ARVO 2015 Annual Meeting Abstracts
287 Meibomian gland, lacrimal gland and tear film, I
Monday, May 04, 2015 3:45 PM–5:30 PM
Exhibit Hall Poster Session
Program #/Board # Range: 2482–2511/D0086–D0115
Organizing Section: Cornea
Program Number: 2482 Poster Board Number: D0086
Presentation Time: 3:45 PM–5:30 PM
Na+-K+-2Cl- Cotransporter in Rabbit Lacrimal Gland Ducts
Plays a Key Role in Lacrimal Secretion
Edit Toth-Molnar1, 4, Mate Katona2, Eszter Vizvari1, Ferenc
Rarosi3, Peter Orvos4, Andrea Facsko1, Viktoria Venglovecz4,
Zoltan Rakonczay2, Chuanqing Ding5, Peter Hegyi2. 1Department
of Ophthalmology, University of Szeged, Szeged, Hungary; 21st
Department of Internal Medicine, University of Szeged, Szeged,
Hungary; 3Department of Biophysics and Informatics, University of
Szeged, Szeged, Hungary; 4Department of Pharmacology, University
of Szeged, Szeged, Hungary; 5Pharmacology and Pharmaceutical
Sciences, Ophthalmology, University of Southern California, Los
Angeles, CA.
Purpose: We recently reported that isolated duct segment from
rabbit lacrimal gland (LG) was able to secrete into the luminal space
in response to secretagogues, which was completely blocked by
bumetanide, suggesting the presence of Cl- transport in these ducts,
especially those mediated by Na+-K+-2Cl- cotransporter (NKCC1).
While NKCC1 was present in the basolateral membranes of duct cells
from rabbit LG, its role remains unclear, and therefore, the aim of the
present work was to further investigate its activity in these ducts.
Methods: Immunofluorescence was used to confirm the localization
of NKCC1. Rabbit LG interlobular ducts were isolated as we
described before. Fluorophotometry with NH4+-pulse technique
was used to elicit pH changes, and the rate of bumetanide-sensitive
cytosolic acidification after addition of NH4+ was used to quantify
the activity of NKCC1. Results were expressed as initial change
of intracellular pH rate calculated for 30 and 60 seconds. A
mixed ANOVA model was applied and p<0.05 was considered as
significantly different.
Results: Immunofluorescence confirmed the presence of NKCC1
in basolateral membranes of duct cells. While the basal activity of
NKCC1 was minimally detectable (0.009±0.006 pH unit/30 sec
and 0.013±0.010 pH unit/60 sec), addition of forskolin (10 μmol/L)
caused a significant increase of its activity to 0.023±0.009 pH
unit/30 sec (p=0.029) and 0.024±0.008 pH unit/60 sec (p=0.045).
However, lower cytosolic [Cl-] had no significant effect on NKCC1
activity in the first 30 sec (0.008±0.003 pH unit/30 sec, p=0.071),
whereas stimulation at 60 sec appeared to be borderline (0.020±0.007
pH unit/60 sec, p=0.053). Hyperosmotic challenge (390 mOsm)
increased NKCC1 activity significantly at both 30 sec (0.023±0.003
pH unit, p=0.0001) and 60 sec (0.024±0.007 pH unit, p=0.025).
Carbachol (100 μmol/L) had no significant effect on NKCC1
activity at either 30 sec (0.001±0.004 pH unit, p=0.867) or 60 sec
(0.006±0.006 pH unit, p=0.388).
Conclusions: Data presented here demonstrated the transmembrane
ionic transport of duct epithelial cells, particularly the central role
of Cl- transport in these cells, which is in part mediated by NKCC1.
These results highlighted the functional involvement of NKCC1
and the pivotal role it may play in LG duct secretion, supporting the
notion that these ducts play a key role in tear production.
Commercial Relationships: Edit Toth-Molnar, None; Mate
Katona, None; Eszter Vizvari, None; Ferenc Rarosi, None; Peter
Orvos, None; Andrea Facsko, None; Viktoria Venglovecz, None;
Zoltan Rakonczay, None; Chuanqing Ding, None; Peter Hegyi,
None
Support: TAMOP-4.2.2.A-11/1/KONV-2013-0035-EU
Program Number: 2483 Poster Board Number: D0087
Presentation Time: 3:45 PM–5:30 PM
Four tear biomarkers distinguish Sjögren’s Syndrome patients
from patients with other autoimmune diseases
Maria C. Edman1, Srikanth Reddy Janga1, Alexander F. Chen2,
Mercy Bechtold2, Alice Kim2, poysophon poysophon2, Wendy J.
Mack2, William Stohl3, Sarah F. Hamm-Alvarez1. 1Pharmacology
and Pharmaceutical Sciences, University of Southern California,
School of Pharmacy, Los Angeles, CA; 2Keck School of Medicine of
USC, Los Angeles, CA; 3Division of Rheumatology, Department of
Medicine, Keck School of Medicine of USC, Los Angeles, CA.
Purpose: Sjögren’s syndrome (SS) is an autoimmune disease (AD)
that commonly affects the lacrimal gland and leads to dry eyes.
Among patients with dry eyes, differentiating patients with SS from
those with other AD or non-AD can be challenging. We recently
identified elevated tear cathepsin S (CTSS) activity as a promising
candidate biomarker for distinguishing SS patients from those with
non-SS AD and non-AD, and we herein evaluate CTSS and three
additional tear proteins, secretory IgA (sIgA), lactoferrin (LF),
and cystatin C (CysC) in SS patients compared to patients with
rheumatoid arthritis (RA) or other non-SS AD
Methods: Female patients with primary or secondary SS (n=28,
age 52±1 yrs), RA (n=27, age 51±2 yrs), or other AD (OAD) (n=18,
age 45±2 yrs) were recruited. An anesthetized Schirmer’s test was
performed on both eyes of each patient, and tear proteins were
eluted and analyzed within 4 hours for CTSS activity and sIgA, LF,
and CysC concentrations using commercial kits. All values were
normalized to total tear protein concentration. Statistical analysis
was performed using one-way ANOVA followed by Bartlett’s test for
equal variance and Tukey’s multiple comparison test, and p ≤ 0.05
was considered significant.
Results: Tear CTSS activity was significantly greater in SS than in
RA (4-fold) or in OAD (8-fold) (SS: 6861±1026, RA: 1826±287,
OAD: 895±136 U/mg). Levels of sIgA and CysC were each 3-fold
lower in SS than in RA or OAD (sIgA, SS: 405±70, RA: 1222±140,
OAD: 1152±145 mg/mL) (CysC, SS 305±40, RA, 944±151, OAD
1048±135 ng/mL), and levels of LF were 3-fold lower in SS than
in RA and 4-fold lower than in OAD (SS: 360±61, RA: 917±92,
AD: 1543±154 mg/mL). There were no significant differences in
CTSS, sIgA and CysC between RA and other OAD, although LF was
significantly lower in RA than in OAD.
Conclusions: Compared to patients with RA or OAD, SS patients
harbor greater tear levels of CTSS activity but lower levels of sIgA,
LF and CysC. These tear proteins may serve as biomarkers and
facilitate earlier diagnosis of SS, thereby enabling more effective
treatment of its ocular pathology.
Commercial Relationships: Maria C. Edman, 13/382,286 (P);
Srikanth Reddy Janga, None; Alexander F. Chen, None; Mercy
Bechtold, None; Alice Kim, None; poysophon poysophon, None;
Wendy J. Mack, None; William Stohl, None; Sarah F. HammAlvarez, 13/382,286 (P)
Support: NH grant EY011386, USC Zumberge Interdisciplinary
Research Grant
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 2484 Poster Board Number: D0088
Presentation Time: 3:45 PM–5:30 PM
Longitudinal Characterization of Tear Fluid Cathepsin S, a
Sjögren’s Syndrome Biomarker, in the male Non-Obese Diabetic
mouse
Srikanth Reddy Janga, Maria C. Edman, Tao Ma, Mihir Shah, Aida
Kouhi, Jingwen Chen, Frances Yarber, Sarah F. Hamm-Alvarez.
Pharmaceutical Sciences, University of Southern California, Los
Angeles, CA.
Purpose: Sjögren’s Syndrome (SS) is an autoimmune disease
characterized by lymphocytic infiltration of lacrimal and salivary
glands and causing decreased glandular function resulting in
subsequent dry eye and dry mouth symptoms. We have previously
shown that activity of the lysosomal protease, Cathepsin S (CTSS),
is elevated in the tear fluid (TF) of male Non-Obese Diabetic (NOD)
mice, a model of SS that spontaneously develops lacrimal gland
exocrinopathy that resembles the human disease. Recently we
confirmed that CTSS is elevated in TF of patients with SS compared
to patients with other autoimmune diseases and healthy controls,
emphasizing its potential as a biomarker for the disease. However,
nothing is known about how CTSS levels are correlated with disease
development and progression. Here, we investigate the changes
in CTSS levels in TF and lacrimal gland (LG) of male NOD mice
monthly starting at 1 month (mth) prior to onset of disease until 7
mths of age, when lymphocytic infiltration is extensive and disease is
prolonged.
Methods: Carbachol-stimulated tear fluid was collected from male
NOD mice at ages 1, 2, 3, 4, 5, 6 and 7 mths (n=4-11 per group),
after which the mice were euthanized and LGs were retrieved for
preparation of lysates. CTSS activity levels were measured in
both tears and lysates using a commercial CTSS activity assay kit
and normalized to total protein concentration. Statistical analysis
was performed through one-way ANOVA with p≤0.05 considered
significant. Values represent Mean±SEM.
Results: TF CTSS activity was 5-fold elevated at 2, 6.6-fold at
3, 7.0 fold at 4, 8.6-fold at 5, 10-fold at 6 and 20-fold at 7 mths
(1;56±0, 2;296±20, 3;340±18 4;400±14, 5; 489±19, 6;571±27 and
7;1166±66) when compared to 1 mth mice, while the activity levels
in lysate showed a 11-fold increase at 2, 17 fold at 3 to 6, and 23.0
fold at 7 mths (1; 387±20, 2; 4468±161, 3; 6593±227, 4;6581±146,
5;6766±238, 6;6388±373, 7;8918±851) when compared to 1 mth
mice. Detectable lymphocytic infiltration of the gland is initiated at 8
weeks in this disease model and ranged from none at 1 mth to 30% at
7 mth.
Conclusions: These results suggest that CTSS activity in LG and TF
continues to increase beyond the early stages of disease. Relative TF
CTSS activity may potentially be used clinically to reflect the extent
of lacrimal gland inflammation and damage.
Commercial Relationships: Srikanth Reddy Janga, None; Maria
C. Edman, 13/382,286 (P); Tao Ma, None; Mihir Shah, None; Aida
Kouhi, None; Jingwen Chen, None; Frances Yarber, None; Sarah
F. Hamm-Alvarez, 13/382,286 (P)
Support: NH grant EY011386
Program Number: 2485 Poster Board Number: D0089
Presentation Time: 3:45 PM–5:30 PM
Tear Fluid Biomarkers: A Comparison of Tear Fluid Retrieval
and Storage Methods
Suzanne Hagan, Eilidh Martin, Katherine Oliver, E.Ian Pearce.
Vision Sciences, Glasgow Caledonian University, Glasgow, United
Kingdom.
Purpose: Increased research using multiplex technology in the field
of ocular surface disease (OSD) has shown its potential in identifying
novel biomarkers. These studies, however, have also highlighted
differences in sampling across laboratories. This preliminary study
compared different tear fluid retrieval and storage methods versus
cytokine expression, using magnetic multiplex bead arrays.
Methods: Pooled tear samples underwent microarray bead analysis
for 7 cytokines (IL1-β, -2, -6, -8, -17, IFN-γ and TNF-α) using
various tear sampling and storage techniques. A standard method
of using 1ml tears in a 50x dilution was used throughout the study,
except when looking at the effect of different tear volumes on
cytokine detection.
Results: Multiple freeze/thaw cycles significantly reduced levels
of IL1-β and -2 (both p<0.05) and showed a trend for a reduction
in the other 5 cytokines. No significant deterioration of cytokines
was noted for samples stored on ice for 5hrs, however storage for
5hrs at room temperature revealed a significant reduction in all 7
biomarkers (p<0.05). Levels of IL1-β and TNF-α were significantly
diminished for tears stored in standard eppendorf tubes, versus
Lo-Bind eppendorfs (p<0.05). Moreover, IL-2, -6, and -17 were
significantly reduced in tears collected with a Schirmer strip versus
a glass microcapillary (p<0.05). A significant reduction in IL-8 and
IFN-g levels was noted in tears retrieved using a minisponge versus
a glass microcap. No significant difference was observed for tears
stored at -20°C versus -80°C, although there was a trend for slight
reductions in cytokine levels at -20°C. There was a general trend
for lower cytokine levels as the tear sample size was increased (1ml
versus 3 and 5 ml), suggesting a possible matrix effect. This reduction
was found to be significant when comparing 1 and 5ml volumes for
IL1-β and 17 (p<0.05).
Conclusions: Differences in tear retrieval and storage methods may
affect cytokine detection by multiplex bead arrays. This may be due
to sample dilution and inherent matrix effects, varying degrees of
protein binding affinities of lab plastics used for storage, and sample
sublimation. A standardised procedure for tear fluid retrieval and
storage would benefit future OSD cytokine analyses. Further work
will be carried out to extend and confirm the present results.
Commercial Relationships: Suzanne Hagan, Allergan (F); Eilidh
Martin, Allergan (F); Katherine Oliver, Allergan (F); E.Ian Pearce,
Allergan (F)
Program Number: 2486 Poster Board Number: D0090
Presentation Time: 3:45 PM–5:30 PM
Changes of matrix metalloproteinases in tears of patients with
diabetes after vitrectomy, and relationship with corneal epithelial
disorder
Takehiro Matsumura, Yoshihiro Takamura, Takeshi Tomomatsu, Yuji
Takihara, Masaru Inatani. University of Fukui, Eiheiji, Yoshida,
Japan.
Purpose: To evaluate the levels of matrix metalloproteinases
(MMPs) in tears of patients with diabetes mellitus, and to investigate
the changes of MMPs levels at perioperative periods and the
relationship with corneal epithelial disorder following vitrectomy.
Methods: The levels of MMPs in tears were measured in the patients
with or without diabetes who were scheduled for vitrectomy. Twentytwo patients with diabetes had proliferative diabetic retinopathy, and
20 patients with epiretinal membrane or macular hole for control
group were recruited. The changes of MMPs levels at perioperative
periods and the relationship with corneal epithelial disorder after
vitrectomy were analyzed.
Results: The mean levels of tear MMPs in the patients with diabetes
before vitrectomy were 6.09 ng/ml, 4.13 ng/ml, and 25.75 ng/ml of
MMP-2, MMP-9, and MMP-10, respectively. The levels of MMP-2,
-9, and -10 at 1 day after surgery in diabetic group were significant
higher than control group (MMP-2, P = 0.008; MMP-9, P = 0.04;
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
MMP-10, P = 0.003). At 1 week after surgery, the level of MMP10 in diabetic group was significant higher than control group (P =
0.004). After vitrectomy, corneal epithelial disorder occurred with 6
patients with diabetic group, while corneal epithelial disorder did not
occur in the control group. Among diabetic group, the MMP-10 level
in tears of patients with corneal epithelial disorder was significantly
higher than that of patients without corneal epithelial disorder (P =
0.04).
Conclusions: The MMPs concentrations in tears of patients
with diabetes were higher than that of non-diabetic patients after
vitrectomy. High MMP-10 levels in tears were observed in patients
of diabetes with corneal epithelial disorder after vitrectomy. Aberrant
level of MMP-10 in tears was suggested to cause corneal epithelial
disorder after vitrectomy.
Commercial Relationships: Takehiro Matsumura, None;
Yoshihiro Takamura, None; Takeshi Tomomatsu, None; Yuji
Takihara, None; Masaru Inatani, None
Program Number: 2487 Poster Board Number: D0091
Presentation Time: 3:45 PM–5:30 PM
Effect of Lid Debridement-Scaling on Dry Eye Signs and
Symptoms in Sjogren’s Syndrome
William Ngo1, 2, Barbara Caffery3, Sruthi Srinivasan1, 2, Lyndon W.
Jones1, 2. 1Centre for Contact Lens Research, Waterloo, ON, Canada;
2
School of Optometry & Vision Science, University of Waterloo,
Waterloo, ON, Canada; 3Toronto Eye Care, Toronto, ON, Canada.
Purpose: To determine the effect of lid-debridement scaling (LBS)
on dry eye signs and symptoms in individuals with Sjogren’s
Syndrome (SS).
Methods: This prospective randomized controlled study enrolled
14 female participants with SS. Diagnosis for SS was confirmed
as per The American College of Rheumatology. Seven individuals
were randomly selected for LBS and the rest served as controls.
LBS was conducted using a stainless steel golf spud (Hilco Wilson
Ophthalmics, Plainville, MA) on both the lower and upper lid
margins of both eyes. Ocular Surface Disease Index (OSDI), ocular
staining score (as per Sjogren’s International Collaborative Clinical
Alliance), fluorescein tear break-up time (FLBUT), meibomian
gland score (using the Meibomian Gland Evaluator on the central 5
glands, MGS), and meibomian gland yielding liquid secretions score
(MGYLS) were assessed prior to LBS, and one month after LBS.
Results: Thirteen participants completed the study. Data from the
right eye only was analyzed.
For the control group (n=6, mean age=62.3±11.6), the pre LBS,
post LBS, and significance level (pre mean±SD vs post mean±SD;
p-value) were: OSDI (58.3±22.1 vs 48.3±29.0; p>0.05), ocular
staining (7.0±4.5 vs 8.2±3.5; p=0.16), MGS (1.3±1.5 vs 1.0±0.9;
p=0.75), MGYLS (0.3±0.5 vs 0.0±0.0; p=0.50), FLBUT (2.99 ±1.54
vs 2.85±1.79; p=0.63).
For the treatment group (n=7, mean age=58.0±8.1), the pre LBS, post
LBS, and significance level were: OSDI (63.2±13.3 vs 46.9±19.4;
p=0.04), ocular staining (6.5±2.9 vs 5.0±3.9; p=0.02), MGS (1.0±1.2
vs 3.1±1.7; p=0.01), MGYLS (0.0±0.0 vs 0.6±1.0; p=0.50), FLBUT
(3.13±0.81 vs 3.45±1.03; p=0.53).
Conclusions: In this pilot study, the group that received LBS showed
statistically significant improvements in OSDI, along with ocular
staining and meibomian gland function. This study indicates that LBS
may help in the management of SS dry eye.
Commercial Relationships: William Ngo, None; Barbara Caffery,
Sjogren’s Society of Canada (S); Sruthi Srinivasan, None; Lyndon
W. Jones, Sjogren’s Society of Canada (F)
Support: Sjogren’s Society of Canada
Clinical Trial: NCT02203188
Program Number: 2488 Poster Board Number: D0092
Presentation Time: 3:45 PM–5:30 PM
Histatin-1 is a Marker for Human Lacrimal Epithelium
Dhara Shah, Marwan Ali, Assraa Jassim Jaboori, Sandeep Jain,
Vinay K. Aakalu. Ophthalmology and Visual Sciences, University of
Illinois at Chicago, Chicago, IL.
Purpose: Study of human lacrimal cell biology is limited by poor
access to tissue samples, small tissue samples, heterogeneous cell
composition of tissue samples and a lack of available cell lines.
In order to develop lacrimal epithelial cell lines and further our
understanding of lacrimal cell biology, we sought to find a better
marker for lacrimal epithelial cells, compared to what is found in the
literature. Previously, we demonstrated that Histatin-1 was highly
and relatively specifically expressed in accessory lacrimal gland
(ALG), using laser capture microdissection of ALG from muller’s
muscle conjunctival resection specimens (MMCR), and Affymetrix ®
microarray analysis.
Methods: We utilized human MMCR specimens and cadaveric main
lacrimal gland (MLG) as sources of lacrimal tissue. These specimens
were analyzed using immunofluorescence antibodies for Histatin-1,
Lactoferrin and Aquaporin-5. Cells from MMCR and MLG were
fixed, permeabilized and incubated with primary antibodies overnight
then with secondary antibodies. Cells were then stained with
mounting medium and analyzed using confocal microscopy.
Results: We found that Histatin-1 is more specific for lacrimal
epithelium in MLG and MMCR compared to the relatively less
specific Aquaporin-5 as we have previously shown. Lactoferrin is
present in conjunctiva and lacrimal epithelium equally.
Conclusions: Histatin-1 is a good, and relatively specific marker
for human lacrimal epithelium in ALG and MLG and can be used to
identify lacrimal cells in future studies.
Commercial Relationships: Dhara Shah, None; Marwan Ali,
None; Assraa Jassim Jaboori, None; Sandeep Jain, None; Vinay
K. Aakalu, None
Support: American Society of Cataract and Refractive Surgery,
Midwest Eye Bank, Fight for Sight Foundation, National Eye
Institute Core Grant, K08EY024339, Research to Prevent Blindness
Unrestricted Grant, EY001792 Departmental Core Grant
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 2489 Poster Board Number: D0093
Presentation Time: 3:45 PM–5:30 PM
Identification and characterization of human tear film
glycoproteins
Carsten Schmelter, Natarajan Perumal, Sebastian Funke, Norbert
Pfeiffer, Franz H. Grus. Department of Ophthalmology, University
Medical Center Mainz, Experimental Ophthalmology, Mainz,
Germany.
Purpose: The glycosylation patterns of tear film proteins have a
major influence on their structure and stability resulting in changes
to various protein properties like signaling or regulatory functions.
Up to date, tear proteomic glycosylation studies are rare and limited
on single or function-related proteins. Therefore, the main objective
of this study was to give a comprehensive overview about the
glycosylation pattern of the human tear proteome by use of a mass
spectrometric platform.
Methods: Tears were pooled from 8 healthy volunteers (mean age:
28 ± 3). Individual tear samples were collected by capillary technique
and Schirmer’s strips. Two-dimensional gel electrophoresis (2-DE)
was performed and the gels were stained with two different dyes:
Glycoprotein staining kit to detect glycoproteins and Colloidal Blue
Staining to visualize the total protein profile. The tear proteome
profiles were analyzed by proteomic based liquid chromatography –
mass spectrometry (LC-MS) strategies. By the usage of bioinformatic
tools based on de novo sequencing, glycoproteins could be revealed
and glycosylation sites characterized.
Results: By 2-DE analysis multiple glycosylation sites of human tear
proteins were supported. Due to buttom-up LC-MS analysis we could
identify up to 30 proteins (false discovery rate < 1%) displaying a
complex glycosylation profile. Thereby, it was possible to detect
these glycoproteins in tear samples collected independently by two
different approaches. By state-of-the-art bioinformatic analyses,
several modification sites could be identified. Thus, for instance,
it was possible to detect new glycosylation sites of abundant tear
proteins like Ig alpha-1 or Ig alpha-2 chains (constant C regions)
as well as modification sites of lesser represented proteins like
Cadherin-6 or Ras-specific guanine nucleotide-releasing factor 2.
Conclusions: Our results further demonstrate that the human tear
film consists of many proteins which show a complex glycosylation
pattern. Aberrant glycosylations of proteins are known to be involved
in various pathophysiological processes, providing the idea that
glycosylation patterns may be of particular interest in biomarker
research. Further studies will be needed to explore the complex
modification pattern of the human tear proteome and to evaluate their
possible role in the formation of eye disorders.
Commercial Relationships: Carsten Schmelter, None; Natarajan
Perumal, None; Sebastian Funke, None; Norbert Pfeiffer, None;
Franz H. Grus, None
Support: Forschungszentrum Translationale Neurowissenschaften
(FTN) Mainz
Program Number: 2490 Poster Board Number: D0094
Presentation Time: 3:45 PM–5:30 PM
Anti-Stress and Basal Tear Stimulatory Tear Factor ‘Lacritin’
Promotes Acetylation of ATG101-Associated Proteins but has No
Apparent Effect on Transepithelial Resistance
Gordon W. Laurie1, Ningning Wang1, Jeffrey Romano1, Robert L.
McKown2. 1Cell Biology, University of Virginia, Charlottesville,
VA; 2Integrated Science and Technology, James Madison University,
Harrisonburg, VA.
Purpose: Lacritin is a multifunctional tear protein whose active
monomer is selectively downregulated in dry eye, and yet is required
to restore health to inflamed epithelia and apparently also stimulates
corneal sensory nerves to promote basal tearing. Our dual purpose
was to further explore lacritin-dependent signaling mechanisms
associated with lacritin mediator ATG101 that rapidly accelerates
autophagy and promotes oxidative phosphorylation to restore health,
and how lacritin might gain contact with corneal sensory nerves,
possibly by transiently loosening tight junctions for lacritin access.
Methods: Cultured human corneal epithelial (HCE) cells were
stressed with dry eye-associated inflammatory cytokines INFG and
TNF in the presence of lacritin or inactive lacritin truncation mutant
C-25. Cell lysates were subjected to affinity precipitation with antiATG101 antibodies. Affinity precipitates were then blotted using
antibodies against acetyl-lysine. To study possible alteration of tight
junctions, transepithelial resistance of HCE or human corneal-limbal
epithelial (HCLE) monolayers or of HCLE stratified cultures in
normal or low calcium media without or with lacritin or C-25 was
measured.
Results: Lacritin, but not C-25, promotes rapid acetylation of several
ATG101 associated proteins in stressed HCE cells. Transepithelial
resistance is greater in HCE cells than in HCLE cells, even of
the latter in stratified culture. Addition of lacritin or C-25 equally
decreased and then increased transepithelial resistance, whereas
positive control low calcium media promoted the progressive loss of
resistance that slowly returned up reintroduction of calcium.
Conclusions: Acetylation of ATG101 associated proteins upon
lacritin, but not C-25 addition, in stressed HCE cells points to
additional signaling mediators of lacritin’s health restorative
activity. Lacritin appears to have no specific affect on transepithelial
resistance, although non-specific loosening of the epithelium may be
sufficient to gain neural access.
Commercial Relationships: Gordon W. Laurie, TearSolutions,
LLC (I), TearSolutions, LLC (P); Ningning Wang, None; Jeffrey
Romano, None; Robert L. McKown, EyeRx (F)
Support: NIH Grant EY024327
Program Number: 2491 Poster Board Number: D0095
Presentation Time: 3:45 PM–5:30 PM
IFN-γ inhibits cell proliferation and outgrowth from cultured
lacrimal gland tissues
Daniela Marcano, Ghanashyam Acharya, Stephen C. Pflugfelder.
Ophthalmology, Baylor College of Medicine, Houston, TX.
Purpose: Increased expression of interleukin-1, interferon-γ (IFN-γ),
and tumor necrosis factor-α has been measured in autoimmune
dacryoadenitis. These pro-inflammatory cytokines stimulate the
production of ROS and RNS species that can induce cell death. This
study tested the hypothesis that IFN-γ inhibits cell outgrowth from
murine lacrimal gland (LG) explants and stimulates production of
pro-apoptotic factors.
Methods: LGs of healthy wild type (WT) and IFN-γ KO C57BL/6
male mice (9-10 weeks old) were removed and used for RT-PCR and
tissue and cell cultures. Total RNA was extracted, cDNA synthesized
and amplified for inducible nitric oxide synthase (iNOS), interleukin1β (IL-1β), and caspase 3. LG cells were obtained by digesting
minced glands in a media supplemented with collagenase type I.
These cells were incubated with WST-1 reagent for 1h at days 3 and
day 14 and absorbance at 440 nm was measured. LG explants were
obtained after digesting small pieces of gland in collagenase for 20
min and cultured on plastic dishes. At day 25, cultures were fixed and
immunofluorescence staining performed for cytokeratins 8 and 14
(K8 and K14), Vimentin, and iNOS.
Results: Compared to IFN-γ KO, higher expression of iNOS, IL-1β,
and caspase 3 was found in WT LGs. Remarkably, iNOS expression
in the IFN-γ KO was 5 times higher than WT. Significantly greater
proliferation was noted in cultured glandular cells from IFN-γ KO
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
compared to WT, suggesting that IFN-γ suppresses it. This finding
is also supported by observed cell outgrowth in 49% of cultured
IFN-γ-KO explants compared to only 34% of WT. Conspicuously,
outgrowth from the IFN-γKO explants is usually greater than the WT
explants. Because fibroblasts are a typical source of contamination
in primary cultures, we used immunofluorescence to characterize
the cellular outgrowth, identifying epithelial cells by K8 and K14
positivity, while spindle-like cells were confirmed by Vimentin
staining. In all cases, cells growing from explants were positive for
either K8 or K14 and negative for vimentin. iNOS was strongly
detected at the leading front of the cell-outgrowth from IFN-γ KO
and WT explants, but immunoreactivity of cells adjacent to the
explants was less in the IFN-γ KO.
Conclusions: IFN-γ suppresses growth and increases production of
pro-apoptotic factors in the LG. Increased iNOS expression in WT
may lead to production of free radicals that reduce cellular viability.
Commercial Relationships: Daniela Marcano, None;
Ghanashyam Acharya, None; Stephen C. Pflugfelder, None
Support: William Stamps Farish Fund, NEI/NIH Core Grant for
vision research EY-002520, Research to Prevent Blindness, the
Oshman Foundation and the Hamill Foundation
Program Number: 2492 Poster Board Number: D0096
Presentation Time: 3:45 PM–5:30 PM
Stem Cell Marker Expression in Accessory Lacrimal Gland as a
Function of Patient Characteristics
Marwan Ali, Dhara Shah, Assraa Jassim Jaboori, Sarmad H. Jassim,
Sandeep Jain, Pete Setabutr, Vinay K. Aakalu. Ophthalmology and
Visual Sciences, University of Illinois at Chicago, Chicago, IL.
Purpose: Lacrimal gland and accessory lacrimal gland (ALG) tissue
contain a variable amount of stem cell marker positive cells. Other
exocrine tissues have been shown to have differential expression
of stem cell marker positive cells related to patient characteristics.
This differential expression may be of value to determine the health,
responsiveness to therapeutic interventions and prognostication for
patients with various disease states. We previously demonstrated
the utility of tissue microarray (TMA) and immunofluorescence
(IF) based analysis of ALG using muller’s muscle conjunctival
resection (MMCR) specimens. We studied the qualitative expression
of the known stem cell and lacrimal cell markers (ABCG2, CD90,
ABCG5, Nestin, CD49f, Aquaporin-5, Histatin-1, Lactoferrin, and
Lacritin) in TMAs and compared these results with relevant patient
characteristics (age, gender, race, status of ocular surface health).
Methods: ALG tissues obtained from MMCR specimens were
created using TMAs. TMAs were constructed using manual tissue
arrayer and blocks were cut into sections using paraffin microtome.
Stem cell markers levels in TMAs were then assessed by two blinded
observers using a standardized scoring system. Patient characteristics
such as age, race, and status of ocular surface health were then
compared against qualitative expression of stem cell markers.
Results: Expression of stem cell markers vary with patient’s
characteristics and disease states.
Conclusions: Thus, differential expression of stem cell markers in
ALG may correlate to patient characteristics. This knowledge can
be used as a basis for further study, and the development of rational
therapeutic interventions.
Commercial Relationships: Marwan Ali, None; Dhara Shah,
None; Assraa Jassim Jaboori, None; Sarmad H. Jassim, None;
Sandeep Jain, None; Pete Setabutr, None; Vinay K. Aakalu, None
Support: American Society of Cataract and Refractive Surgery,
Midwest Eye Bank, Fight for Sight Foundation, National Eye
Institute Core Grant, K08EY024339, Research to Prevent Blindness
Unrestricted Grant, EY001792 Departmental Core Grant
Program Number: 2493 Poster Board Number: D0097
Presentation Time: 3:45 PM–5:30 PM
Angiotensin II infusion induces high blood pressure and decrease
in tear secretion
Shigeru Nakamura, Ryuji Hisamura, Toshihiro Imada, Yusuke Izuta,
Kazuo Tsubota. Ophtalmology, Kei University, Tokyo, Japan.
Purpose: The systemic renin–angiotensin system (RAS) plays an
important role in the endocrine regulation of blood pressure and of
salt and water balance. Tissue-specific RAS is present in the lacrimal
gland and might be involved in tissue function of regulating tear
secretion in the lacrimal gland. The purpose of this study was to
investigate the effect of angiotensin II (Ang II) on tear secretion.
Methods: Female C57BL/6 mice at an age of 8 weeks were used in
this study. Mice were anesthetized by pentobarbital, and an osmotic
minipump (Alzet model 2004) was implanted to deliver Ang II
subcutaneously at a dose of 1.5 mg/kg/day for 4 weeks. Systolic
blood pressure was measured with an automated tail-cuff device.
Change in tear secretion was measured by the cotton thread test for
15 seconds. After Ang II treatment, the mice were killed by cervical
dislocation. The lacrimal glands were removed for histological
examination.
Results: A significant difference was not observed in body weight
gain among Ang II treatment and vehicle groups. Through the
treatment period, a significant increase in systolic blood pressure we
observed in Ang II treatment group (116 ± 0.55 vs 140 ± 31, P<0.001
vs vehicle, n=5, day 14). Tear secretion was lower in the Ang II
treatment group than the initial value through the treatment period
and significant decrease were observed compared to the vehicle group
from day 3 to day14 (3.06 ± 0.88 vs 2.21 ± 0.45, P<0.001 vs vehicle,
n=5, day 14). Reduction of lacrimal achiner cell area were apparent
in the Ang II treatment group.
Conclusions: These data suggest that activation of angiotensin
receptor by Ang II reduces tear secretion and RAS play an important
role in regulation of tear secretion.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Commercial Relationships: Shigeru Nakamura, Ophtecs (E);
Ryuji Hisamura, None; Toshihiro Imada, None; Yusuke Izuta,
None; Kazuo Tsubota, None
Program Number: 2494 Poster Board Number: D0098
Presentation Time: 3:45 PM–5:30 PM
Tear Film Dynamics and Imaging in Tear Break-up
Richard J. Braun1, Carolyn G. Begley2, Peter E. King-Smith3,
Javed Siddique4. 1Dept of Mathematical Sciences, University of
Delaware, Newark, DE; 2School of Optometry, Indiana University,
Bloomington, IN; 3College of Optometry, The Ohio State University,
Columbus, OH; 4Department of Mathematics, Pennsylvania State
University, York, PA.
Purpose: The purpose of this project is to use mathematical models
to visualize tear film (TF) flows and match them with detailed tear
break up (TBU) imaging methods using fluorescence (FL). We
determine the correspondence between FI images for different kinds
of TBU (circular spots and linear grooves) and compute local changes
in tear film osmolarity (c) and FL during and after TBU.
Methods: Math models were solved for local changes tear film
thickness (h), osmolarity and FL (f) concentrations inside the tear
film for localized breakup. FL concentration was converted to FL
intensity I for the thinning TF depending on h and the full range of f
as described by Braun et al (IOVS 2014; 55:1133-1142).
Results: The computed c from the model recovers locally elevated
osmolarity c within areas of TBU as in, e.g., Peng et al (ACIS
2014; 206:250-264) but extends those results significantly. The
model identifies a critical hole size L=[σ/(mv)]1/4d, depending on the
surface tension σ, the viscosity m, the evaporation (thinning) rate v
and the characteristic TF thickness d. For hole radii rw≥L, the TBU
area occurs in the same time d/v and with the same elevated c as for
a flat film. For rw <L, the time to TBU is increased due to inward
surface tension driven flow and the maximum c is decreased due to
(outward) diffusion. The model predicts elevated f as well but its
smaller diffusion rate makes it more susceptible to transport by TF
flow. As a result, f increases much more than c, yielding FL intensity
distributions that are narrower than h distributions, particularly for
rw <L. The computed FL intensity patterns are very important for
properly interpreting TBU with FL imaging and we quantify this for
different initial values of f.
Conclusions: The model explains why small enough holes in the
lipid layer do not lead to TBU, and quantifies FL imaging vs. TF
thickness dynamics in TBU areas. The model is closely compared
with experimental data, matches well with in vivo observations and is
instrumental in understanding TF and TBU dynamics.
TBU time (TBUT, relative to d/v) as a function of hole radius rw
(relative to L) for different peak thinning rates with background
1micron/min rate. Fixed thinning rate distributions are roughly
piecewise constant (dashed) and Gaussian (solid). TBUT increases
dramatically for rw <L.
Commercial Relationships: Richard J. Braun, None; Carolyn G.
Begley, None; Peter E. King-Smith, None; Javed Siddique, None
Support: NIH EY021794 (CGB), EY017951 (PEK-S) and NSF
1412085(RJB), Simons Foundation Grant 281839 (JS)
Program Number: 2495 Poster Board Number: D0099
Presentation Time: 3:45 PM–5:30 PM
Fluorescein tear breakup time measurement disrupts tear film
stability
Meng C. Lin1, 2, Andrew D. Graham3, Thao Yeh2, Tiffany Yuen3.
1
School of Optometry, University of California, Berkeley, Berkeley,
CA; 2Vision Science Graduate Program, University California,
Berkeley, Berkeley, CA; 3Clinical Research Center, School of
Optometry, University of California, Berkeley, Berkeley, CA.
Purpose: We aim to provide insights into the discrepancies between
results from non-invasive (NI) and invasive methods of measuring
tear breakup time (TBUT). We hypothesize that an invasive method
can potentially perturb the tear lipid layer causing a previously
stable tear film (TF) to break up prematurely; a greater proportion of
TFs would be expected to be assessed as unstable with an invasive
method, compared with a NI method.
Methods: Tear film stability (TFS) was assessed on 101 subjects,
with measurement of NITBUT using a Medmont E300 corneal
topographer followed by invasive TBUT using NaF dye (FTBUT) on
strips. A second cohort of 137 subjects was similarly measured, but
with 2 μl of 0.35% NaF instilled by micropipette.
Results: A stable TF was defined as TBUT>10s. In the cohort
whose NaF was applied by strips, 55% of eyes exhibited unstable
NITBUT and FTBUT; 8% stable NITBUT and FTBUT; 35% stable
NITBUT but unstable FTBUT; 2% unstable NITBUT but stable
FTBUT. A higher proportion of TFs was assessed as unstable with
the invasive method, compared with the NI method (p<0.001). In
the cohort whose volume of NaF dye was controlled, 33% of eyes
exhibited unstable NITBUT and FTBUT; 36% stable NITBUT and
FTBUT; 19% stable NITBUT but unstable FTBUT; 12% unstable
NITBUT but stable FTBUT. No significant difference was found in
the proportion of unstable TF between the invasive and NI methods
(p=0.101). There was a higher proportion of stable TF assessed as
unstable with NaF strips than with 2ml NaF (35% vs. 19%; p=0.001).
There was a higher proportion of unstable TF assessed as stable with
2ml NaF than with NaF strips (12% vs. 2%; p=0.001).
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Conclusions: Discordance between NITBUT and FTBUT occurred
37% and 31% of the time with F strips and 2ml NaF, respectively.
NaF dye delivered using a strip, presumably more invasive compared
with 2ml NaF delivered by micropipette, further decreased TBUT,
thus contributing to the discordance between results from the
different methods of TFS assessment. Conversely, cases of short
NITBUT but long FTBUT may be due to an uneven TF that appears
stable with NaF (i.e., no dark spots but areas of NaF intensity appear
differently) but unstable by distortion of the mires with the NI
method. Further investigation is warranted to determine the optimum
volume of NaF dye required to induce minimum disruption to the TF,
and the optimum endpoint for NITBUT assessment.
Commercial Relationships: Meng C. Lin, None; Andrew D.
Graham, None; Thao Yeh, None; Tiffany Yuen, None
Support: UC Berkeley Clinical Research Center Unrestricted Fund;
Roberta Smith Research Fund
Program Number: 2496 Poster Board Number: D0100
Presentation Time: 3:45 PM–5:30 PM
The Pre-Corneal Fluid Shell. What is the Effect of Drop
Instillation?
Anthony J. Bron1, 2, Norihiko Yokoi3, Yang Zhenghao3, 4, Georgi A.
Georgiev5. 1Nuffield Lab Ophthalmology, University of Oxford,
Oxford, United Kingdom; 2Vision and Eye Research Unit (VERU),
University of Anglia Ruskin, UK, Cambridge, United Kingdom;
3
Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,
Japan; 4Department of Biomedical Engineering, Faculty of Life and
Medical Sciences, Doshisha University, Kyotanabe, Japan; 5Faculty
of Biology, University of Sofia “St. Kliment Ohridski”, Sofia,
Bulgaria.
Purpose: In an earlier study we found that the precorneal tear film
(PTF), comprising its tear film lipid layer (TFLL) and mucoaqueous
subphase (MAS), behaves as a “Fluid Shell”, integrated as one body
and moving with the cornea during horizontal saccades (Yokoi et al.
Ocul Surf. 2014). We further showed that the fluorescein-stained film
is imprinted by meniscus-induced thinning (MIT) in pauses at the end
of any saccade, made visible as dark arcs on return from horizontal
saccades and dark bands after vertical saccades. Refractile, bright
bands are revealed on return from a vertical saccade, resulting from
corneal indentation by the lid margins. Here, we studied the effect of
a saline drop on PTF behavior after horizontal or vertical saccades.
Methods: The effect of 25 μL drops of saline on PTF behavior
was studied in the left eyes of 17 subjects without ocular surface
disease, [8 males, 9 females, age: 31.4 ±8.4 (SD) years], The
fluorescein-stained MAS and the TFLL were studied after vertical or
horizontal saccades, using video-biomicroscopy and DR-1, videointerferometry, respectively.
Results: In most instances the fluorescein-stained PTF and lower
meniscus were visibly increased in volume. During blinking, the
thickened MAS was dragged upwards by the upstroke of the blink but
flowed back in the blink interval. When the eye was paused in upgaze
or downgaze, a horizontal, dark band of PTF thinning, imprinted
on the MAS, was revealed when the eye returned to the primary
position. The PTF, expanded by drop instillation, was seen to flow
following the return saccades, the dark bands sometimes forming
a barrier to this flow. After drop instillation, video- interferometry
demonstrated that the TFLL interference pattern after a horizontal
and return saccade no longer resembled the primary pattern as it does
in the untouched normal eye. Also, the return pattern continued to
move, in the direction of the original saccade. This suggests a loss of
cohesion between the TFLL and the MAS in this situation.
Conclusions: The mucoaqueous subphase of the tear film and its
lipid layer are transiently dissociated after the instillation of a saline
drop. Future studies will explore the behavior of tear substitutes on
PTF integrity in normal subjects and in patients with ocular surface
disease.
Commercial Relationships: Anthony J. Bron, None; Norihiko
Yokoi, None; Yang Zhenghao, None; Georgi A. Georgiev, None
Program Number: 2497 Poster Board Number: D0101
Presentation Time: 3:45 PM–5:30 PM
A novel tear analytical method based on surface enhanced raman
spectroscopy
Liying Zhang1, 2, Dulei Zou1, 2, Juan Li1, 2, Shangkun Ou1, Yangluowa
Qu1, Tingting Liu1, Sanming Li1, Bin Ren3, Zuguo Liu1, 2, Wei Li1,
2 1
. Eye Institute of Xiamen University, Fujian Provincial Key
Laboratory of Ophthalmology and Visual Science, Xiamen, China;
2
Xiamen University affiliated Xiamen Eye Center, Xiamen, China;
3
State Key Laboratory of Physical Chemistry of Solid Surfaces,
Xiamen, China.
Purpose: Surface-enhanced raman spectroscopy (SERS) has been
suggested as a quick, noninvasive and sensitive analytical tool. This
study was to investigate the utility of SERS in tear analysis.
Methods: A minimum of 1.5μL tear was collected from each healthy
volunteer. A comparative manner was used to establish an optimal
tear SERS analytical method. The enhanced effects and spectra
reproducibility were tested by raman spectroscopy, and the signals
based on silver nanoparticles (Ag NPs) and gold nanoparticles (Au
NPs) were compared. SERS spectra variation of tear-Ag NP mixture
during evaporation was also evaluated. Spectra of different crystal
zone was observed and photographed after the mixture deposit and
crystallized. The optimal effect of above established methods were
further confirmed by detecting three main tear proteins lysozyme,
lactoferrin and albumin and infectious pyocanine tear.
Results: Compared with the Au NPs enhanced SERS, stable and
reproducible spectra could be obtained in the Ag NPs enhanced
SERS. In addition, better signal to noise (SNR) and more peaks were
shown in the SERS spectra. The Raman shift intensity increased
gradually during the evaporation of tear-nanoparticles mixture and
reaches the maximum at transitional phase, and highly reproducible
spectra were obtained; The reproducibility, relative intensity and
the SNR of SERS spectra observed at central none crystal zone
were higher than those of central square-dendritic crystal zone and
edge zone. The three main tear proteins, i.e. lysozyme, lactoferrin
and albumin were detected with the same method and showed their
specific raman shift. Among the three proteins, lysozyme showed
major contribution to normal tear SERS spectrum. Pyocyanine tear
showed specific peaks compared with that of healthy tear.
Conclusions: Surface enhanced raman spectroscopy of human tear
exhibits specific raman spectra, SERS may be applied in tear analysis
for the diagnosis of ocular surface diseases.
Commercial Relationships: Liying Zhang, None; Dulei Zou,
None; Juan Li, None; Shangkun Ou, None; Yangluowa Qu, None;
Tingting Liu, None; Sanming Li, None; Bin Ren, None; Zuguo
Liu, None; Wei Li, None
Program Number: 2498 Poster Board Number: D0102
Presentation Time: 3:45 PM–5:30 PM
Instrument for simultaneous assessment of tear film surface
quality and subjective vision
David Alonso-Caneiro, Stephen J. Vincent, Brett A. Davis, Ross
Franklin, Michael J. Collins. Contact Lens and Visual Optics
Laboratory, Queensland University of Technology, Brisbane, QLD,
Australia.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Purpose: To develop and test a custom-built instrument to
simultaneously assess tear film surface quality (TFSQ) and subjective
vision quality.
Methods: An infra-red illuminated Placido disk reflects a series of
concentric rings from the tear film on the ocular surface to a camera
that acquires a video recording at 10 fps. Simultaneously, the subject
observed a static target (Siemens star) presented on a micro-display
monitor via a beam splitter. The subject continuously adjusts an
audible scale [0 (bad) – 10 (good)] to quantify their perceived image
quality over time. By simultaneously recording the videokeratoscopic
images and the subjective vision quality score, we were able
to quantify TFSQ over the pupil region, while simultaneously
acquiring the relative quality of vision. To test the system, a set of
measurements were acquired for 3 subjects under controlled blinking
(a blink every 10 seconds for 60 seconds duration) and suppressed
blinking (no blinking for 30 seconds duration) conditions. For each
condition, an initial practice session was undertaken, followed by
two sets of measurements. The measurements were taken during soft
hydrogel contact lens wear.
Results: A correlation analysis between the TFSQ and the subjective
vision quality score during the inter-blink interval reveals a strong
correlation during the suppressed blinking conditions (mean
Pearson’s r=0.91, range 0.58 to 0.95, p<0.05). This correlation was
still significant, although weaker, during the controlled blinking
conditions at 6 blinks per minute (mean Pearson’s r=0.62, range 0.51
to 0.71, p<0.05).
Conclusions: An apparatus and software methods to simultaneously
record and analyze TFSQ and subjective vision was developed. The
infra-red Placido disk light ensures that the pupil is not constricted
during the measurements and that subjective vision quality scores
reflect normal pupil sizes. The method may contribute to our
understanding of the influence of tear film stability upon vision
quality.
Figure 1. Estimated TFSQ and the subjective vision score
during suppressed blinking conditions (top). Representative
videokeratoscopic images for a good (A) and poor (B) TFSQ during
the inter-blink interval, the red regions mark the areas of severe
break-up (bottom).
Figure 2. Estimated TFSQ and the subjective vision quality score
during control blinking conditions. The subject is prompted (with a
tone) to blink every 10 seconds.
Commercial Relationships: David Alonso-Caneiro, None; Stephen
J. Vincent, None; Brett A. Davis, None; Ross Franklin, None;
Michael J. Collins, None
Program Number: 2499 Poster Board Number: D0103
Presentation Time: 3:45 PM–5:30 PM
High Resolution Color Micrographs and Analysis of Tear Film
Breakup
Peter E. King-Smith1, Kathleen S. Reuter1, Carolyn G. Begley3,
Richard J. Braun2. 1Optometry, Ohio State University, Columbus,
OH; 2Mathematical Sciences, Univerisity of Delaware, Newark, DE;
3
Optometry, Indiana University, Bloomington, IN.
Purpose: To study and analyze high resolution color micrographs of
non-invasive tear film breakup.
Methods: Over 10,000 high resolution color micrographs from
126 subjects were examined for signs of tear film breakup. Images
covered an area of 200 μm diameter with a resolution of 1 μm.
Blur from movement was eliminated by stroboscopic illumination.
As described in the results and figures, breakup was recognized
as an area bounded by low contrast, colored, contour-like fringes
corresponding to the surrounding tear film. Breakup was observed
in six subjects. To emphasize these colored fringes, “chromaticity
images” were generated to show color information after discarding
the luminance information from the lipid layer. Contrast of the
recorded and chromaticity images was increased to show details.
Results: Figs. 1 and 2 give examples of high resolution images of
breakup; panels A and B show the recorded and chromaticity images.
In both figures, the regions of breakup have a pale orange-red color
compared to the surrounding tear film. Fig. 1B shows a breakup area
(between arrows) of about 50 μm diameter with additional breakup
areas at the edge of the image. In Fig. 1A, the reflectance over the
breakup areas is similar to that over the tear film whereas the breakup
area has high reflectance in Fig. 2A.
Conclusions: The orange color of breakup may be due to interference
between reflections from the peaks and valleys of the of the tear
surface as it is “draped” over the rough surface of the epithelium.
Fluid dynamics simulations indicate that localized evaporation could
cause the large breakup area in Fig. 2 but not the smaller area in
Fig. 1B. In the latter case, circular objects (perhaps lipid droplets),
indicated by arrows in Fig. 1A, may contribute to breakup. In Fig.
2A, arrows indicate dark areas which may be gaps between epithelial
cells caused by osmotic shrinkage. The high reflectance in Fig. 2A
may be caused by increased refractive index of the ocular surface,
again due to osmotic shrinkage.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Fig. 1. Recorded (A) and “chromaticity” (B) images of tear film
breakup, 54 year old white female, normal Ocular Surface Disease
Index, OSDI, score. See text for details.
Fig. 2. Recorded (A) and chromaticity (B) images of tear film
breakup, 62 year old white female, normal OSDI score. See text for
details.
Commercial Relationships: Peter E. King-Smith, None; Kathleen
S. Reuter, None; Carolyn G. Begley, None; Richard J. Braun,
None
Support: EY 017951 (PEK-S), EY021794 (CGB), NSF 1022706
(RJB)
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 2500 Poster Board Number: D0104
Presentation Time: 3:45 PM–5:30 PM
Involvement of the Extrinsic and Intrinsic Pathways in UBVInduced Apoptosis of Corneal Epithelial Cells
John L. Ubels1, Courtney D. Glupker1, Mark P. Schotanus1, Jodie
T. De Vries1, Loren D. Haarsma2. 1Department of Biology, Calvin
College, Grand Rapids, MI; 2Physics and Astronomy, Calvin College,
Grand Rapids, MI.
Purpose: Investigating the possible role of the high [K+] in tears
(25 mM) in protecting the cornea from UVB, we have reported that
exposure of human corneal limbal epithelial (HCLE) cells to UVB
results in loss of K+ from the cells, activation caspases-8 and -3, and
apoptosis. This induction of apoptosis is inhibited by incubation
of the cells in medium with elevated [K+] (Exp Eye Res. 92:425,
2011). The present study investigated whether the response to UVB
is mediated via Fas ligand-independent activation of Fas (extrinsic
pathway) or via caspase-9 (intrinsic pathway).
Methods: HCLE cells were exposed to 150 mJ/cm2 UVB (302 nm)
followed by incubation in medium containing 5.5, 25 or 50 mM K+
for up to 6 hours. Activation of caspases-3, -8 and -9 was measured
using fluorometric assays. Control cells, not exposed to UVB, were
incubated in medium with 5.5 mM K+. Knockdown of Fas and
caspase-8 by siRNA was confirmed by western blot. Activation of
Kv3.4 channels by UVB (80 mJ/cm2), determined using BDS-1, was
measured by patch-clamp recording in the whole cell, perforated
patch mode.
Results: Knockdown of Fas to <15% of control levels reduced
UVB-induced activation of caspase-8 by only 15% and had no effect
on caspase-3 activity or activation of K+ channels. Knockdown
of caspase-8 to <17% of control levels had no effect on caspase-3
activation by UVB, but complete inhibition of caspase-8 by Z-IEDTFMK prevented activation of caspase-3 by UVB. Exposure of HCLE
cells to UVB activated caspase-9 by 12-fold over control levels
within 4 hr. This activation was inhibited in medium with high [K+].
UVB–induced caspase-9 activity is completely inhibited by Z-LEHDFMK. UVB caused a 6.9-fold increase in caspase-8 activity and a
45.8-fold increase in caspase-3. This activation was reduced to 0%
and 12% of maximum, respectively, by inhibition of caspase-9.
Conclusions: Knockdown of Fas has minimal effect on downstream
UVB-induced activation of apoptotic mechanisms, while UVBinduced activation of caspases-8 and -3 is highly dependent on
caspase-9 activation. It appears that the Fas-mediated extrinsic
pathway is less important than the intrinsic pathway in the response
of HCLE cells to UVB. The suppression of UVB-induced caspase-9
activity by high extracellular [K+] supports our overall hypothesis
that high [K+] in tears protects the corneal epithelium from UVB by
reducing K+ efflux when K+ channels are activated.
Commercial Relationships: John L. Ubels, None; Courtney D.
Glupker, None; Mark P. Schotanus, None; Jodie T. De Vries,
None; Loren D. Haarsma, None
Support: NIH R15EY023836, Den Ouden Summer Research
Fellowship, Calvin Research Fellowship
Program Number: 2501 Poster Board Number: D0105
Presentation Time: 3:45 PM–5:30 PM
Classification system of clearance of lipids in human tears
according to optical coherence tomography parameters
Maria Silvana Galantuomo, Pietro E. Napoli, Franco Coronella,
Giovanni M. Satta, Maurizio Fossarello. Ophthalmology, University
of Cagliari, Cagliari, Italy.
Purpose: To establish a classification system for clearance of lipids
(CoL) in human tears according to optical coherence tomography
(OCT) parameters. The purpose of this classification system is to
provide a uniform and objective assessment of the dynamics of tear
lipids with a novel technique of contrast-enhanced optical coherence
tomography imaging in evaluation of CoL.
Methods: The CoL appearance grading scale contains a set of OCT
images standards illustrating a range of various concentrations of
lipids in human tears (0.5%, 0.25%, 0.125%, 0.06%, 0%). These
standards consist of OCT scans of the lower tear meniscus (LTM)
at baseline and after instillation of a lipid-based tracer (containing
different concentrations of lipids) or saline. Fifty-one OCT images
were evaluated and scored by three cornea subspecialists in a masked
fashion according to the scale.
Results: High inter-observer agreement was found using the scale to
classify the CoL, with respect to the appearance of OCT reflectivity
(CoL +0.90, interclass correlation coefficient for consistency using a
2-way mixed effect model).
Conclusions: The CoL appearance grading system is a simple,
reproducible system for classifying the OCT appearance of turnover
of lipids in human tears.
Commercial Relationships: Maria Silvana Galantuomo, None;
Pietro E. Napoli, None; Franco Coronella, None; Giovanni M.
Satta, None; Maurizio Fossarello, None
Program Number: 2502 Poster Board Number: D0106
Presentation Time: 3:45 PM–5:30 PM
Meibum color and fatty acid in patients with meibomian gland
dysfunction
Reiko Arita1, 2, Naoto Mori3, Rika Shirakawa2, Kei Asai3, Takahiro
Imanaka3, Yasufumi Fukano3, Masatsugu Nakamura3, Shiro Amano4.
1
Itoh Clinic, Bunkyo-ku, Japan; 2Ophthalmology, University of
Tokyo, Tokyo, Japan; 3Santen Pharmaceutical Co., Ltd., Osaka,
Japan; 4Inouye Eye Hospital, Tokyo, Japan.
Purpose: Posterior blepharitis, defined as inflammatory condition
of the posterior lid margin, is a common disease encountered in
ophthalmic clinics. The major cause of posterior blepharitis is
meibomian gland dysfunction (MGD). We measured the meibum
fatty acid (FA) composition in the patients with MGD and control
subjects, and analyzed the correlation between meibum FA
composition and clinical parameters.
Methods: Thirty eight MGD subjects (13 men, 25 women; mean
± SD of age, 66.9 ± 15.0 years) and 20 control subjects (8 men, 12
women; 64.5 ± 6.7 years) were enrolled. Ocular symptoms score,
corneal and conjunctival staining score, tear film breakup time and
Schirmer value were evaluated. Lid margin abnormalities, meibomian
gland morphology and meibum qualities were evaluated at six
sites; three sites in upper lid (nasal, central and temporal) and three
sites in lower lid (nasal, central and temporal). FA composition in
collected meibum was analyzed by LC-FTMS system. Principal
component analysis (PCA) and Volcano plot analysis were performed
to determine relationships between FA composition and clinical
parameters.
Results: The MGD group had significantly higher mean of most lid
margin findings score, most meibomian gland findings score and the
degree of ease in expressing meibum than those of the control group.
The distribution of meibum color and viscosity saw a significant
difference between MGD and control group. Upper meibum color
score was significantly correlated with epiphora and sticky sensation
in MGD group (epiphora; r=0.49 p=0.020, sticky sensation; r=0.45
p=0.038). There were no significant differences in the grading,
color and viscosity of meibum between upper and lower eyelid and
among nasal, center and temporal sites. One hundred species of FA
were detected in the meibum. Very long chain FAs with 36 and 37
carbon chains were detected in human meibum. Also, FAs with oddnumbered carbon chain such as 17, 19 and 21 carbons were detected.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Clear and yellow meibum groups have a different characteristic to FA
composition. Volcano plot analysis indicated that most unsaturated
FAs had a tendency to increase from clear to colored meibum groups,
especially FAs (30:4), (32:4) and (32:5) showed a tendency to
increase clearly (p<0.00001).
Conclusions: FA composition in human meibum was correlated with
the color grading of meibum classified with a slit-lamp. This finding
is helpful to elucidate the pathogenesis of MGD.
Commercial Relationships: Reiko Arita, Santen Pharmaceutical
company (F); Naoto Mori, Santen Pharmaceutical company
(E); Rika Shirakawa, Santen Pharmaceutical company (F); Kei
Asai, Santen Pharmaceutical company (E); Takahiro Imanaka,
Santen Pharmaceutical company (E); Yasufumi Fukano, Santen
Pharmaceutical company (E); Masatsugu Nakamura, Santen
Pharmaceutical company (E); Shiro Amano, Santen Pharmaceutical
company (F)
Program Number: 2503 Poster Board Number: D0107
Presentation Time: 3:45 PM–5:30 PM
Effects of insulin and high glucose on human meibomian gland
epithelial cells
Juan Ding, Yang Liu, David A. Sullivan. Harvard Medical School,
Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston,
MA.
Purpose: Type II diabetes is a risk factor for meibomian gland
dysfunction (MGD). We hypothesize that this diabetic impact
is due, at least in part, to the direct effects of insulin resistance
and hyperglycemia on human meibomian gland epithelial cells
(HMGECs). To begin to test this hypothesis, we examined whether
insulin and high glucose influence immortalized (I) HMGECs. More
specifically, we evaluated whether insulin promotes cell proliferation,
differentiation and AKT signaling, in a manner similar to that of
insulin-like growth factor-1 (IGF-1). We also determined whether
high glucose is toxic to IHMGECs, and affects signaling molecules
such as insulin receptor (IR), IGF-1R, AKT and ERK, as well as
the lipogenesis regulator sterol-regulatory element binding protein
(SREBP)-1.
Methods: IHMGECs were cultured in serum-containing medium
and treated with insulin, IGF-1, IGF-1R blocking antibody, glucose
or mannitol for varying time periods. Specific proteins were detected
by Western blots, cell proliferation was evaluated by manual cell
counting and lipids were assessed with LipidTOX staining and high
performance thin layer chromatography.
Results: We found a dose-dependent increase in p-AKT after insulin
treatment. However, this signaling occurred at supra-physiological
levels of insulin and appeared to be mediated via IGF-1R. Antibody
blocking of the IGF-1R diminished the p-AKT signaling induced
by insulin. Insulin stimulated cell proliferation and increased
the accumulation of neutral lipids, specifically, triglycerides, in
IHMGECs. High glucose induced a progressive cell loss, which was
accompanied by significantly reduced levels of p-AKT, IGF-1R and
SREBP-1. High glucose had no effect on IR or ERK signaling.
Conclusions: Our data show that insulin activates p-AKT via IGF1R in IHMGECs, and promotes their proliferation and neutral lipid
accumulation. Our data also demonstrate that high glucose is toxic to
IHMGECs, and decreases the cellular content of p-AKT, IGF-1R and
SREBP-1. These results support our hypothesis that insulin resistance
and hyperglycemia have a negative effect on HMGECs and may help
explain why type II diabetes is a risk factor for MGD.
Commercial Relationships: Juan Ding, Schepens Eye Research
Insitute has filed a patent around this technology (P); Yang Liu,
Schepens Eye Research Insitute has filed a patent around this
technology (P); David A. Sullivan, Schepens Eye Research Insitute
has filed a patent around this technology (P)
Support: Supported by NIH grants 1K99EY023536-01A1,
R01EY05612, the Margaret S. Sinon Scholar in Ocular Surface, and
Guoxing Yao Research Fund
Program Number: 2504 Poster Board Number: D0108
Presentation Time: 3:45 PM–5:30 PM
Do cyclosporine A, anakinra, uridine triphosphate, rebamipide
or bimatoprost influence the proliferation, differentiation or
signaling of human meibomian gland epithelial cells?
David A. Sullivan, Yang Liu, Juan Ding, Wendy R. Kam. Schepens
Eye Res Inst/Harvard Med School, Boston, MA.
Purpose: Researchers have hypothesized that treatment with
cyclosporine A (CyA), interleukin-1 receptor antagonists (e.g.
anakinra), P2Y2 receptor agonists (e.g. uridine triphosphate; UTP)
and rebamipide may alleviate human meibomian gland dysfunction
(MGD) and/or dry eye disease. Investigators have also proposed that
prostaglandin analogues (e.g. bimatoprost) may induce MGD. Our
goal was to determine whether these compounds directly influence
human meibomian gland epithelial cell (HMGEC) function.
Methods: Multiple concentrations of each compound were tested
for effects on immortalized (I) HMGEC morphology and survival.
Non-toxic dosages were used for our studies. IHMGEC were cultured
in the presence of vehicle, CyA (8 nM), anakinra (10 mg/ml), UTP
(100 mM), rebamipide (1 nM) or bimatoprost (10 mM) for up to 6
days in various media. Experiments were repeated at least twice,
and included positive controls for proliferation (epidermal growth
factor and bovine pituitary extract), differentiation (azithromycin)
and signaling pathways (insulin-like growth factor 1). Cells were
analyzed for neutral lipid staining (LipidTox), lysosome accumulation
(LysoTracker), lipid composition (high performance thin-layer
chromatography) and AKT phosphorylation.
Results: Our findings demonstrate that CyA, anakinra, UTP,
rebamipide and bimatoprost had no effect on the proliferation,
neutral lipid content, lysosome number, or levels of free cholesterol,
triglycerides or phospholipids in IHMGEC. CyA, anakinra,
rebamipide and bimatoprost significantly reduced the phosphorylation
of AKT, as compared to control. Of interest, tested doses of CyA
above 8 nM (i.e. 10 nM and 0.5 mM) killed the IHMGEC.
Conclusions: Our results show that CyA, anakinra, UTP, rebamipide
and bimatoprost do not influence the proliferation or differentiation of
IHMGEC. However, with the exception of UTP, these compounds do
decrease the activity of the AKT signaling pathway, which is known
to promote cell survival.
Commercial Relationships: David A. Sullivan, None; Yang Liu,
None; Juan Ding, None; Wendy R. Kam, None
Support: Supported by NIH grants EY05612 & 1K99EY02353601A1, the Margaret S. Sinon Scholar in Ocular Surface Research
fund, and the Guoxing Yao Research Fund
Program Number: 2505 Poster Board Number: D0109
Presentation Time: 3:45 PM–5:30 PM
Meibomian gland dysfunction and hypercholesterolemia
Yousef A. Alghamdi2, 1, Nabeel M. Shalabi2, 1, Allison L. McClellan2, 1,
Anat Galor2, 1. 1Ophthalmology, Bascom Palmer Eye Institute, Miami,
FL; 2Ophthalmology, Miami Veterans Administration Medical Center,
Miami, FL.
Purpose: To study the relationship between meibomian gland
parameters and systemic lipid levels in an elderly, predominantly
male population.
Methods: Patients with normal eyelid and corneal anatomy were
prospectively recruited from the Miami VAMC eye clinic. Patients
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
underwent a complete ocular surface examination including
assessment of meibomian gland (MG) parameters. The main outcome
measures were the correlations between meibomian gland parameters
and systemic lipid levels.
Results: The mean age of the 136 patients was 65 (standard
deviation 11); 90% were male, 52% white, and 30% Hispanic. A
large percentage of the population (86%, n=117) had abnormal MG
parameters defined as a score of 2 or greater (in the more severely
affected eye) on any of the studies MG parameters (by subtype:
vascularity 19%, plugging 69%, drop-out 48%, abnormal quality
60%). All associations between MG parameters and systemic lipid
layers (total cholesterol, high-density lipoprotein, low-density
lipoprotein, triglycerides) were weak and most were not significant.
The only significant association was a negative one between
triglyceride levels and meibum quality (Spearman’s rho -0.19,
p-value=0.03).
Conclusions: Meibomian gland dysfunction is a frequent finding in
an elderly, predominantly male population and does not seem to be
related to systemic lipid levels.
Commercial Relationships: Yousef A. Alghamdi, None; Nabeel M.
Shalabi, None; Allison L. McClellan, None; Anat Galor, None
Program Number: 2506 Poster Board Number: D0110
Presentation Time: 3:45 PM–5:30 PM
A new method for determining the effectiveness of a novel
mucomimetic formulation on tear film in an adverse dry
environment
Ali Abusharha1, E.Ian Pearce2. 1Optometry and Vision Sciences, King
Saud University, Riyadh, Saudi Arabia; 2School of health and life
sciences, Glasgow Caledonian university, Glasgow, United Kingdom.
Purpose: To assess the ability of two different treatments techniques
to manage tear film disruption that results from exposure to a low
relative humidity (RH).
Methods: Two environmental conditions (40%RH/210C and 5%RH
/210C) were created using a controlled environmental chamber. At
5% RH,Rohto Dry Eye Relief drop was instilled in two different
modalities, Protection and Relief. At protection visit, Rohto drop
was instilled before the exposure, while in relief technique, subjects
were exposed to low RH for 15 minutes, and then the Rohto drop was
instilled. Rohto was selected for its novel formulation as it contains
a biopolymer of hyaluronic acid and tamarind seed polysaccharide.
Evaporation rate, lipid layer thickness (LLT), noninvasive tear breaks
up time (NITBUT), osmolarity and ocular comfort were assessed in
normal and dry environmental conditions with the use of Rohto in
two different treatment modalities.
Results: All tear film parameters (except Osmolarity, p=0.055) were
adversely affected by the exposure to low RH (p<0.05). The use
of Rohto drops in both techniques resulted in improvement in LLT,
NITBUT and ocular comfort during exposure to 5%RH (p<0.05).
Tear evaporation was significantly improved in protection technique
(p=0.008) but not in the relief method (p=0.09) in compare to 5%.
Conclusions: Protection and relief techniques were shown to be
effective. Using Rohto eye drops before exposure to dry environment
(Protection) was superior to relief for tear evaporation. Therefore,
for maximum effect, it is recommended to use Rohto prior to
exposure to desiccating environment such as this found in aircraft.
The present study demonstrates that using CEC has the potential
to provide researchers with a readily available method to evaluate
rapidly the efficiency of tear supplementation. By using the CEC,
tear film parameters that are typical to those with dry eye patients
could be simulated easily in the laboratory environment. This new
method allows further evaluation of tear film parameters and dry eye
treatment protocols in labs before try it on dry eye patients.
Commercial Relationships: Ali Abusharha, None; E.Ian Pearce,
None
Support: n/a
Program Number: 2507 Poster Board Number: D0111
Presentation Time: 3:45 PM–5:30 PM
Ductal dilation with Meibum retention and decreased expression
of PPARγ in Meibomian gland following ocular surface alkali
injury in mice
Shin Mizoguchi1, Yuka Okada1, Reiko Arita2, Geraint J. Parfitt3, Yilu
Xie3, James V. Jester3, Shizuya Saika1. 1Ophthalmology, Wakayama
Medical University, Wakayama, Japan; 2Itoh Clinic, Saitama, Japan;
3
University of California, Irvine, CA.
Purpose: We previously reported Meibomian gland duct is dilated
following ocular surface alkali injury and that this finding is
independent of Smad3 signaling in mice. The purpose of this study
was to examine the morphological changes within the Meibomian
gland by using 3D image and expression pattern of PPARγ.
Methods: Ocular surface alkali burn was produced by topical
application of 1N NaOH in one eye of adult C57BL/6 mice.
Following healing intervals of 5, 10 and 20 days, 5, the animals were
killed and the upper and lower eyelids were excised. Meibomian
glands were observed in both upper and lower eyelids under
binocular microscope. Meibomian glands were embedded in BMMA
plastic and serially section of immunofluorescent tomography (IT)
and 3 dimensional reconstruction using software Amira®. Another
sets of specimens were processed for cryosectioning and oilred
O staining or for paraffin sections for ommunoshitochemistry for
PPARγ.
Results: As early as day 5 post-alkali burn, marked dilation of
Meibomian gland duct was observed in 3D reconstructions. Oil
red O staining showed the substance in the dilated duct to contain
neutral lipid, presumably Meibum. Dilation was also associated with
a marked reduction in PPARγ of meibocytes of alkali-burned mice as
compared to uninjured normal Meibomian glands.
Conclusions: Ocular surface alkali injury leads to Meibomian gland
ductal dilation and down regulation of PPARγ suggesting loss of
meibocyte differentiation. These findings suggest that alkali injury
leads to obstruction of the meibomian gland orifice and down stream
dilation and atrophy of meibocytes.
Commercial Relationships: Shin Mizoguchi, None; Yuka Okada,
None; Reiko Arita, None; Geraint J. Parfitt, None; Yilu Xie, None;
James V. Jester, None; Shizuya Saika, None
Support: NEI EY021510
Program Number: 2508 Poster Board Number: D0112
Presentation Time: 3:45 PM–5:30 PM
The Prevalence of Meibomian Gland Dysfunction in a Caucasian
Clinical Population
David K. Murakami1, Caroline A. Blackie1, Donald R. Korb1, 2.
1
TearScience, Boston, MA; 2Korb & Associates, Boston, MA.
Purpose: The purpose of this study was to evaluate the prevalence
of Meibomian gland dysfunction (MGD) amongst Caucasian patients
using a standardized metric for MG function and to see whether
various genetic factors (skin type, eye and hair color) increase the
likelihood of MGD in this population.
Methods:
A retrospective observational analysis was performed on de-identified
data from consecutive, fully consented, Caucasian patients (n=168)
at a single clinical center in Boston, MA between June and October
2014. Inclusion criteria: willingness to participate in the study, over
the age of 18, no lid abnormalities, no current ocular inflammation/
disease, no ocular surgery within the last 6 months. Patient’s skin
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
type was graded with the Fitzpatrick Scale (Types I to VI). Eye color
and original hair color data was also collected. Any symptoms of
dry eye were scored using the SPEED Questionnaire. The number
of functional glands in the lower eyelids was assessed with the Korb
Meibomian Gland Evaluator. Meibomian gland dysfunction was
defined as having 6 or fewer functional meibomian glands on the
lower eyelid (approximately 25% of the total number of meibomian
glands present).
Results: Only data for right eyes are presented. The mean age and
symptom score of the patients was 53.1±16.0 years (49 males; 119
females) and 8.4±5.8 respectively. All patients fell within Types I
to IV on the Fitzpatrick Scale, with the majority being Type II or
III, 34% and 48% respectively. The majority of these patients had
either blue or brown eyes, 38.0% or 35.7% respectively, with 68.5%
having brown hair. The prevalence of MGD (25% or fewer functional
MGs) was 70.2%. Using a more conservative cut off of 4 or fewer
functional meibomian glands (17%), the prevalence of MGD was
43%. The variables of skin type, eye color and hair color had no
statistically significant impact of the likelihood of MGD in this
population (p>0.5 for all analyses).
Conclusions: The prevalence of MGD in this population is 70.2%.
This is significantly higher than previous reports of the prevalence
of MGD in Caucasian populations. This is the first report of MGD
prevalence in a Caucasian population using data gathered with a
standardized metric for MG function.
Commercial Relationships: David K. Murakami, TearScience (E),
TearScience (F); Caroline A. Blackie, TearScience (E), TearScience
(F); Donald R. Korb, TearScience (C), TearScience (C), TearScience
(I), TearScience (I), TearScience (P), TearScience (P)
Program Number: 2509 Poster Board Number: D0113
Presentation Time: 3:45 PM–5:30 PM
Epidemiology and genetics of TBUT in a Sardinian genetic isolate
Roberta Farci1, Maria Pina Concas2, Ginevra Biino3, Vaccargiu
Simona2, Cristina Malloci1, Gianfranca Cappai1, Pietro E. Napoli1,
Mario Pirastu2, Maurizio Fossarello1. 1Ophthalmology, University of
Cagliari, Cagliari, Italy; 2National Research Council of Italy, Insitute
of Population Genetics, Sassari, Italy; 3National Research Council of
Italy, Institute of Molecular Genetics, Pavia, Italy.
Purpose:
Evaluation of tear break-up time (TBUT) is a simple tool to assess
the stability of the preocular tear film. To date, most research on
tear film stability focused on environmental risk factors, and little
is known about the role of genetics. Our aim is to find some of the
factors influencing tear film stability in a Sardinian isolate taking
advantage of the high environmental and genetic homogeneity of its
population of which we have complete genealogical records.
Methods:
A multidisciplinary epidemiologic survey was conducted in two
secluded villages of the central eastern Sardinia (Ogliastra).
Participants (n=2593) underwent a complete eye examination
including TBUT test. Subjects were genotyped using Illumina Human
Exome BeadChip array. Correlation of TBUT with more than 100
traits was investigated using linear regression models. GWAS and
linkage analysis were performed on both TBUT as a quantitative
trait (n=2262, mean value of both eyes) and as a binary trait: cases
(TBUT≤5s in both eye, n=133) and controls (TBUT≥10s, n=1468).
After quality controls about 48k SNVs (MAF >0.01) were used
for GWAS assessing genotype-phenotype association by linear
regression analysis under an additive effect model, adjusting for
age, gender and relatedness as implemented in GenABEL. Variance
component and nonparametric linkage analyses were performed using
MERLIN and a subset of SNVs (~20K after clustering for linkage
disequilibrium).
Results:
Standardized prevalence of TBUT ≤5s was 8% (95% CI: 7.1%9.0%), with no significant gender differences. On average, TBUT
decreases of about 0.8s for each 10 years increase in age, it is 0.8s
lower in women, it decreases with increasing BMI and cell mean
corpuscular volume, but it rises 0.12s for each 1D increase in
spherical equivalents (P<0.05). TBUT heritability was about 10%.
GWAS of quantitative TBUT (Bonferroni significant threshold
1.1x10-6) revealed several suggestive loci: i.e. PRL (P=5.4x10-5),
FBLN2 (P=6.7 x10-6), AKAP13 (P=8.98 x10-5). In GWAS of binary
trait, we found a suggestive signal in DNAH10 gene (P=1.67x10-5).
Linkage analysis showed a signal for quantitative TBUT on chr
1p32.2 (LOD=2.39) and one for the binary trait on chr 11q22.3
(LOD=2).
Conclusions:
This is the first population based study on genetics of TBUT. Using
different approaches we identified several suggestive genes/loci some
of which already been described associated with eye physiology and
pathologies.
Commercial Relationships: Roberta Farci, None; Maria Pina
Concas, None; Ginevra Biino, None; Vaccargiu Simona, None;
Cristina Malloci, None; Gianfranca Cappai, None; Pietro E.
Napoli, None; Mario Pirastu, None; Maurizio Fossarello, None
Program Number: 2510 Poster Board Number: D0114
Presentation Time: 3:45 PM–5:30 PM
Correlation between Infrared Meibography and Ocular Surface
Disease signs and symptoms
Nallely Ramos-Betancourt, Laura A. González-Dibildox, Carla Rocío
Robles-Gutiérrez, Dalia Marylin Rojo-Zamudio, Jorge OzornoZarate, Jaime D. Martinez, Everardo Hernandez-Quintela. Cornea
and Refractive Surgery, Asociación Para Evitar la Ceguera, I.A.P.,
Mexico City, Mexico.
Purpose: To determine the correlation between infrared meibography
characteristics and ocular surface disease signs and symptoms.
Methods: Thirty-six eyes from 18 patients were included. Fourteen
(77.8%) patients were female; mean age was 40.83 years old (+/17.2, range 21-82). All patients underwent Ocular Surface Disease
Index (OSDI) and Dry Eye Questionnaire (DEQ-5) questionnaires.
Corneal and conjunctival staining was evaluated by fluorescein
and, lisamine green, then graded by the Oxford Schema. Tear Film
Break Up Time (TFBUT), Schirmer I test with anesthesia and,
margin lid characteristics were recorded. All patients were evaluated
with an Infrared Meibography (Keratograph 4, OCULUS, Wetzlar,
Germany). The following data was analyzed: meibomian gland area,
meibomian gland area loss (MGAL), tortuosity and presence of white
patches of upper and lower eyelid. Pearson and Spearman correlation
coefficients were used as statistical analysis.
Results: Mean OSDI score was 23.74 (+/-18.79, range 2.5-23.74),
and mean DEQ-5 score 7.0 (+/-3.96, range 0-14). A positive
correlation was found between: MGAL of upper lid and fluorescein
corneal staining (r= 0.331, p = 0.048), MGAL of lower lid and
TFBUT (upper lid r= -0.335, p = 0.046; lower lid r= -.342, p =
0.041), and MGAL and Schirmer test (upper lid r = -0.455, p =
0.007, lower lid r= -607, p= <0.001). Tortuosity was found in 36.1%
of upper lid and 2.8% in lower lid. Two (5.6%) eyes have MGAL
greater than a 33% in the upper lid, and 6 (16.7%) in the lower lid.
No white patches were found in any patient. No correlation was
found between symptoms (by OSDI and DEQ-5 questionnaires) and
infrared Meibography characteristics (r = .120, p = .648, and r=.088,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
p = .737, respectively). Asymmetric disease by infrared Meibography
was found in 38.8% (7) subjects.
Conclusions: At list a third of subjects had an asymmetric disease.
Positive correlation was found between infrared meibography and
ocular surface disease signs. No correlation was found between
meibography and symptoms.
Commercial Relationships: Nallely Ramos-Betancourt, None;
Laura A. González-Dibildox, None; Carla Rocío RoblesGutiérrez, None; Dalia Marylin Rojo-Zamudio, None; Jorge
Ozorno-Zarate, None; Jaime D. Martinez, None; Everardo
Hernandez-Quintela, None
Program Number: 2511 Poster Board Number: D0115
Presentation Time: 3:45 PM–5:30 PM
Blepharitis and thin corneal thickness : An unexpected
association
Naïla HOUMAD, Fanny Tréchot, Mathilde Boiché, Jean-Marc
Perone, Louis Lhuillier, Oualid Guechi. Ophthalmology, CHR Metz
Thionville, Villers-les-nancy, France.
Purpose: The aim of this study is to measure central corneal
thickness in patients with blepharitis associated with meibomian
gland dysfunction. Local inflammation and alteration of the tears film
might result to corneal thinning.
Methods: All consecutive patients seen in consultation with
blepharitis in our Department of Ophthalmology in September 2014
were included. Blepharitis clinic criteria were those reported by
MGD workshop. Meanwhile a control group was set up. Patients
with ophthalmic associated pathology, or recent history of ophthalmic
surgery were excluded. The central corneal thickness was measured
with a non contact pachymeter (NT 530P,Nidek, Jp).
Results: The study group was made of 40 eyes of 20 patients (11
men) with blepharitis, mean age was 58.5±14.4. Forty eyes of 20
healthy patients (9 men) were used as a control group, mean age was
56.75±13.39. The study group and the control group were comparable
in gender and in age. In the study group, the mean central thickness
was 527.5mm±29.8 and in the control group mean central corneal
thickness was 554.9mm±24.7. There was statistically significant
difference between the two groups using a Z normal distribution test.
Conclusions: Blepharitis may be associated with a thinner corneal
thickness. Increased of osmolarity in the tear fluid and ocular surface
inflammation are likely to be the cause of this decrease.
Commercial Relationships: Naïla HOUMAD, None; Fanny
Tréchot, None; Mathilde Boiché, None; Jean-Marc Perone, None;
Louis Lhuillier, None; Oualid Guechi, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].