDIA

Presenting Author:

JAYANTI JHA

Email - jayantijha0210@gmail.com

Corresponding Author:

SEEMA KASHYAP

Email - dr_skashyap@hotmail.com

Abstract Body

Introduction: Uveal melanoma (UM) is the most common intraocular malignancy commonly

arising from the choroid. There is a lack of effective therapy to detect and treat early metastatic

spread in these patients. Therefore, it is important to find new biomarkers that help in early

detection of metastasis of this disease. There is no current study available on UM with respect to

ATM (Ataxia Telangiectasia Mutated) protein that induces DNA damage response. Loss of

nuclear ATM (nATM) in pancreatic, colorectal, gastric cancer was found to be a prognostic

biomarker for cancer progression.

Aims & Objectives: The aim of the study is to determine the efficacy of ATM protein as a

prognostic biomarker in UM. Our objectives are to detect the expression and localization of ATM

protein and correlate its association with clinicopathological parameters and prognostic outcome.

Materials and methods: Expression of nATM was investigated on 69 formalin fixed paraffin

embedded choroidal melanoma samples by immunohistochemistry and validated by western

blotting. Results were then correlated with clinical and histopathological parameters. To determine the prognostic significance, Kaplan–Meier analysis and multivariate analysis by Cox’s Proportional Hazards Model was performed. Results: There was a male preponderance in our study. Histopathological high-risk factors were identified in 30/69 (43.5%) cases. Loss of nuclear ATM was found in 65.2% of the cases. This was statistically significant with epithelioid cell type, high pigmentation, Large Tumor Diameter >10mm, High-Risk Factors>1, tumor height and advanced tumor staging (p<0.05). On multivariate analysis, advanced tumor staging was found out to be an independent prognostic factor. Conclusion: Our data suggest that loss of nuclear ATM protein might serve as a potential prognostic marker in the pathogenesis of uveal melanoma and leads to increased risk of metastasis. These findings demonstrate an important role of ATM protein and may have therapeutic potential in uveal melanoma. However, further studies are required in a larger cohort of patients with longer follow up and translational validation needs to be performed.

Phd-129

Identifying proteomic signatures in aqueous humor of diabetic rat

Bhattacharyya Sukrit 1, Halder Nabanita

1, Singh Basker S.

2, Chawla Rohan

3, Velpandian Thirumurthy

1

1Ocular Pharmacology & Pharmacy Division, Dr. R P Centre for Ophthalmic Sciences,

2Department of Biophysics,

3Department of Ophthalmology, Dr. R P Centre for Ophthalmic Sciences,

Presenting Author:

Name: Sukrit Bhattacharyya

Email: sukritbhattacharyya31@gmail.com

Corresponding Author:

Name: Prof. Thirumurthy Velpandian

Email: tvelpandian@hotmail.com

Introduction: Disease progression related changes in the overall proteome in diabetic

retinopathy has not been monitored. Proteomics being a high-throughput, analytical discipline

to analyze complex biological data sets is well suited to the study of the pathogenic

mechanisms and the biomarker identification of diabetic retinopathy.

Objective: The aim of the present study was to analyse the early stage proteomic profile of

diabetes in the aqueous humor of streptozotocin-induced diabetic rat model.

Materials and Methods: Male Wistar rats(250-300g) were injected with

streptozotocin(45mg/kg) i.p. followed by measurement of blood glucose to characterize the

diabetic state. Fundus imaging was done to assess the retinal structure and vasculature. At

periodic intervals, aqueous humor was collected and subjected for LC-MS/MS based global

proteomics approach to map various proteins.

Results: At the 7th

day post diabetes induction, fundus imaging showed normal retinal

vasculature. Proteomics analysis revealed a greater number of peptide spectral matches in the

diabetic group(400-800) than the control group(300-400). Defence response proteins were

higher in the diabetic group(40 counts), than the control group(22 counts). Counts of proteins

involved in metabolic processes were also more in the diabetic group(70 counts), as

compared to the control group(49 counts).

Conclusion: The study shows that, even though, there are no physiological changes

observed in the early stage of diabetes, changes at the overall protein profile begin to appear,

which is expected to contribute to the later stage diabetic complications. Further studies are in

progress to evaluate and understand the late stage proteomic changes in diabetic rat model.

Key words: Diabetic retinopathy, qualitative proteomics, aqueous humor, Orbitrap, rat

model

Phd-133

QSPR MODELING OF 8 CONGENERIC ANTIFUNGAL AND ANTIPROTOZOAL

AGENTS IN RABBITS

Das U1, Titiyal JS

2, Singh B

3, Sreenivas V

4, Jayaram B

5, Halder N

1, Velpandian T

1

1Ocular Pharmacology and Pharmacy,

2Department of Ophthalmology,

3Department of Biophysics,

4Department of Biostatistics, AIIMS, New Delhi, India

5Department of Chemistry, IIT, New Delhi, India

Presenting Author:

Name: Ujjalkumar S Das

Email: ujjwal87kumar@gmail.com

Corresponding Author:

Name: Prof. Thirumurthy Velpandian

Email: tvelpandian@hotmail.com

Introduction:Prediction of intraocular penetration of novel compounds is an important goal

for ocular drug discovery. In the present study, we attempted to quantitate intraocular

penetration of 8 congenericcompounds belong to antifungals (AFs) and antiprotozoals

(APs)to derive quantitative structure-property relationship (QSPR).

Objectives:The objectives of the present study were 1) to evaluate ocular dispositionof AFs

and APs after systemic administration in rabbits; 2) to derive and correlate vitreous

penetration with physicochemical and molecular dynamic properties.

Materials and Methods:Twelve New Zealand albino rabbits of either sex weighing 1.8-

2.2kg were administeredcassette of 8AFs and APsintravenously at a dose of 10µmoL/kg and

sacrificed using CO2 at predetermined time points (30, 60 and 120min; n=4).AFs and APs

were quantified in plasma, ocular fluidsand tissue samplesusingLC-MS/MS.Percentage

vitreous penetration and penetration index (logVP) for each analyte were calculated and

correlated with calculated molecular properties. Multiple linear regression (MLR) was

employed to generate in-silico prediction model to calculate logVP.

Results:Cassette dosing derived mean percentage vitreous penetration of tinidazole

(71.6±12.9%) was found to be highest followed by fluconazole (67.6±4.7%) > metronidazole

(45.7±6.1%) > voriconazole (44.8±4.9%) > clotrimazole (22.1±9.5%) > miconazole

(13.3±5.2%) > ketoconazole (8.7±1.9%) > itraconazole (5.3±1.1%).MLR model was

generated using parameters viz., band-gap and ionization constant showed best prediction

(R2=0.989, Q

2=0.920, RMSE=0.063, n=8) with respect to plasma concentration.

Conclusion:This study successfully modeled vitreous penetration of experimental

compounds in-vivo in rabbits which further needs to be validated using external set of non-

congeneric compounds.

Key words: Cassette dosing,antifungals,antiprotozoals, mass spectrometry, QSPR

Phd-140

Aqueous humor metabolomics of glaucomatous patients

Gowtham.L1, Nabanita Halder

1, Tanuj Dada

3, Rama jayasundar

2, Baskar singh

4, Velpandian T

1

1Ocular pharmacology & pharmacy, All India Institute of Medical Sciences, New Delhi, India.

2Department of NMR, All India Institute of Medical Sciences, New Delhi, India.

3Department of Ophthalmology, All India Institute of Medical Sciences, New Delhi, India.

4Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.

E mail: gowthamkpm@gmail.com

Introduction: Intraocular pressure is primarily regulated by the endogenous metabolites

which maintain the homeostasis of aqueous humor dynamics. Therefore, understanding the

metabolomic profile of aqueous humor is expected to explain pathophysiology to diagnose

(via biomarkers) and to help optimizing the pharmacological drug management involved in

glaucoma.

Aim &Objective: To determine variations in the levels of both primary and secondary

metabolites(neurotransmitters&autocoids)of glaucomatous patients using NMR and LC-

MS/MS.

Methods:Institutional human ethics committee approval was obtained. This preliminary case

control study involved with two glaucomatous study group of primary open angle & primary

angle closure glaucoma with cataract patients as control. 15 patients were recruited for each

group according to the inclusion & exclusion criteria.70 to 100μL of aqueous humor was

collected by paracentesis during the surgery which was stored at -80°C within 1 hour. For

NMR studies, peak assignments were carried out using NMR Shift database library. The

concentration (mM) of metabolites was determined using Trimethyl silyl propionic acid

(TSP) as reference and their percentage changes were calculated. A sensitive Liquid

chromatography tandem mass spectrometry method was developed and neurotransmitters

were detected through electrospray ionizer using multiple reaction monitoring operated in

positive ion mode. The developed method is having the linearity, accuracy and precision

according to the USFDA bioanalytical method validation guidelines and hence applied

successfully for estimation of metabolite levels.

Results: In thisstudy, NMR spectrumshowed the altered levels of several important primary

metabolites like leucine, alanine, glucose, tyrosine and phenylalanine. Interestingly, LC-

MS/MS analysis revealed the significant elevation in the levels of both excitatory (Glutamic

acid) and inhibitory neurotransmitters (GABA) in both of the glaucomatous patient groups in

comparison to the cataract group (control). Concentrations of acetyl choline, histamine and

1-methyl histamine of glaucomatous group were found significantly declined.

Conclusion: This finding suggests that the impaired levels of both primary precursor

metabolites as well as their product neurotransmitter and autocoid metabolites may play an

important role in altering the homeostasis of aqueous humor dynamics and contribute disease

pathogenesis.

Key words: Metabolomics, Amino acids, Biomarkers, Primary metabolites, Aqueous humor,

glaucoma.

(Corresponding author - Dr. T. Velpandian, Email: tvelpandian@hotmail.com)