Optometry & Vision Science:
Protein Deposition and Clinical Symptoms in Daily Wear of Etafilcon Lenses
Subbaraman, Lakshman N.*; Glasier, Mary-Ann†; Varikooty, Jalaiah‡; Srinivasan, Sruthi§; Jones, Lyndon‖
*PhD, BSOptom, FAAO
§PhD, BSOptom, FAAO
‖PhD, FCOptom, FAAO
Centre for Contact Lens Research, School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.
Received January 3, 2012; accepted June 18, 2012.
Lakshman N. Subbaraman Centre for Contact Lens Research, School of Optometry University of Waterloo 200 University Avenue West Waterloo, Ontario Canada e-mail: firstname.lastname@example.org
Purpose. To determine the relationship between clinical signs and symptoms and protein deposition over 8 h of wear of etafilcon A lenses in symptomatic and asymptomatic contact lens wearers.
Methods. Thirty adapted soft contact lens wearers (16 symptomatic and 14 asymptomatic) were fitted with etafilcon A lenses. In vivo wettability, non-invasive tear break-up time, and subjective symptoms (vision, comfort, and dryness) were assessed at baseline and after 2, 4, 6, and 8 h. After 2, 4, 6, and 8 h time points, lenses were collected, and total protein, total lysozyme, and active lysozyme deposition were assessed.
Results. There was a significant reduction (p = 0.032) in the non-invasive tear break-up time at 8 h in both groups. In the symptomatic group, there was a significant reduction in subjective comfort and dryness ratings at 6 and 8 h measurement with respect to baseline (p < 0.05). There was a significant increase in total lysozyme and total protein deposition (p = 0.027) across all time points in both groups; most of the lysozyme remained active (>94% at 8 h). Pearson's correlations between subjective symptoms and protein deposition showed poor correlations for total protein/lysozyme and any subjective factor (r < 0.3; p > 0.05), and only weak correlations between dryness and % active lysozyme (r = 0.3 to 0.5 for all time points). However, stronger correlations were found between active lysozyme and subjective comfort (r = 0.6 to 0.7; p < 0.001).
Conclusions. In addition to investigating total protein deposited on contact lenses, it is of significant clinical relevance to determine the conformational state of the deposited protein.
Dryness and discomfort are considered to be one of the primary reasons for contact lens intolerance and discontinuation of lens wear.1–3 Contact lens-related dry eye has often been associated with changes in functional visual acuity,4,5 reduced lens wearing time,6 increased risk of bacterial adhesion, ocular surface desiccation, and infection.7,8 Several studies have attempted to determine the potential mechanisms for contact lens-related dry eye, and it has been observed that increased evaporation of the tear film,9 rapid pre-lens tear film thinning,10 limbal injection,10 inflammation,11–13 reduced lacrimation with concurrent increased osmolality,14,15 and an increase in tear film osmolality10 are all significant factors that may be associated with contact lens-related dry eye. In addition, higher water content contact lenses,10,16 reduced wettability of the lens surface,17–19 or any of the aforementioned factors could also be associated with contact lens-related dry eye, confirming that this condition is multifactorial. Several studies have demonstrated that dryness and discomfort ratings become worse independently of the amount of dehydration or water content of the lens material.2,20,21
The tear film is by far the most dynamic unit in the lacrimal functional unit, which consists of a variety of components, including proteins, lipids, mucins, peptides, electrolytes, and salts. Using a proteomic technique, 97 proteins have been identified in the tear film,22 and many of these proteins are known to sorb onto contact lens materials.23,24 Protein deposition on contact lens materials is highly material dependent, with water content and surface charge having significant impacts on the amount of protein deposited.25–36 One of the major tear proteins that is recovered from group IV contact lens materials is lysozyme.28,34,36–40 Lysozyme is a bacteriolytic enzyme with a relatively small molecular weight (14 kDa) and a positive charge at neutral pH. Once lysozyme firmly adsorbs onto contact lens materials, it tends to undergo conformational changes,28,40–42 which might potentially result in a variety of immunological responses, including contact lens-associated papillary conjunctivitis.43–46
Several studies have determined changes that occur in tear film protein or lipid levels in contact lens wearers, with some of these studies classifying contact lens wearers as being either “tolerant” or “intolerant.”11,13,47–61 However, these studies did not quantify the protein deposited on the lens materials per se; therefore, it was not possible to determine the relationship between various clinical parameters and the amount of protein deposited on the lenses. Another study38 that quantified the protein deposited on the lens material determined the effect of overnight eye closure on the rate and composition of protein deposition on the probable change in the rate of reflex-type tear secretion associated with eye closure. Some studies have determined the link between protein deposition on contact lenses and subjective symptoms reported by lens wearers.62–66 However, all these studies determined the deposition on lenses by using relatively insensitive techniques, such as visible deposition or video image analysis.64–66
Although studies have speculated that the conformational state of the deposited protein could have an influence on various subjective symptoms in contact lens wearers,62,67 to date, no study has determined the relationship between subjective symptoms and the conformational state of the deposited protein, or indeed the differences in these factors in symptomatic and asymptomatic contact lens wearers. Thus, the main purpose of this study was to investigate the impact of lens wearing time on clinical signs, subjective symptoms, and the quantity of total protein and lysozyme deposition and the conformational state of the lysozyme deposited over an 8 h wear period of a high water content ionic lens material (etafilcon A; Acuvue; Johnson & Johnson, Jacksonville, FL) in a group of symptomatic and asymptomatic contact lens wearers and to determine whether there is any association between the clinical signs and symptoms and the protein deposition measured on the lenses.
MATERIALS AND METHODS
Ethics clearance was obtained from the Office of Research Ethics at the University of Waterloo before commencement of the study. The study was carried out in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all participants before enrollment. The study was conducted at the Centre for Contact Lens Research, School of Optometry, University of Waterloo. This was a non-dispensing randomized study involving previously adapted soft contact lens wearers. Participants were classified as being “symptomatic” or “asymptomatic” based on their responses to a prescreening questionnaire, and the examiners were masked to the presence of symptoms. Participants who were classified as being symptomatic with their soft lenses were those subjects who reported reduced comfortable lens wear with their existing soft lens materials after a minimum of 6 h of lens wear and who needed to resort to ocular lubricants to sustain their lens wear. Participants were eligible to participate in the study if they were at least 16 years or older. Participants who were <18 years (and >16 years of age) were eligible to participate with the parent's or guardian's permission and after reading the Information and Consent Letter for adolescents. Self-consent was applicable for those participants who were >18 years of age. All participants were fitted with etafilcon A contact lenses (group IV lens material; Acuvue; Johnson & Johnson), and the participants were not permitted to use any rewetting drops during the course of the study. The participants were advised not to wear any contact lenses or use any form of lubricants for 5 days before the initial lens-dispensing visit.
Each participant attended on two consecutive days, with a baseline and two study visits on each day. On each day, the first visit was the baseline visit, and this was followed by second and third visits anytime between 2 and 8 h, and all the study visits were randomly determined based on a randomization table. During the baseline visit on day 1, contact lenses were inserted into both eyes, and, after the lenses had settled, objective and subjective measurements were determined. During the first study visit (which was randomly determined) on day 1, objective and subjective measurements were determined, and at the end of this visit, one lens was randomly removed from one of the participant's eyes for protein analysis. A new lens was reinserted into that eye to ensure the subject was binocularly corrected. During the second study visit later that day (which was again randomly determined), objective and subjective measurements were taken, and at the end of this visit, the lens from the other eye was collected for protein analysis. On the following day, the same procedures were repeated, for the remaining two time points, which were determined using a randomization table. Thus, each participant had lenses collected for analysis after four time periods (two per day), with the time periods being after 2, 4, 6, or 8 h of lens wear.
The objective measurements were performed on the participants at baseline and after 2, 4, 6, and 8 h of lens wear. Tear film stability was assessed by determining the non-invasive tear break-up time (NITBUT) using the ALCON Eyemap model EH-290 topography system (ALCON Inc., Forth Worth, TX). Participants were asked to blink three times before each measurement was taken. NITBUT was determined by measuring the time taken for distortions or discontinuities to appear in the reflected image of the concentric ring pattern. The time (in seconds) for the tear film to rupture (and thus distort the rings) was measured to the nearest 0.1 of a second. Three measurements were taken on each eye, and the average of these was used for analysis purposes.
Overall wettability of the contact lenses was assessed in vivo using the grid viewed on the ALCON Eyemap (ALCON Inc.). The image of the Placido disc was viewed on the monitor of the Eyemap, and the in vivo wettability of the contact lenses was graded on a five-point scale (0 to 4), where “0” related to a lens exhibiting “severely reduced” wettability and “4” a lens with “perfect” wettability.68 In vivo wettability of the contact lenses was assessed according to the following schema: Grade 0: One or more non-wetting areas >0.5 mm in size; Grade 1: Several non-wetting areas, 0.1 to 0.5 mm in size; Grade 2: Single non-wetting area 0.1 to 0.5 mm in size; Grade 3: Small (<0.1 mm), discrete non-wetting areas; Grade 4: 100% of anterior lens surface wettable.
Assessment of Subjective Symptoms
Participants completed visual analog scales at baseline, 2, 4, 6, and 8 h study visits. Participants rated the subjective symptoms of vision, comfort, and dryness on a scale of 0 to 100 (0 = worst rating, 100 = best rating).
After 2, 4, 6, and 8 h of lens wear, lenses were collected by a gloved examiner, and the lenses were briefly rinsed with saline to remove any residual loosely adhered tear film and placed in individual sealed glass vials containing a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile (ACN/TFA), as described previously.37,42 The vials were incubated in the dark at room temperature for 24 h, after which the aliquots of lens extracts were transferred to sterile Axygen microcentrifuge tubes and evaporated to dryness in a Savant Speed Vac (Halbrook, NY). Dried protein pellets were stored at −80°C for up to 2 weeks before reconstitution.
Reagents and Materials
Immuno-Blot polyvinylidene difluoride membranes were purchased from Bio-Rad Laboratories (Mississauga, ON, Canada). Polyclonal rabbit anti-human lysozyme was purchased from Cedarlane Laboratories (Hornby, ON, Canada), and goat anti-rabbit IgG-HRP was purchased from Sigma (St. Louis, MO). Human lysozyme (neutrophil) and lyophilized Micrococcus lysodeikticus cells were also purchased from Sigma. Bovine serum albumin standard was obtained from Pierce Biotechnology Inc (Rockford, IL). All other reagents purchased were of analytical grade.
Measurement of Total Lysozyme Deposition—Electrophoresis and Immunoblotting
Before electrophoresis/Western blotting and lysozyme activity analysis, lyophilized protein pellets were reconstituted in modified reconstitution buffer—MRB (10 mM Tris-HCl; 1 mM EDTA, with 0.9% saline), pH 12.0, and BioStab Biomolecule Storage Solution (Sigma-Aldrich).69 Human lysozyme standard curves were run on each Western blot so that four points falling within the linear range of detection were produced, to facilitate regression analysis of sample extracts. Lysozyme standards were prepared fresh on the day of analysis from a 1.0 μg/μl frozen stock of purified human neutrophil lysozyme with modified reconstitution buffer, pH 8.0, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blotting to polyvinylidene difluoride membranes. Lysozyme was identified using a rabbit anti-human lysozyme polyclonal antibody (Calbiochem), followed by a peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich). Individual standard curves of purified human neutrophil lysozyme (Sigma-Aldrich) were run on each gel to facilitate regression analysis. The entire procedure is described in detail elsewhere.42,69,70
Negative Control—Extraction and Western Blot Analysis of Unworn Lenses
Three new unworn etafilcon A lenses were extracted in ACN/TFA solution and were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting, as described earlier.
Measurement of Lysozyme Activity
The contact lens extracts were assayed for lysozyme activity using a fresh suspension of M lysodeikticus for each sample, as described previously.40,42,70 Micrococcal cells were suspended in 50 mM sodium phosphate buffer (pH 6.3) to an initial optical density of 1.0 at 450 nm (Multiskan Spectrum ELISA Plate Reader, fitted with a microcuvette, Thermo Labsystems). Human neutrophil lysozyme standard (2.5, 5, 12.5, 50, 150, 250 ng) was run concurrently with the samples. The mass of active lysozyme in contact lens extracts was extrapolated from the native lysozyme standard curve, as described previously.40,42,70 The final calculation was the percent of active lysozyme: % active lysozyme = (active lysozyme/total lysozyme) × 100.
Measurement of Total Protein Deposition
The total protein extracted from the lenses was determined using the Micro-BCA assay. Manufacturer's instructions were followed for the Micro-BCA Protein Assay Reagent Kit (Pierce Biotechnology). Phosphate buffered saline was used as the buffer. Each data point was the average of three determinations.
Statistical analysis was conducted using Statistica 7 software (StatSoft Inc., OK). All data are reported as mean ± standard deviation and range, unless otherwise indicated. A two-way repeated measures analysis of variance was performed, with time course and groups as the factors, and post hoc multiple comparison testing was undertaken using the Tukey-HSD test. Pearson's correlations were performed to determine the relationship between various clinical signs and symptoms vs. the analytical measures. In all cases, a p value of <0.05 was considered significant.
Based on the participants' responses to the prescreening questionnaire, 16 participants (mean age, 24.73 ± 5.31 years, 9 female and 7 male) were classified as symptomatic and 14 participants (mean age, 25.31 ± 4.78 years, 13 female and 1 male) were classified as asymptomatic.
Fig. 1 shows the NITBUT values over time for the symptomatic and asymptomatic groups. There was no significant difference in the NITBUT values between the two groups at any time point (p > 0.05), but the 8 h time point was significantly lower than the baseline measurement in both the symptomatic and asymptomatic groups (p = 0.032). Fig. 2 shows the in vivo wettability over time for the symptomatic and asymptomatic participants. Although in vivo wettability is seen to reduce over the course of the day for both groups, this reduction was not statistically significant for either group (p > 0.05). There was also no significant difference between the two groups at any time point (p > 0.05).
Subjective Symptom Ratings
Fig. 3 shows that there was no significant difference for subjective vision ratings for both groups over time (p > 0.05) and also between the two groups at any time (p > 0.05), although the symptomatic group showed lower ratings at all time points.
Fig. 4 shows that there was no significant decrease in comfort over time in the asymptomatic group (p > 0.05); however, in the symptomatic group, the 6 and 8 h ratings were significantly lower than the baseline measurement (p = 0.013). The symptomatic group had significantly lower comfort ratings than the asymptomatic group at the 6 and 8 h time points (p = 0.035). However, there was no significant difference between the two groups at other time points (all p > 0.05), although the symptomatic group had lower comfort ratings at these times.
Fig. 5 shows that there was no significant reduction in dryness ratings over time in the asymptomatic group (p > 0.05); however, in the symptomatic group, the 6 and 8 h ratings were significantly lower than the baseline measurement (p = 0.012). The symptomatic group reported significantly more dryness than the asymptomatic group at the 6 and 8 h time points (p = 0.024). However, there was no significant difference between the two groups at other time points (all p > 0.05), although the symptomatic group had lower dryness ratings at these times.
Table 1 shows the total protein, total lysozyme deposition, and percentage active lysozyme recovered from the lenses at various time points in the asymptomatic and symptomatic participants. There was a gradual increase in total protein deposition and total lysozyme deposition on the lenses across the four time points (p < 0.05) both in the asymptomatic and symptomatic group of participants. However, there was no significant difference between the two groups at any time point (p > 0.05). There was a gradual reduction in the activity of lysozyme deposited across the four time points, albeit it was not statistically significant (p > 0.05). The percentage active lysozyme recovered from the symptomatic lens wearers was lower at all time points, although this was not statistically significant (all p > 0.05).
Pearson's correlations between clinical signs and any of the protein deposition measures showed poor (r < 0.2) insignificant correlations (p > 0.05). Pearson's correlations between subjective symptoms and protein deposition showed poor correlations for total protein/total lysozyme and any subjective factor (r < 0.3; p > 0.05), and only weak correlations between dryness and active lysozyme (r = 0.3 to 0.5 for all time points) as shown in Table 2. However, significant linear correlations were found between the active lysozyme and subjective comfort (r = 0.6 to 0.7; p < 0.001) as shown in Table 2.
To date, this is the only study that reports on the relationship between clinical signs, subjective symptoms, and the conformational state of lysozyme deposited on contact lenses. These results clearly suggest that there is a linear correlation between lysozyme activity recovered from contact lenses and subjective comfort, even over short periods of lens wear. Previous studies have shown that tolerant contact lens wearers have fewer symptoms of discomfort and a more stable tear film (as measured by a higher maximum forced interval between blinks, tear meniscus height and volume, and NITBUT).71 In the past, studies have also shown that the tear film of tolerant lens wearers showed lower levels and activity of secretory phospholipase A2, lower concentration of lipocalin, and lower levels of peroxidized lipids.47 It has also been shown that in the absence of lens wear, there were no differences between tolerant and intolerant lens wearers in conjunctival or limbal redness, lipid layer appearance, tear flow rate, tear film osmolality, and total protein, lactoferrin, lysozyme, or secretory immunoglobulin A concentrations in the tear film.71
Non-invasive Tear Break-Up Time
When a contact lens is inserted into the eye, the tear film is disturbed and tear film break-up time reduces significantly.72,73 Previous studies have shown the NITBUT of soft contact lens wearers to be in the range of 3 to 10 s,59,74,75 which is similar to that found in the current study. A study by Guillon et al. showed that there was no difference in the tear film stability between asymptomatic and symptomatic contact lens wearers, although they found a significant difference between asymptomatic and symptomatic non-contact lens wearers.73 However, another study by Glasson et al. showed that contact lens wear affected the stability of the tear film in tolerant contact lens wearers more than in intolerant contact lens wearers.76 In their study, NITBUT decreased more dramatically in the tolerant contact lens wear group, and it was also shown that the NITBUT of intolerant subjects was significantly lower initially before lens wear and remained low over 6 h of lens wear. Another study by Fonn et al. also demonstrated a statistically significant decrease in pre-lens NITBUT in symptomatic lens wearers during a 5 h period, regardless of soft lens type, compared with no significant change in asymptomatic subjects.20
In Vivo Wettability
Although high levels of total protein and total lysozyme deposited on etafilcon lens materials within a few hours of lens wear (Table 1), it is clear that these deposits do not modify the measured in vivo wettability of these lenses to any appreciable extent. In the past, studies that determined the wettability of etafilcon lens materials over short periods of lens wear using indirect methods such as pre-lens NITBUT also showed similar findings.77 Previous in vitro and ex vivo studies that determined the influence of tear proteins on wettability of etafilcon lens materials also found that these deposits do not reduce the wettability of etafilcon lens materials over short periods of lens wear or over short periods of in vitro incubation,78–80 and this can be attributed to the fact that lysozyme penetrates into the bulk of the etafilcon lens material rather than remaining on the surface of these lens materials.81
As expected, symptomatic contact lens wearers in this study showed a significant reduction in subjective comfort and dryness ratings over 8 h of lens wear, whereas the ratings of asymptomatic lens wearers remained relatively constant (Figs. 4 and 5). These results are consistent with those from previous studies where symptomatic lens wearers showed a decrease in comfort and dryness ratings using visual analog scales over time.2,20,71
Protein Deposition on Lenses
It is clear from Table 1 that etafilcon lenses attracted substantial quantities of total protein and total lysozyme even after 8 h of lens wear in both the symptomatic and asymptomatic participants. This finding is in accordance with other previous in vitro and ex vivo studies that evaluated protein and lysozyme deposition on etafilcon contact lenses.30,31,36,39–42,82–87 The increased affinity of lysozyme to the etafilcon material occurs because methacrylic acid imparts a negative charge to the material and thus thermodynamically favors the deposition of lysozyme, which is a positively charged species at physiological pH.
After 8 h of lens wear, the percentage active lysozyme recovered from etafilcon lenses worn by symptomatic and asymptomatic lens wearers was >94% (Table 1), which is significantly higher than those seen from silicone hydrogel lens materials.40–42,70,88,89 Previous ex vivo41,42 and in vitro40,89 studies have also demonstrated that the percentage active lysozyme recovered from conventional hydrogel group IV etafilcon lens materials is significantly higher than those seen in novel silicone hydrogel and other groups of conventional hydrogel lens materials. Denaturation of a protein on any polymeric surface is dependent on several factors, including contact time of the protein with the substrate, chemical composition of the substrate, surrounding pH, type of protein, temperature of the surrounding medium, and also the location of the protein in a polymer.28,67,82,90–93 Using confocal microscopy, it has recently been shown that lysozyme is primarily located within the bulk of etafilcon lens materials, with relatively little surface-located lysozyme,81 resulting in significantly increased levels of lysozyme remaining active.
Relationship between Protein Deposition and Subjective Symptoms
A previous study by Lever et al. that investigated the relationship between total protein deposition and patient-rated lens comfort found that there was no statistical correlation between these two factors.62 This was the only study that attempted to determine the relationship between protein deposition on contact lenses and subjective comfort by quantifying the total protein deposited on lenses using biochemical techniques62; other studies estimated the relationship by evaluating the visible deposition/video image analysis of deposits on the lenses.4,63–66 Most studies that used visible deposition/video image analysis showed that there was an association between visible deposition and comfort4,64–66; however, one study that surveyed 50 comfortable and uncomfortable contact lens wearers did not show a difference in the amount of visible deposition on the lenses between the two groups.63 A recent study that looked at correlating clinical responses during contact lens wear with the amount of protein or cholesterol extracted from lenses after wear suggested that the quantity of protein that deposits onto contact lenses during wear may have more effect on lens performance on eye94; however, this study did not look at the conformational state of the protein deposited on these contact lenses. Furthermore, protein deposition has a significant potential to cause problems, as these deposits do play a significant role in modulating microbial adherence to lens materials.95,96 Therefore, it is important that practitioners advise their patients regarding the importance of lens disinfection and cleaning and appropriate lens replacement schedules.
This study is the first to demonstrate that a significant correlation exists between subjective comfort and active lysozyme recovered from etafilcon lens materials, even over short periods of lens wear. However, these results should be interpreted with caution, as it would be erroneous to conclude that denatured lysozyme on contact lenses is solely responsible for the symptoms experienced by symptomatic contact lens wearers. Rather, they should be interpreted as lysozyme deposited on the contact lenses of symptomatic lens wearers tends to denature more than that seen in asymptomatic lens wearers. This is likely to happen because of the biochemical changes that occur in the tear film of symptomatic lens wearers,71,76 resulting in altered properties of the lens material, potentially leading to a change in the conformational state of the deposited lysozyme. Therefore, in addition to the other factors mentioned earlier, the conformational state of the deposited protein, inflammatory and subinflammatory mediators, and the secretomotor response of the lacrimal system could also be significant factors in contact lens-induced dry eye, reiterating that this condition is multifactorial.
In conclusion, the results from this study suggest that even over a short period of contact lens wear, a significant correlation exists between subjective symptoms of comfort and dryness and the activity of lysozyme recovered from etafilcon contact lenses, with little correlation being shown with total amounts of either total protein or total lysozyme. Therefore, in addition to investigating the total quantity of the deposited protein, it is of significant clinical relevance to study the conformational state of the deposited protein. These results have tremendous implications, in that the novel contact lens materials that are being developed should possess properties that can retain the activity of the deposited protein, in addition to being deposit resistant. Care regimens and multipurpose solutions should be capable of removing denatured proteins that are deposited on the lens materials or be manufactured to maintain protein activity at the material surface.
Further work is required to determine whether this important clinical finding is transferable to those patients who use silicone hydrogel lens materials. It would also be of interest to determine whether there is a difference in the activity of lysozyme recovered from the tears of symptomatic and asymptomatic contact lens wearers. Although it is interesting to note that a significant correlation exists between the conformational state of the deposited protein and clinical symptoms, further studies with a better sample size and validated instruments that clearly identify symptomatic and asymptomatic groups are warranted. In addition to determining the activity of lysozyme, it would also be of interest to determine the activity of lactoferrin and other tear proteins with high cationicity, such as the defensin peptides.
Lakshman N. Subbaraman
Centre for Contact Lens Research, School of Optometry
University of Waterloo
200 University Avenue West
This work was funded by a Collaborative Research and Development Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) and Alcon Research Limited, U.S.
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comfort; contact lens; deposition; lysozyme; protein; protein activity; tears
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