Neha Gadaria-Rathod

Tear cytokine profile as a noninvasive biomarker of inflammation for ocular surface diseases: standard operating procedures.

PURPOSE To provide standard operating procedures (SOPs) for measuring tear inflammatory cytokine concentrations and to validate the resulting profile as a minimally invasive objective metric and biomarker of ocular surface inflammation for use in multicenter clinical trials on dry eye disease (DED). METHODS Standard operating procedures were established and then validated with cytokine standards, quality controls, and masked tear samples collected from local and distant clinical sites. The concentrations of the inflammatory cytokines in tears were quantified using a high-sensitivity human cytokine multiplex kit. RESULTS A panel of inflammatory cytokines was initially investigated, from which four key inflammatory cytokines (IL-1β, IL-6, INF-γ, and TNF-α) were chosen. Results with cytokine standards statistically satisfied the manufacturers quality control criteria. Results with pooled tear samples were highly reproducible and reliable with tear volumes ranging from 4 to 10 μL. Incorporation of the SOPs into clinical trials was subsequently validated. Tear samples were collected at a distant clinical site, stored, and shipped to our Biomarker Laboratory, where a masked analysis of the four tear cytokines was successfully performed. Tear samples were also collected from a feasibility study on DED. Inflammatory cytokine concentrations were decreased in tears of subjects who received anti-inflammatory treatment. CONCLUSIONS Standard operating procedures for human tear cytokine assessment suitable for multicenter clinical trials were established. Tear cytokine profiling using these SOPs may provide objective metrics useful for diagnosing, classifying, and analyzing treatment efficacy in inflammatory conditions of the ocular surface, which may further elucidate the mechanisms involved in the pathogenesis of ocular surface disease.

HLA-DR EXPRESSION AS A BIOMARKER OF INFLAMMATION FOR MULTICENTER CLINICAL TRIALS OF OCULAR SURFACE DISEASE

There are currently no validated minimally invasive objective metrics for the classification and evaluation of ocular surface diseases and/or for evaluating treatment efficacy. We thus sought to establish a standardized methodology for determining the relative amount of the inflammatory biomarker HLA-DR on the ocular surface and to evaluate the precision, reliability and repeatability of its use for large multicenter clinical trials and translational research studies of ocular surface disease. Multiple studies were conducted to establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a minimally invasive, objective, ocular surface inflammatory biomarker. The established SOPs provide specific guidelines for HLA-DR collection and analysis, in order to incorporate it reliably into multicenter clinical trials and/or translational research. Duplicate cell samples from impression cytology (IC) samples of both normal and dry eye individuals were collected and split to assess repeatability (between the splits and between the duplicate samples). To determine storage capability, one duplicate was stained immediately and the other after 30 days cold storage. To demonstrate the feasibility of the use of the SOP for a multicenter clinical trial, clinicians out-of-state were trained to collect IC samples, and the samples shipped to our Biomarker Laboratory, logged, processed and analyzed. Demonstration of the ability to incorporate of IC into a randomized double masked clinical trial of dry eye disease (DED) was performed. In all cases, processing and analyses were performed by a masked independent observer. The validity/viability of the SOPs was established by demonstrating that: 1) sufficient numbers of cells can be collected via IC; 2) the precision/repeatability of the relative biomarker expression quantified in samples; 3) personnel at distant sites can be taught to collect, store and ship samples successfully; 4) samples can be stored for up to 30 days (refrigeration) before processing without affecting results; 5) IC can be incorporated into a double blind randomized clinical trial (RCT) of DED; and 6) the Biomarker Laboratory can track a large number of masked samples reliably. In conclusion, our standard operating procedure for impression cytology analysis of HLA-DR expression appears to be repeatable and reproducible for use in multicenter clinical trials, providing a minimally invasive objective biomarker of inflammation of the ocular surface.

Precision and Accuracy of TearLab Osmometer in Measuring Osmolarity of Salt Solutions

Abstract Purpose: The purpose of this study was to examine the inherent precision and accuracy of TearLab Osmolarity System using salt solutions, including solutions of very high osmolarity (>360 mOsm/L). Materials and methods: Ten salt solutions with osmolarity between 286 mOsm/L and 394 mOsm/L (increments of 12 mOsm/L) plus an additional solution of 400 mOsm/L were tested twice on both the TearLab osmometer and a freezing point depression osmometer. For precision, we compared the two repeated osmolarity measurements of 11 solutions obtained from TearLab. For accuracy, we compared the averaged osmolarity measurements obtained from TearLab to those from the freezing point depression osmometer. For both precision and accuracy, Bland–Altman test of agreement was used. Results: For precision, the upper 95% limit of agreement was 4.7 mOsm/L, and the lower 95% limit of agreement was −7.1 mOsm/L. The repeatability coefficient was 5.9 mOsm/L. For accuracy, the upper 95% limit of agreement was 4.8 mOsm/L and the lower 95% limit of agreement was −5.3 mOsm/L. Conclusions: The present study is the first study to demonstrate that the TearLab in situ osmometer can precisely and accurately measure osmolarity of salt solutions, including those with very high osmolarity. Future studies to evaluate the precision and the accuracy of the machine in measuring complex fluids, such as tears, need to be done, and the clinical significance of measuring tear osmolarity in patients needs to be further determined.

Emerging drugs for the treatment of dry eye disease

Introduction: Dry eye disease (DED) is a common, age-related ocular condition that in its mildest forms causes bothersome symptoms of ocular discomfort, fatigue, and visual disturbance that interfere with quality of life and in its more severe forms causes chronic pain and fluctuating vision. Though it is highly prevalent and costs billions of dollars to manage, current treatments have largely been inadequate, making it a frustrating condition, both for physicians and patients alike. Areas covered: This article will cover the recently discovered pathophysiology of DED that has prompted investigators to explore new molecules that target the core mechanisms that drive DED. These include anti-inflammatory/immune-modulatory drugs, secretagogues, lubricant, hormones, and autologous serum. Their potential mechanism of action and data from recent trials on efficacy/safety will be reviewed. Expert opinion: The emerging drugs have a vast range of putative mechanisms of action that may not only provide symptomatic relief but may potentially break the vicious cycle of DED and provide long-lasting cure. Current and future research may change our perspective on DED and redefine its treatment algorithms.

Red Blood Cell Fatty Acid Analysis for Determining Compliance with Omega3 Supplements in Dry Eye Disease Trials

PURPOSE To evaluate pill counts and red blood cell (RBC) membrane fatty acid profiles as measures of compliance with oral omega3 polyunsaturated fatty acids (ω3 PUFAs) and to compare the two techniques. METHODS Sixteen dry eye disease subjects were given oral ω3 PUFA or placebo for 3 months. Compliance was measured by pill counts and blood tests at baseline and 3 months. The Wilcoxon signed-rank tests and rank-sum tests were used to compare changes from baseline and the difference between the two groups; Spearman correlation coefficients were used to assess the relationship of pill counts to changes in blood FAs. RESULTS Pill counts for the ω3 (n=7) and placebo (n=9) groups showed a mean consumption of 4.39 and 4.76 pills per day, respectively. In the ω3 group, the median change from baseline was +1.46% for eicosapentaenoic acid (EPA) (P=0.03), +1.49% for docosahexaenoic acid (DHA) (P=0.08), and -1.91% for arachidonic acids (AA) (P=0.02). In the placebo group, median changes in all measured FAs were small and not statistically significant. The difference in change in FA levels between the two groups was significantly greater for EPA (P=0.01) and AA (P=0.04). The correlations between pill counts and changes in EPA (r=0.36, P=0.43) and DHA (r=0.17, P=0.70) were not strong. CONCLUSIONS RBC FA analysis can be used to measure compliance in the active group and also monitor the placebo group for nonstudy ω3 intake. Low correlation of pill counts with blood levels suggests that pill counts alone may be inaccurate and should be replaced or supplemented with objective measures.

New insights into infectious keratitis.

Staphylococcus aureus isolates that are resistant to all available penicillins and other b-lactam antimicrobial drugs are said to be methicillinresistant S. aureus (MRSA). Although predominantly considered to be a nosocomial infection, the incidence of community-acquired infections is rising. A study on S. aureus data submitted to The Surveillance Network by >200 laboratories in the United States from January 2000 to December 2005 showed that the proportion of MRSA in serious ocular S. aureus infections increased from 29.5% in 2000 to 41.6% in 2005. The changing epidemiology of MRSA infections is also evident in cases with microbial keratitis after keratorefractive surgery. In ASCRS surveys, although the number of postrefractive surgery keratitis cases decreased, from 116 cases in 2001 to 48 cases in 2004 to 19 cases in 2008, the incidence of MRSA increased from nil in 2001 and 2004 to 28% in 2008, with MRSA being the most common organism cultured. The pattern of antibiotic sensitivity in MRSA is also changing. Data from the Ocular Tracking Resistance in US Today, which annually evaluates in vitro antimicrobial susceptibility of 4 common bacteria including S. aureus to ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, penicillin, azithromycin, tobramycin, trimethoprim, and polymyxin B revealed that out of 197 ocular isolates of S. aureus 16.8% were MRSA. The susceptibility of MRSA to all fluoroquinolones was just 15.2%. Trimethoprim was the only agent tested with high activity against MRSA. In another study it was shown that although all MRSA isolates were

In Vivo Efficacy of Histatin-1 in a Rabbit Animal Model

ABSTRACT Purpose/Aim: Corneal abrasions and nonhealing corneal epithelial defects are common conditions that cause pain and sometimes are slow to heal. Histatins, a family of histidine-rich peptides, have been implicated in oral and skin epithelial wound healing, and have been shown to be effective in vitro in human corneal epithelial cells. The objective of this study was to test the efficacy of histatin-1 on corneal epithelial wound healing in rabbits. Materials & Methods: Twenty-two (22) rabbits were separated into four treatment groups, each containing 3–7 rabbits. Treatments included three histatin-1 formulations (0.1 ug/ml. 1 ug/ml, and 10 ug/ml) and one inactive vehicle, one drop given three times per day. Eight (8) mm circular wounds were created using 0.5 ml of 20% ethyl alcohol in the right eye of each rabbit. A masked observer photographed each eye twice daily using slit-lamp biomicrophotography. Wound area was analyzed by using ImageJ. Statistical analysis was conducted using Graphpad Prism. Results: Wound recovery was faster in animals given 0.1 ug/ml, 1 ug/ml, and 10 ug/ml when compared to the vehicle solution at 6, 24, and 30 hours after wound creation (p < 0.01). No adverse events were observed in any eyes. When analyzing area under the curve, % recovered area was higher overall in the 0.1 ug/ml (p < 0.01), 1 ug/ml (p < 0.01), and 10 ug/ml (p < 0.001) groups when compared to the vehicle solution. Hourly healing rate was also observed to be faster in the 0.1 ug/ml, 1 ug/ml, and 10 ug/ml groups (p < 0.001) at 24 hours postinjury suggesting an accelerated healing process as compared to the vehicle group. Conclusion: This study represents the first in vivo experiment evaluating and confirming the efficacy of topical histatin on the corneal epithelium wound healing. Further studiesare warranted to better understand the mechanism and safety of topical histatin-1 in corneal epithelial wound-healing and its potential role for human disease treatment.