DNA damage response proteins and its role in tumor progression of uveal melanoma with patient outcome.

BACKGROUND: The role of DNA damage response (DDR) proteins is poorly understood in uveal melanoma. ATR belongs to one of those proteins that induce DDR by arresting the cell cycle which leads to DNA repair. ATR is localized at position 23 on the same chromosome 3 where BAP1 is located at position 21.1 which is a known poor prognostic marker of UM. The aim of our study is to detect the expression of ATR at the protein and RNA levels and determine its prognostic significance. METHODS: Expression of nuclear ATR was investigated on sixty-nine UM patients. Formalin-fixed paraffin-embedded choroidal melanoma samples were taken to evaluate the expression of ATR. Fifty samples were also validated by real-time PCR. Results of both protein and mRNA were then correlated with clinicopathological parameters. To determine the prognostic significance, Kaplan-Meier and multivariate analyses were performed. RESULTS: Loss of ATR protein was seen in 72% cases which was statistically significant with epithelioid cell type (p = 0.005), tumor thickness (p = 0.016), mitotic figures (p = 0.001) and BAP1 loss (p < 0.001). At the transcriptional level loss of ATR was seen in 76% cases which were statistically significant with metastasis (p = 0.046), staging (0.044) and loss of BAP1 (p = 0.022). On multivariate analysis loss of ATR and tumor staging came out to be independent prognostic parameters. CONCLUSION: Our data suggest that ATR might serve as a potential prognostic marker in UM patients and could serve as a potential therapeutic target.

A study of changes in levator muscle in congenital ptosis.

PURPOSE: To study microscopic and ultrastructural changes of levator palpebrae superioris (LPS) muscle in congenital ptosis. METHODS: In this prospective observational study, LPS muscle was studied in 77 eyelids with congenital ptosis; 35-simple congenital ptosis (SCP), 12-Marcus Gunn jaw winking phenomenon (MGJWP), and 30-blepharophimosis epicanthus inversus syndrome (BPES). Light microscopy, enzyme histochemistry, immunohistochemistry and electron microscopy were performed, and results were analyzed. RESULTS: Muscle fibers were detected in 83.33% of MGJWP, 22.86% of SCP and 16.67% of BPES eyelids. Fibers were detected significantly more in individuals with moderate ptosis, LPS action > 4 mm, present eyelid crease and eyelid fold. Severe endomysial and perimysial fibrosis was seen significantly more in individuals with MGJWP. Fat infiltration and nuclei internalization were seen in all three groups. The absence of degenerating or regenerating fibers and inflammatory cells, normal staining pattern on immunohistochemistry and absence of accumulation of any abnormal substance were found in all three groups. Abnormal mitochondrial staining pattern was seen occasionally in three groups. On electron microscopy, muscle was detected in 1 SCP eyelid and 8 MGJWP eyelids out of which 4 had myofibrillary disruption. All other eyelids where muscle fibers were not detected had only fibrocollagenous tissue. CONCLUSION: Fibrocollagenous tissue predominated in all the cases, and muscle fibers detected correlated inversely with the severity of ptosis. The absence of degenerating, regenerating fibers and inflammatory cells supported the theory of dysgenesis of muscle. However, internalization of nucleus seen in all the subtypes is a feature favoring dystrophy.

Ultrasound biomicroscopy image patterns in normal upper eyelid and congenital ptosis in the Indian population.

PURPOSE: To study the features of upper eyelid in healthy individual and different types of congenital ptosis in the Indian population using ultrasound biomicroscopy (UBM). METHODS: This was a prospective observational study at a tertiary care center. Eyelid structure of healthy individuals with no eyelid abnormalities (n = 19); simple congenital ptosis (n = 33) cases; Marcus Gunn jaw-winking ptosis (MGJWP, n = 7) cases, and blepharophimosis-ptosis-epicanthus inversus syndrome (BPES, n = 20) cases were studied on a vertical UBM scan using 50-MHz probe. Lid-thickness, tarsal-thickness, orbicularis oculi and levator-Muller-orbital septum-conjunctival (LMSC) complex were measured in primary gaze. Comparison was made between four groups and results were statistically analyzed using ANOVA test. In normal individuals, LMSC measurements were repeated in down-gaze imaging. RESULTS: Skin with subcutaneous tissue, LMSC complex and pre-aponeurotic fat-pad appeared echodense while orbicularis oculi and tarsus appeared echolucent. In primary gaze, mean thickness (± standard deviation) of the eyelid, tarsus, orbicularis oculi and LMSC, respectively, were: 1.612 ± 0.205, 0.907 ± 0.098, 0.336 ± 0.083, and 0.785 ± 0.135 mm in normal individual. LMSC showed 46.64% increase in thickness on down-gaze. The mean eyelid thickness and LMSC were thicker in MGJWP and BPES as compared to normal. In different types of congenital ptosis cases, various patterns of UBM imaging were observed. CONCLUSION: UBM allows noninvasive imaging of eyelid structures with good anatomical correspondence in normal eyelids and study the structural alterations of eyelids in different types of congenital ptosis. UBM can be used to highlight the anatomical difference in normal eyelids that may help modify the surgery for better cosmetic outcomes. Furthermore, it has the potential to be used in preoperative evaluation and operative planning in certain types of acquired ptosis, which needs to be evaluated.

S2'-subsite variations between human and mouse enzymes (plasmin, factor XIa, kallikrein) elucidate inhibition differences by tissue factor pathway inhibitor -2 domain1-wild-type, Leu17Arg-mutant and aprotinin.

Essentials Current antifibrinolytics - aminocaproic acid and tranexamic acid-can cause seizures or renal injury. KD1L17R -KT , aprotinin and tranexamic acid were tested in a modified mouse tail-amputation model. S2'-subsite variations between human and mouse factor XIa result in vastly different inhibition profiles. KD1L17R -KT reduces blood loss and D-dimer levels in mouse with unobserved seizures or renal injury. SUMMARY: Background Using tissue factor pathway inhibitor (TFPI)-2 Kunitz domain1 (KD1), we obtained a bifunctional antifibrinolytic molecule (KD1L17R -KT ) with C-terminal lysine (kringle domain binding) and P2'-residue arginine (improved specificity towards plasmin). KD1L17R -KT strongly inhibited human plasmin (hPm), with no inhibition of human kallikrein (hKLK) or factor XIa (hXIa). Furthermore, KD1L17R -KT reduced blood loss comparable to aprotinin in a mouse liver-laceration model of organ hemorrhage. However, effectiveness of these antifibrinolytic agents in a model of hemorrhage mimicking extremity trauma and their inhibition efficiencies for mouse enzymes (mPm, mKLK or mXIa) remain to be determined. Objective To determine potential differences in inhibition constants of various antifibrinolytic agents against mouse and human enzymes and test their effectiveness in a modified mouse tail-amputation hemorrhage model. Methods/Results Unexpectedly, mXIa was inhibited with ~ 17-fold increased affinity by aprotinin (Ki ~ 20 nm) and with measurable affinity for KD1L17R -KT (Ki ~ 3 µm); in contrast, KD1WT -VT inhibited hXIa or mXIa with similar affinity. Compared with hPm, mPm had ~ 3-fold reduced affinity, whereas species specificity for hKLK and mKLK was comparable for each inhibitor. S2'-subsite variations largely accounted for the observed differences. KD1L17R -KT and aprotinin were more effective than KD1WT -VT or tranexamic acid in inhibiting tPA-induced mouse plasma clot lysis. Further, KD1L17R -KT was more effective than KD1WT -VT and was comparable to aprotinin and tranexamic acid in reducing blood loss and D-dimer levels in the mouse tail-amputation model. Conclusions Inhibitor potencies differ between antifibrinolytic agents against human and mouse enzymes. KD1L17R -KT is effective in reducing blood loss in a tail-amputation model that mimics extremity injury.

Impression cytology diagnosis of ulcerative eyelid malignancy.

OBJECTIVE: The utility of impression cytology in ocular diseases has predominantly been restricted to the diagnosis of dry eye, limbal stem cell deficiency and conjunctival neoplasias. Its role in malignant eyelid lesions remains largely unexplored. Although scrape cytology is more popular for cutaneous lesions, impression cytology, being non-traumatic, has an advantage in small and delicate areas such as the eyelid. The present study has been designed to evaluate its role in the diagnosis and management of malignant eyelid lesions. METHODS: Thirty-two histopathologically proven malignant eyelid lesions diagnosed over a 2-year period, including 13 basal cell carcinomas, 11 sebaceous carcinomas, four squamous cell carcinomas, two malignant melanomas and two poorly differentiated carcinomas, formed the study group. RESULTS: The results of impression cytology were compared with those of histopathology in the study group and with an age- and sex-matched group of benign cases as controls. The sensitivity of impression cytology was 84% (27/32) for the diagnosis of malignancy and 28% (9/32) for categorization of the type of malignancy. CONCLUSIONS: Impression cytology is a simple, useful, non-invasive technique for the detection of malignant ulcerative eyelid lesions. It is especially useful as a follow-up technique for the detection of recurrences.

Epigenetic inactivation of the E-cadherin gene in eyelid sebaceous gland carcinoma.

BACKGROUND: E-cadherin and ß-catenin are crucial components of the cell-cell adhesion complex. Their loss has often been associated with tumour metastasis and poor clinical outcome. Both loss of E-cadherin at the cell membrane and a stabilizing mutation in CTNNB1 (ß-catenin gene) have been associated with ovarian, colorectal, hepatocellular and nonmelanoma skin cancer, such as squamous and basal cell carcinomas. Absence of E-cadherin may be caused by promoter hypermethylation of the E-cadherin gene (CDH1). OBJECTIVES: To determine the role of E-cadherin promoter hypermethylation and CTNNB1 gene mutation in the aggressive behaviour of sebaceous gland carcinoma of the eyelid. METHODS: Thirty-six cases of sebaceous gland carcinoma were subjected to E-cadherin methylation-specific polymerase chain reaction and mutational analysis for the CTNNB1 gene. E-cadherin and ß-catenin staining was evaluated by immunohistochemistry. Results were correlated with the clinicopathological features of sebaceous gland carcinoma. RESULTS: nMethylation of the E-cadherin promoter region was detected in 72% of eyelid sebaceous gland carcinoma cases and loss of E-cadherin immunostaining in 83%. E-cadherin promoter hypermethylation showed a significant association with the loss of membranous E-cadherin (P = 0·038) and it was of borderline significance with reduced disease-free survival (P = 0·05). It was also found to be associated with advanced age (73%), tumour size ≥ 2 cm (77%), orbital invasion (83%), lymph node metastasis (60%), tumour recurrence (60%) and poor histological differentiation (90%). DNA sequencing revealed no stabilizing ß-catenin gene mutation in sebaceous gland carcinoma. Loss of membranous ß-catenin was observed in 61% cases, which associated significantly with both E-cadherin promoter methylation (P = 0·0262) and loss of E-cadherin membranous localization (P=0·0015). CONCLUSION: Epigenetic inactivation of the E-cadherin gene causes loss of membrane-bound E-cadherin and could contribute to the reduced disease-free survival in eyelid sebaceous gland carcinoma. Mutations in the ß-catenin gene do not seem to be involved in the pathogenesis of eyelid sebaceous gland carcinoma.

Retinoblastoma.

Retinoblastoma is the most common intraocular malignancy in children and one of the very few life-threatening ophthalmic conditions. Genetically, the disease may be heritable or non-heritable. It can have unilateral or bilateral involvement and can present either sporadically or with a positive family history. Leukocoria and strabismus are the most common presentations. Diagnosis is made by indirect ophthalmoscopy aided by imaging techniques. Multidisciplinary management is aimed at saving lives, salvaging the globe and maintaining good vision. The use of neoadjuvant chemotherapy and focal treatments, such as cryotherapy, laser photocoagulation, transpupillary thermotherapy, brachytherapy and periocular chemotherapy, form the mainstay of globe preserving treatment in retinoblastoma. In developing countries, retinoblastoma is unfortunately accompanied by a high mortality rate due to delayed diagnosis made at advanced stages of the disease. Early diagnosis and timely management are vital for a good prognosis.

Visual function alterations in orbital tumors and factors predicting visual outcome after surgery.

PURPOSE: To study the effect of orbital tumors on visual functions and highlight the factors predictive of visual outcome after surgery. METHODS: A prospective interventional study compared visual function parameters and fundus changes, before and after surgery, in eyes having well-defined orbital tumors with the normal fellow eye. These included visual acuity (VA), refractive error, keratometry changes, color vision, Goldmann visual field (GVF), and visual evoked response (VER). RESULTS: In total 28 cases (age range 7-56 years), of which the majority of tumors were vascular (46%) and lacrimal (18%) in origin, had a mean VA of 0.54±0.33 in the affected eye, which improved postoperatively to 0.66±0.31 (P=0.002). The affected eye had a median refractive error of +0.00 DS (-2.00 to 5.13), which was significantly more hyperopic than the normal eye (median +0.00 DS; range -1.25 to +1.63 DS) and normalized postoperatively. Keratometry showed higher astigmatism in the involved eye (P=0.004). The fundus showed disc pallor, edema, and/or choroidal folds, of which disc edema resolved in all cases after surgery. In all, 40% of the affected eyes had a deficient color vision and this partially improved postoperatively (P=0.25). GVF had abnormalities in 10 cases, half of which normalized postoperatively (P=0.04). The VER of affected eyes had a mean amplitude of 8.91±4.59 µv and latency of 116.3±14.7 ms, with improvement after surgery (P=0.005 and 0.001, respectively). CONCLUSION: Orbital tumors adversely affected visual functions. The presenting acuity depended on disc changes, color vision abnormalities, and prolonged VER latency. The postoperative VA depended on VA at presentation, amount of proptosis, degree of hyperopia, and clinically significant VER abnormalities.

Fibronectin-adherent monocytes express tissue factor and tissue factor pathway inhibitor whereas endotoxin-stimulated monocytes primarily express tissue factor: physiologic and pathologic implications.

BACKGROUND: Monocytes are critical cells in initiating physiologic and/or pathologic tissue factor (TF)-induced intravascular and extravascular coagulation. Monocytes constitutively express small amounts of TF and tissue factor pathway inhibitor (TFPI). Non-adherent lipopolysaccharide (LPS)-stimulated monocytes express significant amounts of TF; however, increased expression of TFPI by these cells is controversial. Further, whether fibronectin-adherent monocytes (mimicking conditions in the extravascular space) express sufficient TFPI to inhibit TF-procoagulant activity (PCA) is unknown. OBJECTIVE: To compare TF and TFPI expression by fibronectin-adherent and LPS-stimulated non-adherent monocytes. METHODS: Monocytes were isolated from normal peripheral blood, adhered to fibronectin or stimulated with lipopolysaccharide (LPS) under non-adherent conditions and examined for expression of TF and TFPI using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), ELISA and factor X (FX) activation. RESULTS: Under LPS-free conditions, the fibronectin-adherent monocyte TF mRNA, antigen and activity were markedly upregulated. Notably, cell and microparticle (MP)-associated TF and alternatively spliced TF (asTF) were all upregulated. TFPI mRNA and antigen were also upregulated in the fibronectin-adherent monocytes, which significantly inhibited TF-PCA. TFPI mRNAs for both alpha and beta forms were detected. The peak in TFPI activity occurred in tandem with the peak in TF-PCA. In contrast, LPS-stimulated monocytes, which expressed cell and MP-associated TF and asTF, demonstrated only minimal expression of TFPI as determined by mRNA, antigen or inhibition of TF activity. CONCLUSION: Both LPS-stimulated and fibronectin-adherent monocytes demonstrate a procoagulant phenotype by expressing TF but only fibronectin-adherent monocytes express significant amounts of TFPI to control thrombin generation and fibrin formation in the context of extravascular space.

Simultaneous ocular and systemic cysticercosis and tuberculosis.

Human cysticercosis and tuberculosis are endemic diseases in developing countries. Both these diseases have certain common factors of origin. We would like to present the co-existence of these infections in a 20-year-old female. She was a known case of pulmonary and ocular tuberculosis and she acquired cysticercosis of the eye and brain.