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» Ongoing & New Research Programmes |
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Ongoing Research Projects
Basic Research
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Pathogenic Mechanism Of Uveitis Associated With Past Leptospiral Infection
| Investigators: |
Dr. VR. Muthukkaruppan
Dr. SR. Rathinam |
| Research Scholars: |
C. Gowri Priya |
| Funded by: |
Aravind Medical Research Foundation |
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Leptospiral aetiology of uveitis has been demonstrated on the basis of presence of anti-leptospiral LPS antibodies in the serum and pathogenic leptospiral DNA in the aqueous humor. The causative factor responsible for the predominant infiltration of neutrophils and the presence of inflammatory cytokines in aqueous humor of these patients has been identified as LPS. On the basis of the following evidences: (a) A significant level of leptospiral LPS was observed in aqueous humor by dot blot using a monoclonal antibody F70-24, (b) The leptospiral LPS in aqueous humor was serovar specific and the same serovar specific antibody was found in serum of the same patient, (c) Leptospiral LPS induced the production of inflammatory cytokines in whole blood cultures and (d) Infiltrating cells from aqueous humor of leptospiral uveitis patients spontaneously produced inflammatory cytokines in vitro. The study confirms that leptospiral uveitis is different from other forms of uveitis based on the aetiology and the pathogenic mechanism.
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“Two-Parameter Analysis” A Precise Corneal Epithelial Stem Cell Marker
| Prinicipal Investigator: |
Dr. VR. Muthukkaruppan
Dr. M. Srinivasan
Dr. NV. Prajna |
| Research Scholars: |
Arpitha Parthasarathy |
| Funded by: |
Aravind Medical Research Foundation |
The novel combination of the two parameters, high expression of p63 combined with a large N/C ratio was used to identify a subset of limbal cells with putative stem cell characteristics (IOVS 2005). The following evidences confirm that the two parameters in combination form a precise corneal epithelial stem cell marker. Isolated limbal basal cells (Known location for stem cells) contained an enriched population of cells positive for the above marker, having high colony forming efficiency. Such cells possess slow-cycling label (Brdu) retaining property.
Evaluation Of Label Retaining Property Of Cultured Limbal Epithelial Cells In Relation To Two-Parameter Analysis
Limbal explant cultures were labeled briefly with BrdU followed by chase for 3-weeks. Cytospin smears of cells from the outgrowth were double immunostained for p63 and BrdU and then subjected to two-parameter analysis. (A) Confocal image after 2D average reconstruction showing 2 cells positive for p63 (green). A small cell (arrow head) with high levels of p63 and high N/C ratio and a large cell with low N/C ratio (arrow). (B) The same field showing BrdU positive (red) LRC (arrow head). Note: The large cell of (A) negative for BrdU.(C) Overlay image of transmitted light and propidium iodide for cells in (A).
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In vitro and in vivo study on the secretion of Gly367Arg mutant Myocilin protein
| Prinicipal Investigator: |
Dr. P. Sundaresan |
| Co-Invetigators: |
Dr. SR. Krishnadas
Dr. S. Krishnaswamy, MKU, Madurai. |
| Research Scholars: |
J. Kanagavalli |
| Funded by: |
Indian Council of Medical Research |
Mutations in the Myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examined the expression of normal and mutant Myocilin (Gly367Arg) in cultured TM cells. Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. Western blot confocal microscopic analysis was used to determine the cellular localization of Myocilin protein. Molecular Modeling and Dynamics for the mutant was demonstrated with the native Myocilin model using GROMACS. Gly367Arg mutation causes accumulation of Myocilin protein within TM cells with extensively reduced secretion, in contrast to wild type Myocilin. The secreted Myocilin in the aqueous humor of patients with Gly367Arg mutation correlated with the in vitro findings, confirming the disease-causing glaucomatous phenotype. Further, Gly367Arg mutation occurs in a hydrophobic region causing aggregation, leading to burial of a charged residue resulting in the conformational change to accommodate the mutation. Our results suggest that Gly367Arg is a potential mutation causing malfunction of TM cells either by dominant negative effect or gain of function of mutant Myocilin. The structural model indicates the aggregation of Myocilin protein confirming the pathogenic significance of Gly367Arg./mutation.
Confocal microscopic image of TM cells transfected with wild type and mutant Myocilin.
A. Untransfected TM cells showed no endogenous expression of Myocilin.
B. The TM cells showed wild type Myocilin expression in the perinuclear region.
C. Intense staining of Gly367Arg mutant Myocilin in the perinuclear region of TM cells.
D-F. Phase contrast images of panels A-C respectively.
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Molecular Study on Congenital Rubella Syndrome in south Indian population
| Investigators: |
Dr. P. Sundaresan
Dr. P. Vijayalakshmi |
| Collaborators |
Dr. David Brown
Dr. Li Jin
Health Protection Agency, London |
| Research Scholar: |
T. Amala Raja Sundari |
| Funded by: |
World Health Organization, Geneva
ORBIS International, New Delhi |
Congenital rubella Syndrome accounts for significant rate of mortality and morbidity in south India since the vaccination is not mandatory. The main objective of the study is to apply molecular technique such as Real-Time (RT) and nested Block-Based (BB) PCR for detecting the rubella viral RNA in clinical specimens from suspected CRS infants with ocular anomalies. Part of the study has been carried out at Health Protection Agency, London by Ms.Amala.
Samples between age group 0-59 months from IgM IgG positive, IgG positive only and IgM IgG negative categories were investigated. The viral RNA was detected in 85% cases of age between 0-11months who were sero-positive for both anti-rubella IgM and IgG. High percentage of positivity was observed in lens material and oral fluid samples. The positive samples were sequenced and the data was analyzed using DNA star and seqman software program. Totally, 41 samples from 26 cases were
sequenced and showed significant diversity.
Training on Rubella virus culture was also carried out in vero and RK13 cell line using Lab adapted positive Judith strain and the confirmation of viral growth was done by Immunofluorescence and nested BB-PCR.
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Identification of candidate genes and screening for polymorphisms of genes associated with Type ii diabetic retinopathy
| Investigators: |
Dr. P. Sundaresan
Dr. P. Namperumalsamy
Dr. R. Kim |
| Research Scholar: |
B. Suganthalakshmi |
| Funded by: |
TIFAC-CORE in Diabetic Retinopathy
Aravind Medical Research Foundation |
India is crowned as a diabetic capital. Diabetic Retinopathy is a most frequent microvascular complication of diabetes and the leading cause of acquired blindness in developed countries. Diabetic patients with microvascular disease have increased profile of gene expression and enzyme activity, which may be due to variants in the genes encoding angiogenic growth factors and the enzymes involved in the biochemical pathway. To identify the polymorphisms in genes associated with type 2 diabetic retinopathy, 176 Diabetic retinopathy (DR) and 120 diabetic without retinopathy (DNR) samples were screened for polymorphisms in aldose reductase gene (ALR) gene.
Two novel promoter polymorphisms have been identified in ALR gene. The heterozygous genotype of one of the polymorphisms showed higher odds ratio and was found to be significantly associated with DR when compared to DNR.
The aldose reductase activity (AR) in erythrocytes was carried out in 15 DR and 15 DNR study subjects with the help of Dr.Bhanuprakash reddy in National Institute of Nutrition (NIN), Hyderabad. The AR activity was higher in both DR and DNR group (>5u/g Hb) when compared with the control samples (2.89u/g Hb, n=500) of NIN. There is no difference in AR activity between DR and DNR groups. Further studies are underway with larger sample size. |
Studies on the proangiogenic and vascular growth factors in relation to the pathogenesis of Eales’ disease and Diabetic Retinopathy
| Investigators: |
Dr. VR. Muthukkaruppan
Dr. P. Namperumalsamy
Dr. R. Kim
Dr. Dhananjay Shukla
Dr. R. Anand Rajendran |
| Research Scholar: |
P. Murugeswari |
| Funded by: |
TIFAC-CORE Department of Science and Technology |
| Giemsa stained cytospin smear of RPE Cells |
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| Cadaver RPE cells | RPE D407 cell line |
Phase Contrast images showing live cultures of Retinal Pigment Epithelium
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| RPE D407 cell line 4-day confluent culture | Cadaver RPE cells 28-day confluent culture |
To understand the pathogenic mechanism of Diabetic Retinopahty, earlier we demonstrated the levels of Proinflammatory cytokines (IL-6, IL-8, IL-1B), Chemokine factor (MCP-1), angiogenic growth factor (VEGF) and antiangiogenic factor (PEDF) in the vitreous, aqueous and serum of Proliferative diabetic retinopathy, Eales’ disease and Macular Hole patients. Diabetic retinopathy is also an inflammatory disease as reported earlier. As PEDF is secreted by RPE cells we are interested in studying the cytokines and growth factors secreted by RPE cells.
Cytokine levels in the conditioned medium of Retinal Pigment Epithelium (RPE)
- In conditioned medium of primary cultures, constitutive expression of IL6, IL8 and VEGF was observed.
- In the RPE D407 cell line constitutive expression of IL6 and VEGF were observed
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Molecular Genetic Analysis Of Autosomal Recessive Congenital Hereditary Endothelial Dystrophy
| Principal Investigators: |
Dr. P. Sundaresan |
| Co-Investigators: |
Dr. M. Srinivasan
Dr. J. Arun Kumar |
| Research Scholar: |
B. Hemadevi |
| Funded by: |
Aravind Medical Research Foundation & Singapore Eye Research Institute |
Pedigree of Family 1
RFLP analysis of R869C mutation
M-Marker, C-Control
Congenital Hereditary Endothelial Dystrophy (CHED) is a heritable disorder of the corneal endothelium characterised by bilateral, diffuse corneal clouding, which impairs visual acuity, occurring at or soon after birth. CHED can be Autosomal dominant (CHED1 [MIM %121700]) or Autosomal Recessive (CHED2 [MIM %217700]) with the later being more common and severe. Both CHED1 and CHED2 map to chromosome 20 at two distinct loci. This collaborative study has identified SLC4A11 as a novel candidate gene for CHED2 (Nat.Genet.2006).
Further, we have been screening CHED2 families for mutations in this gene. As a result of screening, in the Family 2, nonsense mutation was identified causing a premature stop at codon 605 (R605X). Two missense mutations R869C and R755Q were identified in Family 1 and Family 3 respectively. In addition R755 and R869 residues were highly conserved among orthologs and paralogs. In family 4, a 4bp deletion (353_356delAGAA) results in an aberrant truncated protein of 128 residues. In Family 5, a deletion insertion in intron 15 (IVS15 -6 _ -16 delins GGCCGGCCGG) was identified. None of these mutations were identified in 50 controls. The identification of mutations in all families analyzed in this study indicates that the recessive CHED is genetically homogeneous.
RFLP analysis was carried out using Msp1 restriction enzyme. The Proband (V-7) and his sister (V-6) were homozygous for the R869C mutation while their parents (IV-7, IV-8)) and grandmother (III-3) were heterozygous for the mutation.
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Genetic and functional analysis of FOXL2 gene in Indian Patients with BPES syndrome
| Investigators: |
Dr. P.Sundaresan |
| Co-Investigators: |
Prof. Reiner A. VEITIA
Génétique-Génomique
Professeur de Université Paris VII
Faculté de Médecine Port Royal/ Hôpital Cochin
24, rue de Faubourg St.Jacques. 75014 Paris
Dr.Usha Kim |
| Research Scholar: |
J. Nallathambi |
| Funded By: |
French Embassy New Delhi and EGIDE France |
The blepharophimosis syndrome (BPES) is an autosomal dominant developmental disorder in which craniofacial/eyelid malformations are associated (type I) or not (type II) with premature ovarian failure (POF). Mutations in the FOXL2 gene, encoding a forkhead transcription factor, are responsible for both types of BPES. Heterozygous polyalanine expansions of +10 residues (FOXL2-Ala24) account for 30% of FOXL2 mutations and are fully penetrant for the eyelid phenotype. Here we describe the first homozygous FOXL2 mutation leading to a polyalanine expansion of +5 residues (FOXL2-Ala19). This novel mutation segregates in an Indian family where heterozygous mutation carriers are unaffected whereas homozygous individuals have the typical BPES phenotype, with proven POF in one female. Expression of the FOXL2-Ala19 protein in COS-7 cells revealed a significantly higher cytoplasmic retention compared to the wild-type protein. This is the first study providing genetic evidence for a recessive inheritance of BPES associated with ovarian dysfunction (published in Human genetics, November 2006).
Pedigree of the Five-generation consanguineous Indian family in which a typical BPES phenotype in homozygous individuals (Individuals I:1 and II:3 were represented as affected based on family history). Bottom; Agarose gel electrophoresis (3%) of a PCR amplicon shows segregation of FOXL2–polyAla19 expansion c.684–698dup15 (p.A228_A232dup) in two affected and five unaffected individuals. M 100 bp DNA marker, C unrelated healthy control; the number symbols refer to the pedigree, Bl negative control for PCR reaction. Unaffected individuals III:5, III:6, III:8, IV:6 and IV:8 carry a wild type and an expanded allele (PCR fragment of 304 and 319 bp, respectively); affected individuals are homozygous for the expanded allele and the control individual only carries a wild type band.
Subcellular localization of FOXL2–Ala19. COS-7 cells were transfected with constructs driving overexpression of FOXL2–Ala14 (wild type), Ala19 and Ala24 fused to the GFP. FOXL2–Ala24 was included for comparison. After 48 h of transfection FOXL2–Ala19 was mislocalized in the cytoplasm in 15% of transfected cells but did not induce significant intranuclear aggregation (compared to the effect of Ala24).
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New Research Projects
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Pathogen host interaction in human mycotic keratitis
| Pricipal Investigator: |
Dr. N. Venkatesh Prajna |
| Co Investigators: |
Dr. K. Dharmalingam
Sr. Professor & Head
School of Biotechnology
Madurai Kamaraj University
Dr. S. Lalitha |
| Research Scholars: |
S. Ananthi |
| Funded by: |
Department of Biotechnology, Government of India |
Fungal keratitis is an important cause of corneal blindness in India. The purpose of this study was to elucidate the alterations in the tear proteins of fungal keratitis patient, which may have a bearing on pathogenesis. Tear samples were collected from culture positive fungal keratitis patients. Tears from the fellow eye and from other healthy individuals served as controls.
Two-dimensional (2D) electrophoresis was used for separation of fractionated infected tear proteins and control tear proteins. MALDI TOF based detection of selected protein spots were performed from the gels. A MASCOT search engine was used for further identification of proteins.
The Glutaredoxin related protein was expressed only in the tears of fungal keratitis patients. Six other normal tear proteins were present in both samples, but with varied expression. Secretory actin-binding protein and Serum albumin precursor were up-regulated in the infected samples. Cystatin S precursor, cystatin SN precursor, cystatin and human tear lipocalin were down regulated in the infected samples.
Glutaredoxin related protein is known to be produced by Aspergillus fumigatus during oxidative stress conditions and presence of this protein in the tears of patients with fungal keratitis is of considerable interest.
250 µg of deoxycholate precipitated Aspergillus infected tear sample was loaded. IEF was performed using IPG pH 3-10 NL strips, followed with 12% SDS-PAGE and Coomassie G250 stained. The position of molecular mass markers on right side and position of pI on the top of the gel is shown. |
Identification of Genetic Defects Occurring In Indian Oculocutaneous (OCA) and Ocular Albinism (OA) Families
| Principal Investigator: |
Dr. P. Sundaresan |
| Co-Investigator: |
Dr. Vijayalakshmi Perumalsamy
Dr. Asim Kumar Sil |
| Research Scholar: |
K. Renugadevi |
| Funded by: |
Department of Biotechnology |
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The purpose of this study is to detect albinism gene exonic mutations and its Co-inheritance in Indian Oculocutaneous (OCA1) or Ocular albinism (OA1) families. We have so far collected 98 DNA samples from patients.
The objectives are
- Linkage analysis using the genetic markers near each known albinism genes on chromosomes 5,6,9,11,15 & X for Oculocutaneous and Ocular albinism patients in India.
- To develop a rapid diagnostic method for albinism (OCA1/OA1) using PCR-RFLP for early carrier detection and genetic counseling for patients.
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Bioinformatics in Ophthalmology
| Investigator: |
Dr. VR.Muthukkaruppan
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| Collaborator: |
Dr. S. Krishnasamy
Madurai Kamaraj University |
| Research Scholar: |
M. Vanitha |
| Funded by: |
Aravind Medical Research Foundation |
Among the “New Kinds of Science” emerging from the convergence of computing and science, a crucial role is played by BIOINFORMATICS. Bioinformatics can provide its support to diagnostic medicine, especially in imaging systems, developing databases on eye diseases. Ophthalmology is no exception to this. The use and advantages of bioinformatics can be utilized.
The purpose of this project is to develop database on Eye diseases and in particular to start with Primary Open Angle Glaucoma. Aravind Eye Care System has enormous number of data in all levels, Clinical, epidemiological and as well genetic analysis. Database can be created using programming skills and softwares. Zope is an open source application server for building content management systems, intranets, portals, and custom applications. Using PostgreSQL and Python scripts a database will be created with Genetic, Clinical and Epidemiological data in a single source. With regard to software development, Electronic Medical Records can be maintained in Aravind Eye Care System, so that database management will be much easier |
Will Cytoskeletal Drugs Prevent Posterior Capsule Opacification?
| Principal Investigators: |
Dr. VR. Muthukkaruppan
Dr. Baohe Tian, Department of Ophthalmology and Visual Sciences,
University of Wisconsin-Madison, (UW), USA
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| Co-Investigators: |
Dr. Hari Priya |
| Research Scholar: |
M. Jeyalakshmi |
| Funded by: |
National Eye Institute, NIH, USA |
The main objective of the project is to determine whether the cytoskeletal drugs LAT-B or H-7 would help in clearing all the lens epithelial cells during cataract surgery in cadaver human eyes. The experiments are designed to evaluate the number/area of residual lens epithelial cells immediately after surgery with and without treatment by cytoskeletal drugs. The second set of experiments will study the effects of cytoskeletal drugs on proliferation and migration of lens epithelial cells in cultured human lens capsular bag after cataract surgery. The PCO and accompanying changes in the cultured lens capsule will be observed under a microscope for several weeks.
If we could demonstrate the efficacy of this drug in removing the residual lens epithelial cells and reducing the levels of PCO in culture, it is possible to have a safe and effective means to reduce incidence of PCO and consequent decrease in post operative vision following cataract surgery in humans.
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Corneal Surface Reconstruction Using Bio-Engineered Autologous Oral (Buccal) Mucosal Epithelium
| Investigators: |
Dr. VR. Muthukkaruppan
Dr. N. Venkatesh Prajna
Dr. Usha Kim
Dr. M. Srinivasan |
| Research Scholar: |
P. J. Jeya Prita |
| Funded by: |
Defence Research and Development Organization –
Life Science Research Board (DRDO – LSRB) |
The purpose of this study is to generate buccal epithelial sheet by culturing patient’s own buccal mucosal epithelium under appropriate culture conditions that will be transplanted onto the cornea of the patient with total limbal stem cell deficiency. This study aims at characterising the cellular profile and to analyze the presence of stem cells in the bioengineered buccal mucosal epithelial sheet on the basis of expression of various markers like p63, Cytokeratin 3, 5, 10, 12, 14, 19, Cx43 and ABCG2 and high N/C ratio and comparing them with the limbal and corneal epithelial stem cells. It is possible to generate large colonies from isolated buccal mucosal epithelial cells. |
Application of multiplex PCR in the diagnosis of infectious retinitis
| Investigators: |
Dr. Lalitha Prajna
Dr. Rathinam SivaKumar
Dr. R.Kim |
| Funded by: |
ICMR |
The main goals of this project are to detect the causative agents like Herpes virus, Cytomegalovirus and Varizella zoster virus in cases of infectious uveitis and retinitis by multiplex PCR for more rapid results.
The Ocular Microbiology Laboratory has established molecular based diagnosis using Polymerase chain reaction techniques for the common infectious agents of the eye. Nested PCR is also used which helps to increase the specificity of the tests. These tests are capable of detecting viral infections like Herpes virus, Cytomegalovirus and Varizella zoster virus, parasitic like toxoplasmosis and common bacterial and fungal infections. Techniques are also available for the detection of Propionibacterium acnes and Mycobacterium tuberculosis. Nested PCR is useful for the diagnosis especially in ocular tuberculosis where routine microbiological culture is not always feasible. We have tested 20 suspected ocular tuberculosis patient’s samples and based on these results it was possible to start specific therapy. The number of samples with suspected viral etiology that was tested by nested PCR was 30.
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