Veerappan Muthukkaruppan

Proinflammatory cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative diabetic retinopathy and eales' disease

Purpose: To investigate the mechanism of angiogenesis in proliferative diabetic retinopathy (PDR) and Eales’ disease (ED) on the basis of the levels of proinflammatory cytokines, angiogenic growth factor, and antiangiogenic factor in the vitreous humor. Methods: Twenty-five patients with PDR, 10 patients with ED, and 25 with macular hole (MH) as control subjects were studied. The concentration of the proinflammatory cytokines interleukin-6 (IL-6), IL-8, IL-1&bgr;; chemokine-monocyte chemoattractant protein-1 (MCP-1); angiogenic factor-vascular endothelial growth factor (VEGF); and antiangiogenic factor-pigment epithelium derived factor (PEDF) in the vitreous fluid obtained from the eyes during vitrectomy were measured by sandwich enzyme linked immunosorbent assay (ELISA). Results: IL-6, IL-8, MCP-1, and VEGF levels in the vitreous were significantly higher in PDR (P < 0.0001) and ED (P < 0.0001) than in MH patients. Conversely, the vitreous level of PEDF was significantly reduced in PDR (P < 0.0001) but not in ED. A significant correlation was observed between VEGF and IL-6 in ED patients. Conclusion: The authors demonstrate the importance of VEGF in retinal neovascularization of ED which is an idiopathic inflammatory venous occlusion. Further study is required to understand the interrelationship between VEGF and inflammatory cytokines in PDR and ED.

LruA and LruB Antibodies in Sera of Humans with Leptospiral Uveitis

ABSTRACT Uveitis can be a serious complication of leptospirosis. Previous studies indicated that the leptospiral lipoproteins LruA and LruB are expressed in the eyes of uveitic horses and that antibodies directed against those proteins show in vitro cross-reactivity with components of equine lens, ciliary body, and/or retina. We now demonstrate that sera from a significant proportion of humans who have leptospiral uveitis also contain antibodies against LruA and LruB. Different categories of nonleptospiral uveitis and autoimmune uveitis were also screened; patients diagnosed with Fuchs uveitis or Behçets syndrome produced antibodies that cross-reacted with LruA and LruB, suggesting similarities of the autoimmune responses in those diseases with those of leptospiral uveitis.

Identification of Human Corneal Epithelial Stem Cells on the Basis of High ABCG2 Expression Combined With a LargeN/C Ratio

Till date there is no exclusive marker for human corneal epithelial stem cells (CESCs). In this study, our strategy is to combine high expression of ABCG2, a putative SC marker with high N/C ratio to develop a specific method for identification of CESCs.

A subset of human limbal epithelial cells with greater nucleus-to-cytoplasm ratio expressing high levels of p63 possesses slow-cycling property

Purpose: The purpose of this study was to evaluate the subset of limbal epithelial cells with greater nucleus-to-cytoplasm (N/C) ratio expressing high levels of p63 for their slow-cycling property, a characteristic feature of stem cells (SCs). Methods: Limbal and peripheral corneal explant cultures were pulse labeled with 5-5-bromo-2′-deoxyuridine (BrdU) for 5 days, followed by a period of 3-week chase. Cultured explants were cryosectioned and stained for BrdU. The epithelial cells in the outgrowth and those remaining on the explant were isolated and subjected to cytospin and double immunostaining for BrdU and p63, followed by identification of label-retaining cells (LRCs) and quantification of p63 expression using confocal microscopy. Results: A distinct population of small cells with large N/C ratio expressing high levels of p63 retained the BrdU label after 21-day chase. Further, this population of LRCs, negative for the differentiation marker K3, was observed in the epithelial outgrowth of limbal but not in that of peripheral cornea. LRCs were seen to migrate along the cut edge of limbal explants in culture and were also observed as clusters of small cells in the outgrowth, which contained cells with the ability to form holoclone colonies. Conclusions: These results demonstrate that the small cells with large N/C ratio and high levels of p63 have BrdU label retaining slow-cycling property, thus confirming that these 2 parameters in combination may serve as a precise marker for identification and quantification of ex vivo-expanded limbal SCs. This method would be useful to standardize the optimal culture conditions that can maintain and expand SCs for therapeutic applications.

A pilot study on the infiltrating cells and cytokine levels in the tear of fungal keratitis patients

AIM To determine the cellular profile and cytokine levels in the tear fluid of fungal keratitis patients. MATERIALS AND METHODS Tear samples were collected from six fungal keratitis patients (Group I) from active stages of the disease up to resolution. Tears collected from the following served as controls: uninfected fellow eye (Group II A) of Group I, patients undergoing cataract surgery (Group II B) and acute conjunctivitis (Group II C). The cellular profile was evaluated. Interleukines (IL-6, IL-8 and IL-1beta) were estimated using sandwich enzyme immunoassay. Statistical analysis was carried out using nonparametric two-sample median test. RESULTS Polymorphonuclear leukocytes (PMN) were the predominant infiltrating cells in Group I. During the initial stages of fungal infection, levels of IL-6 and IL-8 in the tear samples were found to be significantly increased when compared with Group II A (P=0.019 for IL-6, P<0.001 for IL-8). This was also true for IL -8 (P=0.008) levels in Group I and Group II B). While IL-6 levels decreased significantly towards healing, IL-8 remained slightly elevated even after healing. These cytokines were at the base level in Group II A. Lymphocytes and PMN were present in equal proportions in Group II C, which showed elevated levels of cytokines but not to the extent of Group I. CONCLUSION This horizontal study indicates that understanding the nature of the inflammatory response in the tears of fungal keratitis patients is of considerable interest and warrants further investigations.

Evidence for Endotoxin as a Causative Factor for Leptospiral Uveitis in Humans

PURPOSE To understand the pathogenic mechanism of leptospiral uveitis by determining the profile of infiltrating cells, the levels of cytokines, and the causative factor in aqueous humor (AH). METHODS AH and blood samples were collected from 22 patients with leptospiral uveitis that was confirmed by microscopic agglutination test (MAT). Nine patients with Behçets uveitis, 10 with phacolytic uveitis, and 13 with age-related cataract were included as control subjects. A cytometric bead array was used to estimate human inflammatory and Th1/Th2 cytokines. The level of endotoxin in AH was estimated by limulus amebocyte lysate (LAL) test and by dot blot analysis using a leptospiral serovar lipopolysaccharide (LPS)-specific monoclonal antibody. RESULTS Except for one patient with leptospiral uveitis, AH from all other patients and control subjects was negative for Gram-negative bacterial endotoxin by LAL test. However, a significant level of serovar Copenhageni LPS was observed in AH of patients with leptospiral uveitis seropositive for the same serovar by MAT, in contrast to its absence in all control subjects. A selective infiltration of neutrophils as well as a significant increase in the levels of protein and cytokines IL-12p70, TNF, IL-6, IL-8, and IL-10 was observed in AH of patients with leptospiral uveitis. Phacolytic uveitis was associated with a high proportion of activated macrophages and increased levels of IL-6 and IL-8, whereas Behçets uveitis was associated with a predominant infiltration of neutrophils and increased levels of IFN-gamma. CONCLUSIONS The results demonstrate the presence of serovar-specific LPS in AH, and thus it is likely that endotoxin is a causative factor in leptospiral uveitis.

Prevalence of eye signs in congenital rubella syndrome in South India: A role for population screening

Purpose: Congenital rubella syndrome (CRS) resulting from maternal rubella infection, especially in the first trimester, affects an estimated 100 000 infants each year worldwide. Immunisation has reduced its occurrence in the developed world, though it remains a problem in countries with poor immunisation coverage. This population-based study was aimed at screening children below 5 years of age for ocular signs suspicious of CRS. Methods: Suspected CRS cases were recruited from hospital and outreach services of the Aravind Eye Care System over a 24-month period. Clinical confirmation was based on the fulfilment of the World Health Organization (WHO) definition, and laboratory confirmation was based on a positive test for IgM antibody. Results: Children under 5 years of age (n = 51 548) with ocular complaints were screened for eye signs suspicious of CRS; CRS compatible signs were detected in 1.92% (1090) children. Of these suspects (299), 27.42% were subsequently confirmed clinically according to WHO definition, and (46) 4.2% were serologically (Laboratory) confirmed. Of all the eye signs evaluated for screening, cataracts were the most sensitive (80.43%). Conclusions: Cataracts among children have a high sensitivity for detecting CRS in India. It is the only clinical eye finding that has a high enough sensitivity and specificity to be useful as a screening tool for CRS.

A method to isolate human limbal basal cells enriched for a subset of epithelial cells with a large nucleus/cytoplasm ratio expressing high levels of p63

The objectives were to develop method of isolating viable human limbal basal cells in order to enrich a subset of small cells with a large Nucleus/Cytoplasm (N/C) ratio expressing high levels of p63, nuclear protein. Limbal tissues were treated with trypsin for 50 min at 37°C in an orbital shaker at 100 rpm with epithelial side down followed by additional 5 min with epithelial side up and then with Dispase II to obtain various epithelial fractions. Isolated cell fractions were assessed for colony forming efficiency and ΔNp63α, connexin (Cx43) mRNA levels. Cytospin smears were double‐immunostained for p63 and any one of the stem cell (SC) related markers and analyzed using a laser scanning confocal microscope and advanced image analysis software (Leica Confocal software, 2.61 build 1537 version) for quantification of fluorescence intensity. The isolated limbal basal cells were highly positive for ΔNp63α mRNA but expressing low Cx43 mRNA. They gave rise to higher number of large colonies with compact morphology in contrast to the limbal suprabasal/superficial (LS/S) colonies. Furthermore, a subset with a large N/C ratio expressing high levels of p63 was observed, as much as 25% among the limbal basal cell fraction, in contrast to only about 4% in the total limbal epithelial cells. Such cells were positive for K5 and negative for Ki67, Cx43, and 14‐3‐3s and were absent in the LS/S fraction. These results collectively substantiate our method of isolation of limbal basal layer cells containing an enriched population of cells with SC phenotype. Microsc. Res. Tech. 2008.

In vivo Confocal Microscopic Analysis of Limbal Stroma in Patients With Limbal Stem Cell Deficiency.

Purpose: Using in vivo confocal microscopy, we established that unique hyperreflective structures in the anterior limbal stroma of healthy individuals represent the limbal stromal niche. The aim of this study was to characterize the limbal stromal microarchitecture in patients with limbal stem cell deficiency (LSCD). Methods: After obtaining informed consent, 10 patients with LSCD and 3 with macular corneal dystrophy were recruited. In vivo confocal imaging of the limbus and cornea of the affected and normal eyes was performed using an HRT III laser scanning microscope, beyond the epithelium deep into the stroma. Results: In the case of LSCD, the limbal epithelium was replaced by conjunctival epithelium. A large number of inflammatory and dendritic cells were identified along with blood vessels from the epithelium to deep stromal layers. The unique hyperreflective niche structures were replaced by homogenously bright fibrous structures in all eyes with total LSCD. In patients with partial LSCD, even the clinically defined normal limbus had fibrotic stroma. In a patient with focal LSCD, only the affected limbal stroma remained fibrotic, whereas the adjacent clinically normal limbus had the unique hyperreflective structures. Although the opaque corneal stroma appeared bright because of proteoglycan deposition, it was possible to identify the normal limbal epithelial and stromal architecture in macular corneal dystrophy. Conclusions: In the case of LSCD, the limbal stromal niche was replaced by bright fibrotic structures indicating persistence of damage several months after injury. Further studies are required to characterize the sequential events occurring in the anterior limbal stroma after injury using this noninvasive method.

Identification of glaucomatous optic nerve head changes in Indian donor eyes without clinical history

Purpose: The purpose of this study is to develop methods to identify glaucoma by examining the optic nerve head (ONH) of donors eyes when information on the preexisting ocular disease is unavailable. Materials and Methods: The ONH of the donors eyes was evaluated under a stereomicroscope for the cup-disc ratio (CDR) and focal retinal rim thinning. The vertical diameter of the cup and disc was also measured using a precalibrated eyepiece micrometer. The suspect eyes were subjected to histological analysis to confirm the presence of specific glaucomatous changes. Results: A total of 202 eyes from 119 donors (68 males and 51 females, aged 42–96) were evaluated for glaucoma. Among them, 190 (94%) eyes showing vertical CDR in the of 0.0–0.6 range were considered nonglaucomatous and the remaining eyes with >0.6 as glaucoma suspect. The calculated mean CDR of the two groups (0.3 ± 0.16, 0.62 ± 0.27) was highly significant (P = 0.0003). Of 12 eyes suspected of glaucoma, 7 eyes from 5 donors showed specific glaucomatous changes by histology. The prevalence of glaucoma was 4.2% among the donors studied. Conclusions: A simple method of screening fresh donor eyes for selecting those with glaucoma features using CDR and histological analysis was reported. This method helps to obtain biologically active human ocular tissue for glaucoma research on gene expression, ultrastructural/proteome changes, and outflow mechanism.