WO2015163821A1

WO2015163821A1 - Monitoring and/or detecting meibomian gland dysfunction

Info

Publication number: WO2015163821A1
Application number: PCT/SG2015/050079
Authority: WO
Inventors: Louis Tong; Markus R WENK
Original assignee: Singapore Health Services Pte Ltd; National University Of Singapore
Priority date: 2014-04-23
Filing date: 2015-04-23
Publication date: 2015-10-29

Abstract

The invention relates to monitoring a subject suffering from meibomian gland dysfunction and/or detecting if a subject is suffereing from and/or at risk of meibomian gland dysfunction by analysing at least one lipid parameter in a tear sample from the subject.

Description

Monitoring and/or detecting meibomian gland dysfunction Field of the invention

The present invention relates to the field of dry eye syndrome (DES). In particular, the invention relates to meibomian gland dysfunction (MGD).

Background of the invention

Any listing or discussion of an apparently prior-published document in this specificaiton should not necessarily be taken as an acknowledgement that document is part of the state of the art or is common general knowledge.

Dry eye syndrome is a prevalent ophthalmic condition adversely affecting up to 80% of the population over the age of 80, with potential debilitating effects on specific segments of the population such as contact lens wearers, people who have undergone refractive surgeries, postmenopausal women, and patients suffering from a variety of autoimmune disorders (Aquavella 2013). DES is a multifactorial disease of the tears and ocular surface caused by a deficiency in tear production or excessive evaporation. Regardless of the initiating causes, chronic dryness and the resultant tear film hyperosmolarity leads to inflammation that jeopardizes the structural and functional integrity of the lacrimal gland, meibomian gland, and corneal and conjunctival epithelial tissues. The gradual destruction of these tissues that are major contributors of various tear film components further disturbs tear film homeostasis and results in a vicious cycle of inflammatory events that represents the major pathological mechanism in DES (Baudouin 2001 ).

Meibomian gland dysfunction (MGD) is a major cause of dry eye and ocular discomfort (Baudouin 2001 ). MGD is a diffuse condition of the eyelids characterized by progressive obstruction of meibomian gland terminal ducts due to ductal hyperkeratinization or inspissation of secretion (Foulks 2007). In MGD, pathological alterations in the compositions of the meibomian gland secretions, also known as the meibum, the predominant source of lipids for the human tear film, result in the thickening of the meibum, subsequently leading to the blockage of the glandular ducts. The occlusion may also be attributed to excessive colonization by bacterial commensals as well as exfoliated skin materials and crusts as a result of hyperkeratinization of the glandular ducts.

Warm compresses currently represent the most frequently prescribed treatment for patients with MGD or glandular obstruction leading to DES. There remains a need for improved therapy for MGD and also monitoring of MGD.

Summary of the invention

According to a first aspect, the present invention provides a method for monitoring a subject suffering from meibomian gland dysfunction comprising comparing at least one lipid parameter of at least one tear sample from the subject at a first time point with the lipid parameter of at least one tear sample from the subject at a second time point.

According to a second aspect, the invention provides a method for detecting if a subject is suffering from and/or at risk of meibomian gland dysfunction a comprising comparing at least one lipid parameter of at least one tear sample from the subject with a control.

The control may be at least one lipid parameter of a tear sample from a typically healthy subject. The control may also be data derived from a population of such healthy subjects. For example, the data may be a profile representing a particular lipid parameter of individual tear samples from a population of such healthy subjects. For example, the data includes the mean or median value of the particular lipid parameter obtained from the population. In particular, each healthy subject is not suffering from meibomian gland dysfunction or dry dye syndrome. Typically, the healthy subject is assessed as not suffering from meibomain gland dysfunction or dry eye syndrome based on standard clinical diagnostic criteria. Brief description of the figures

Figure 1 shows an illustration of the study design in Example 2, Eligible MGD patients were randomly assigned into one of the three treatment arms (i.e. warm towels, Blephasteam, Eyegiene). Patients in each treatment arm carried out routine treatment using the assigned eyelid warming modality for 10 minutes each time, and for two times a day.Tear samples were collected at Week 0 and at Week 12 for lipid analyses.

Figure 2 shows the visual analogue scale for recording ocular symptoms at Week 0.

Figure 3 shows the visual analogue scale for recording ocular symptoms at Week 12.

Figure 4 Changes in tear lipid levels with routine eyelid-warming treatment. Bar plots illustrate the changes in absolute concentration of total lipids (pmol ml^"1) (A) and molar fractions of lysophospholipids classes (B), phospholipid classes (C), nonpolar lipid classes (D), and OAHFA, CS, and sphingolipid classes (E) in tears of MGD patients (n = 32) after routine eyelid-warming treatment for a total therapeutic window of 12 weeks. Values were plotted as mean ± SEs. # 0.05< PO.10, ^*P<0.05, ^** PO.01 , ^**^* PO.001. CE, cholesteryl ester; Cer, ceramide; CHO, free cholesterol; CS, cholesteryl sulfate; DAG, diacylglyceride; GluCer, glucosylceramide; GM3, ganglioside mannoside 3; LPS, lysophosphatidylserines; OAHFA, O-acyi-o>hydroxy-FA; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglyceride; WE, wax ester.

Figure 5 Changes in lysophospholipid classes in tears with eyelid-warming treatment. Dot plots illustrate the molar fractions of total LpPE (A), LPE (B), LPC (C), and LPI (D) in individual MGD patients (n = 32) before and after the 12- week eyelid-warming treatment. # 0.05<P<0.10, ^* P<0.05, ^** P<0.01 , *** p<0.001.

Figure 6 Changes in individual lysophospholipid species with eyelid-warming treatment. Heat maps illustrate molar fractions of individual species of LpPE (A), LPE (B), LP! (C), and LPC (D) at week 0 and week 12 of the study. # 0.05<P<0.10, ^* PO.05, ** P<0.01 , *^** P< 0.001.

Figure 7 Changes in individual phospholipid species with eyelid-warming treatment. Bar plots illustrate the molar fractions of individual species of PE (A), PC (B), PS (C), and PI (D) in the tear samples of MGD patients (n = 32) at week 0 and week 12 of the study. Values were plotted as mean ± SEs. # 0.05<P<0.10, ^* PO.05, ^** P<0.01 , ^*** P<0.001 .

Figure 8 Clinical relevance of amphiphilic lipids to MGD. Dot plots illustrate the molar fractions of total CS (A) and OAHFA (B) in individual MGD patients (n = 32) before and after the 12-week eyelid-warming treatment. Following 12-week eyelid-warming treatment, increases in the levels of total OAHFA and numerous individual OAHFA species were significantly and negatively correlated with the alleviation of ocular discomfort (C); increases in total OAHFA and numerous OAHFA species were significantly and negatively correlated with improvement in ocular evaporation rate (D); and reductions in total LpPE and numerous LpPE species were significantly correlated with decreasing rate of ocular evaporation (E). Ellipse indicates region within 95% confidence interval of the correlating parameters. #0.05 < P < 0.10, * P < 0.05, ^** P < 0.01 , *** P < 0.001.

Definitions

As used herein, lipidome refers to the totality of lipids in a biological sample, for example a tear sample.

As used herein, the term "comprising" or "including" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term "comprising" or "including" also includes "consisting of. The variations of the word "comprising", such as "comprise" and "comprises", and "including", such as "include" and "includes", have correspondingly varied meanings.

Lipid parameter of a sample includes the amount or level and/or type of lipids in the sample. Lipid parameter includes the amount or level of each type of lipid in the sample. Lipid parameter also includes the amount or level of a combination of different types of lipid in the sample. Abbreviations: CS, cholesteryl sulfate; DAG, diacylglyceride; DES, dry eye syndrome; GIIAPLA₂, group HA phospholipase A₂; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; LpPE, lyso-plasmalogen PE; MGD, meibomian gland dysfunction; OAHFA, O-acyl- o-hydroxy-FA; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PLA₂ , phospholipase A₂; PLC, phospholipase C; PS, phosphatidylserine; Schir I, Schirmer's I test; TBuT, tear breakup time.

Detailed description of the invention

According to a first aspect, the present invention provides a method for monitoring a subject suffering from meibomian gland dysfunction comprising comparing at least one lipid parameter of at least one tear sample from the subject at a first time point the lipid parameter of at least one tear sample from the subject at a second time point.

For example, a change in the lipid parameter comprising:

(i) a decrease in the level of at least one lysophopholipid;

(ii) an increase in the level of at least one phospholipid;

(iii) a decrease in the level of at least one polyunsaturated fatty acid- containing diacylglycerol; and/or (iv) an increase in the level of cholesteryl sulphate and/or O-acyl-ω- hydroxy-fatty acid (OAHFA); in the tear sample from the subject at the second time point compared to the first time point is indicative that the subject has improved in symptoms of meibomian gland dysfunction at the second time point compared to the first time point.

It will be appreciated that the subject has improved in symptoms of meibomian gland dysfunction at the second time point compared to the first time point means that the symptoms of meibomian gland dysfunction in the subject has aiieviaied ai the second time point compared to the first time point.

On the other hand, a change in the lipid parameter comprising:

(v) an increase in the level of at least one lysophopholipid;

(vi) a decrease in the level of at least one phospholipid;

(vii) an increase in the level of at least one polyunsaturated fatty acid- containing diacylglycerol; and/or

(viii) a decrease in the level of cholesteryl sulphate and/or O-acyl-ω- hydroxy-fatty acid (OAHFA); in the tear sample from the subject at the second time point compared to the first time point is indicative that the subject has deterioriated in symptoms of meibomian gland dysfunction at the second time point compared to the first time point.

More in particular, an increase or a decrease in the level of the lysophospholipid, phospholipid, polyunsaturated fatty acid-containing diacylglycerol, cholesteryl sulphate and/or O-acyl-c -hydroxy-fatty acid (OAHFA) as the case may be is a significant reduction or a significant increase.

It will be appreciated that no significant change in:

(ix) the level of at least one lysophopholipid;

(x) the !eve! of at least one phospholipid;

(xi) the level of at least one polyunsaturated fatty acid-containing diacylglycerol; and/or

(xii) the level of cholesteryl sulphate and/or O-acyl-co-hydroxy-fatty acid (OAHFA); in the tear sample from the subject at the second time point compared to the first time point is indicative that there is no change in the symptoms of meibomian gland dysfunction at the first and second time points.

In particular, the first time point is before and the second time point is after treatment for meibomian gland dysfunction. Accordingly, it will be appreciated that the method according to the first aspect may be adapted to assess if a subject is responding positively or negatively to the treatment. The treatment may comprise warming therapy of the eyelid and/or steroid therapy. The warming therapy may be a warm compress. The warm compress may be in the form of a hot towel or a warm compress system, for example EyeGiene from Eyedetec Medical Inc. The warming therapy may be a eyelid-warming device which can be worn. An example is an eyelid-warming device in the form of a pair of goggles from Blaephasteam.

The method according to the first aspect may also be adapted to assess efficacy of a method of treatment for meibomian gland dysfunction by comparing a lipid parameter before and after administration of method of treatment. The method may also be adapted to compare a new method of treatment for meibomian gland dysfunction with warming therapy of the eyelid and/or steroid therapy.

In the comparison, a difference in at least one lipid parameter in the subject compared to the control may be regarded as suggestive that the subject may be suffering from and/or at risk of meibomian gland dysfunction. In particular, the difference should be a significant difference. For example, a change in the lipid parameter comprising:

(xiii) a decrease in the level of at least one lysophopholipid;

(xiv) an increase in the level of at least one phospholipid;

(xv) a decrease in the level of at least one polyunsaturated fatty acid- containing diacylglycerol; and/or

(xvi) an increase in the level of cholesteryl sulphate and/or O-acyl-o hydroxy-fatty acid (OAHFA); in the tear sample from the subject compared to the control may be indicative that the subject is suffering from and/or at risk of developing meibomian gland dysfunction.

It will be appreciated that no significant change in:

(xvii) the level of at least one lysophopholipid;

(xviii) the level of at least one phospholipid;

(ixx) the level of at least one polyunsaturated fatty acid-containing diacylglycerol; and/or

(xx) the level of cholesteryl sulphate and/or O-acyl-co-hydroxy-fatty acid (OAHFA); in the tear sample from the subject compared to the control is indicative that the subject is not suffering from and/or at risk of meibomian gland dysfunction.

The control may be at least one lipid parameter of a tear sample from a typically healthy subject. The control may also be data derived from a population of such healthy subjects. For example, the data may be a profile representing a particular lipid parameter of individual tear samples from a population of such healthy subjects. For example, the data includes the mean or median value of the particular lipid parameter obtained from the population. In particular, each healthy subject is not suffering from meibomian gland dysfunction or dry dye syndrome. Typically, the healthy subject is assessed as not suffering from meibomain gland dysfunction or dry eye syndrome based on standard clinical diagnostic criteria.

The method according to the second aspect may be useful in complementing the diagnosis of meibomian gland dysfunction based on standard clinical diagnostic criteria.

It will be appreciated that the method for any aspect of the invention may be an in vitro method. For example, the in vitro method utilises isolated tear sample(s).

The present invention may be useful in assessing the severity of meibomian gland dysfunction. A comparison of the lipid parameters of the tear samples at the first time point and the second time point may be performed. Alternatively, a comparison of the lipid parameter(s) of tear sample(s) from the subject may be performed with the control. It will be appreciated that the lipid parameters of tear samples of the subject at the first and second time points may be compared with the control.

Examples of the lysophophospholipid applicable for the method of any aspect of the present invention include but are not limited to at least one lysophosphatidylcholine, at least one lysophosphatidylethanolamine, at least one lyso-plasmologen phosphatidylethanolamine, at least one lysophosphatidylinositol and/or at least one lysophosphatdidylserine.

Examples of the phospholipid applicable for the method of any aspect of the present invention include but are not limited to at least one polyunsaturated fatty acid-containing phospholipid, at least one phosphatidylcholine, at least one phosphatidylethanolamine, at least one plasmalogen phosphatidylethanolamine, at least one phosphatidylinositol and/or at least one phosphatidylserine. In particular, the phospholipid may comprise comprises total phosphatidylethanolamine.

Examples of the polyunsaturated fatty acid-containing diacylglycerol applicable for the method of any aspect of the present invention include but are not limited to DAG 16:1/22:5; DAG 18:0/20:4; DAG 18:0/22:6; DAG 16:0/22:5 or DAG 16:0/22:6. Examples of the O-acyl-oo-hydroxy-fatty acid (OAHFA) include but are not limited to OAHFA 18:1/26:1 ; OAHFA 18:1/30:2, OAHFA 18:1/34: 1 ; OAHFA

18:1/25:0, OAHFA 18:1/28:1 , OAHFA 16:1 /32:1 , OAHFA18: 1/30:1 ; OAHFA 18:1/31 :0; OAHFA 18:2/32:1 ; OAHFA 18:1/32:2 or OAHFA 18:1/34:2.

It will be appreciated that the tables and figures provide further examples of lipids applicable for the present invention.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

Example 1 A method for diagnosing and treating tear instability using determination of O-acvl-omeqa hvdroxyl fatty acids and replacement of this using the topical therapy or dietary supplementation.

Purpose: Meibomian gland dysfunction (MGD), a major ocular surface condition, is managed by lid warming. We aim to evaluate treatment-induced alteration of tear lipids and evaporation in MGD.

Methods: 32 patients, mean age 54.7 years (SD:10.4) and 65.6% females, were analysed. 53.1 % had some aqueous deficiency (Schirmers I <= 8 mm/5 minutes), whereas 75% had tear instability (break up time <3 sec). 10 patients had hot towel compress, 10 had Eyegiene and 12 had Blephasteam. Tear collected at baseline and 12 weeks were analysed, and thermography performed. The relative levels of the lipid species were obtained using mass spectrometry.

Results: Treatment-induced change (p<0.001) among 550 lipids/classes was observed for 30 lipids (7 increased and 23 reduced). Most lipids increased were O-acyl hydroxyl ω fatty acids (OAHFAs). Only 2 of the 30 lipids that changed were correlated to a reduction in dry eye symptoms. (OAHFA 18:1/32:2, r=-0.371 , p=0.036 and OAHFA 18:1/33:1 , r=-0.355, p=0.046).

The drop in 3 lipids LPC18:2 (r=0.38) , LPE18:0p (r=0.41) and LPE20:1 p (r=0.36) was associated with decrease in tear evaporation. Lipids increased with treatment OAHFA 18:1/30:1 (r=-0.43) and OAHFA 18:1/31.0 (r=-0.38) were also associated with reduced tear evaporation. The change in the OAHFA class was inversely correlated to the change in the evaporative rate (r=-0.39) and the change in LpPE was directly correlated to the change in evaporative rate (r=0.41 ).

Conclusions: The lipids deficient in dry eye such as OAHFAs are increased by lid warming. Lipid changes may underpin the effect of treatment in MGD. These findings show that the specific lipids are useful as either diagnostic markers of dry eye or therapeutic entities, which they will be tested through supplement strategies in animal experiments. Example 2 Longitudinal changes in tear fluid lipidome brought about by eyelid warming treatment in a cohort of meibomian gland dysfunction

Materials and method

Clinical cohort and study design

This study involves patients (n = 32) from a 3-month longitudinal study evaluating the effects of eyelid warming in a cohort of MGD patients. Written informed consent was obtained from all participants in the current study. The clinical procedure was specifically approved by the Singhealth Centralised Institutional Review Board (CIRB Ref No.: 201 1/197/A) and registered at the ClinicalTrials.gov database (NCT01448369). The tenets of the Declaration of Helsinki for all human research conducted in this study. Withdrawal from the study followed the usual good clinical practice in clinical trials. For withdrawn subjects, no data were obtained after the date of the withdrawal. Withdrawn subjects were not replaced.

Patients at the Singapore National Eye Centre dry eye clinic who met the eligibility criteria (Table 1 ) were invited for screening. Table 1 Inclusion and exclusion criteria for recruitment of participants

Participants were then enrolled with written informed consent obtained by the clinical trial coordinator. Eligible patients (Table 2) were randomly assigned into three respective treatment arms each utilizing a different treatment modality (Figure 1 ): traditional method of warm compresses using a hot towel (n = 10); Blephasteam (n = 10); and EyeGiene (n = 12). Blephasteam (Spectrum Thea, France) is an eyelid-warming device available in Europe that can be conveniently used at home. The goggles provide standardized heat of ~38°C to liquefy lipids and humidify the chambers with mineral water to ensure optimal moisture levels. EyeGiene (Eyedetec Medical Inc.) is a self-contained, convenient warm compress system for the eyes. The system is composed of a reusable eye mask and one-time use warmers that are inserted into the eye mask during usage. The warming units are activated by squeezing just prior to usage and deliver 40°C heat for up to 5 min within 30-60 s. The production of heat is based on a sustained thermochemical reaction.

Table 2 Demographic and clinical information of recruited patients at Week 0.

Subiect Gender Race Age TBut Schir I

A1 F Chinese 65 1.7 5

F Chinese 64 o n

A3 M Chinese 53 1.4 1

A4 M Chinese 65 1.5 1 1

A5 F Chinese 59 1 .5 19

A6 F Chinese 81 2.6 27

A7 F Chinese 34 5.8 6

A8 F Chinese 42 5.1 18

A9 F Malay 58 2 12

A10 F Chinese 60 0.8 7

B1 M Chinese 65 2.4 12

B2 M Chinese 68 1 .5 10

B3 M Chinese 46 2.4 5

B4 F Chinese 57 1 .5 1

B5 M Chinese 61 5.4 1

B6 F Chinese 46 2.6 1

B7 F Chinese 52 3 9

B8 F Chinese 49 9 3

B9 F Chinese 47 2.1 21

B10 F Chinese 48 2.3 1 1

C1 F Chinese 51 1 .3 7

C2 M Chinese 61 2.1 7

C3 F Chinese 51 0.9 0

C4 F Chinese 61 4.1 2

C5 M Chinese 56 2.5 19 C6 F Chinese 54 3 0

C7 M Chinese 65 1.5 7

C8 M Chinese 29 1 24

C9 F Chinese 57 1.5 7

C10 F Chinese 43 0.8 16

C1 1 F Malay 54 1.1 32

C12 M Chinese 48 1.6 2

The patient cohort was predominantly Chinese with mean age = 54.7 years old, mean starting tear breakup time (TBuT) = 2.5 s and mean Shirmer^'s 1 test (Schir I) = 9.8 mm. Group A; Warm towel; Group B: Blephasteam; Group C: Eyegiene.

Screening visit was performed at the regular dry eye clinic, and baseline examination was subsequently carried out. Follow-up visits were conducted after 12 weeks of treatment. A window period of ±3 days was permitted for this visit. Tear fluid samples were collected from the right eye of each participant at baseline visit (week 0) and at the end of the treatment period (week 12) using Schirmer's strips as described previously (Lam et ai, 2014a). Tear samples collected were frozen immediately and kept at 80°C until further analyses.

Treatment regimes

Patients in each treatment arm carried out routine treatment using the assigned eyelid-warming modality for 10 min each time and for two times a day. All patients were allowed to continue their regular management of MGD in the form of lid scrub with Blephagel. The frequency of use of these measures was monitored in a daily diary, and other types of MGD treatment such as omega-3 tablets, antibiotics or steroid ointments, and the manual expression of meibomian glands were prohibited. Outcome parameters evaluated

A visual analog scale (VAS) was applied to evaluate DES symptoms as previously described (Figures 2 and 3) (Schaumberg et ai, 2007). The outcome was taken as the change in the global score at week 12 from that at week 0, which was calculated from the discomfort frequency and severity as previously described (Schaumberg et al., 2007). Other outcome measures include differences in the VAS of visual outcomes (i.e., blurred vision and light sensitivity) (Figures 2 and 3), tear breakup time (TBuT), Schirmer's I test (Schir I), and corneal fluorescent staining. Details on the clinical procedures have been reported previously (Lam et al., 201 1 ).The severity of MGD was also graded in this study. Microscopic signs of MGD including the presence of misdirected lashes, fragility of lashes, scurf formation, irregularity of meibomian gland orifices, loss of meibomian gland expressibility, formation of plaques, and the number of blocked meibomian gland orifices (i.e., plugs) were recorded. The Yamaguchi grading scheme was used to identify microscopic signs of MGD (Tong et al., 2010) and essentially indicates the position of the Marx's line relative to the meibomian gland orifices, which has been previously shown to correlate strongly with meibomian gland function (Yamaguchi et al., 2006). Ocular evaporation rate was measured based on infrared thermography in a clinical room setting as reported previously (Petznick et al., 2013). Lipid extraction and HPLC/multiple-reaction-monitoring analyses

Lipids were extracted from the Schirmer's strips using a modified version of the Bligh and Dyer's method as optimized previously (Lam et al., 2014a). Polar lipids and neutral lipids were analyzed using an Agilent HPLC 1200 system coupled with ABSciex QTRAP 4000 and ABSciex 3200, respectively. Lipidomic analyses were chiefly based on the principle of HPLC/multiple-reaction- monitoring of individual lipid species. The detailed lipidomic platform optimized for human tear fluid analyses have been previously described in details elsewhere (Lam et ai, 2014a; Lam et ai, 2013 and Lam et al., 2014b).

Statistical analyses

One-way ANOVA with post hoc Tukey was first performed to compare the differences in the changes of clinical indices before and after treatment among the three groups (i.e., week 12 - week 0). Following this, paired-r comparisons were performed on the tear lipid profiles of a combined group of patients from the three individual treatment arms, obtained at week 0 and week 12 of the study. This would reveal lipid alterations under an extended period of routine eyelid warming. False discovery rate was controlled for based on q values calculated using R 3.0.1 (Table 3). Correlation analyses between the changes in individual lipid species/classes with changes in clinical signs following 12- week treatment was performed using Spearman^'s correlation. Ellipse demarcates 95% confi dence region of correlating parameters and lipid species/classes.

Table 3 p- and q-values for paried-t test between lipid levels at Week 0 and

Lipid p-value q-value Lipid p-value q-value

Total WE 0.05 0.04 PC34:le,34:0p 0.32 0.16

Total CE 0.27 0.14 PC34:0e 0.44 0.18

CHO 0.54 0.22 PC34:4 0.38 0.17

CS 0.07 0.06 PC34:3 0.04 0.05

Total TAG 0.95 0.35 PC34:2 0.04 0.05

Total DAG 0.10 0.07 PC34:1 0.18 0.11

Total OAHFA 0.00 0.00 PC34:0 0.23 0.13

Total SM 0.31 0.14 PC36:5e,36:4p 0.87 0.30

Total Cer 0.19 0.11 PC36:4e,36:3p 0.47 0.19

Total GluCer 0.79 0.31 PC36:3e,36:2p 0.37 0.17

Total G 3 0.21 0.11 PC36:2e,36:lp 0.49 0.20

Total PC 0.09 0.07 PC36:le,36:0p 0.54 0.20

Total PE 0.03 0.03 PC36:0e 0.52 0.20

Total PS 0.03 0.03 PC36:5 0.08 0.06

Total PI 0.17 0.10 PC36:4 0.02 0.03

Total LPC 0.00 0.00 PC36:3 0.01 0.03

T ι θ- +ια -.ιI 1 D C

i_r i_ 0.01 0.02 PCS 5: 2 0.03 0.04

Total LpPE 0.00 0.00 PC36:1 0.13 0.09

Total LPS 0.29 0.14 PC36:0 0.04 0.05

Total LPI 0.00 0.00 PC38 6e,38:5p 0.30 0.16

LPE16 .1 0.29 0.10 PC38 5e,38:4p 0.86 0.30

LPE16 Ό 0.00 0.00 PC38 4e,38:3p 0.51 0.20

LPE18 2 0.23 0.08 PC38 3e,38:2p 0.52 0.20

LPE18 1 0.96 0.30 PC38 2e,38:lp 0.43 0.18

LPE18 0 0.00 0.00 PC38:7 0.78 0.28

LPC16:0e 0.00 0.00 PC38:6 0.01 0.03

LPC16:1 0.00 0.00 PC38:5 0.02 0.04

LPC16:0 0.00 0.00 PC38:4 0.01 0.03

LPC18:0e 0.00 0.00 PC38:3 0.01 0.03

LPC18 2 0.00 0.00 PC38:2 0.08 0.06

LPC18 1 0.00 0.00 PC38:1 0.16 0.11

LPC18 0 0.00 0.00 PC38:0 0.34 0.16

LPC20:4 0.00 0.00 PC40:6e,40:5p 0.91 0.31 LPC22:6 0.45 0.15 PC40:5e,40:4p 0.26 0.14

LPI16:0 0.00 0.00 PC40:7 0.06 0.06

LPI18:0 0.00 0.00 PC40:6 0.01 0.03

LPI20:4 0.00 0.00 PC40:5 0.02 0.03

LPE16:0p 0.00 0.00 PC40:4 0.03 0.04

LPE18:lp 0.00 0.00 PC40:3 0.16 0.11

LPE18:0p 0.00 0.00 PC40:2 0.44 0.18

LPE20:lp 0.00 0.00 PC40:1 0.82 0.29

LPE20:0p 0.00 0.00 PC40:0 0.83 0.29

PI 32:1 0.20 0.12 PE32:2 0.43 0.18

PI 34:2 0.26 0.14 PE34:2p,34:3e 0.35 0.16

PI 34:1 0.21 0.12 PE34:lp,34:2e 0.15 0.11

PI 36:4 0.34 0.16 PE34:2 0.01 0.03

PI 36:3 0.02 0.04 PE34:1 0.01 0.03

PI 36:2 0.17 0.11 PE36:4p,36:5e 0.09 0.07

PI 36:1 0.34 0.16 PE36:3p,36:4e 0.03 0.04

PI 36:0 0.15 0.11 PE36:2p,36:3e 0.89 0.30

PI 38:6 0.06 0.06 PE36:lp,36:2e 0.79 0.28

Pi 38:5 0.06 0.06 PE36:4 0.04 0.05

PI 38:4 0.35 0.16 PE36:3 0.01 0.03

PI 38:3 0.05 0.05 PE36:2 0.00 0.00

PI 40:6 0.03 0.04 PE36:1 0.06 0.06

PI 40:5 0.16 0.11 PE38:6p,38:7e 0.00 0.01

PI 40:4 0.91 0.31 PE38:5p,38:6e 0.02 0.04

PS 34:2 0.02 n n i 0.34 0.16

PS 34:1 0.02 0.04 PE38:6 0.08 0.06

PS 36:2 0.00 0.02 PE38:5 0.01 0.03

PS 36:1 0.20 0.12 PE38:4 0.02 0.04

PS 38:4 0.03 0.04 PE38:3 0.09 0.07

PS 38:3 0.00 0.01 PE38.-1 0.05 0.05

PS 40:6 0.00 0.03 PE40:6p,40:7e 0.07 0.06

PS 40:5 0.32 0.16 PE40:5p,40:6e 0.07 0.06

PS 40:4 0.21 0.12 PE40:4p,40:5e 0.60 0.22

PC32:2e_/32:lp 0.96 0.32 PE40:3p,40:4e 0.56 0.21

PC32:le,32:0p 0.41 0.18 PE40:2p,40:3e 0.78 0.28

PC32:2 0.04 0.05 PE40:6 0.32 0.16

PC32:1 0.08 0.06 PE40:5 0.25 0.14

PC32.0 0.36 0.16 PE42:6p,42:7e 0.28 0.15

PC34:2e,34:lp 0.44 0.18 PE42:5p,42:6e 0.28 0.15 Results

No appreciable difference was observed in the clinical outcomes among the three treatment arms at the end of the 12-week period (Table 4), except for a marginal difference (P = 0.06) in the improvement of ocular discomfort using EyeGiene over Blephasteam (Table 4). The evaluation was based on changes in clinical parameters before and after routine eyelid warming using the respective modality, for a total duration of 12 weeks. The foregoing results thus imply that participants in all three treatment arms essentially received a comparable degree of lid warming throughout the course of treatment considered in terms of clinical outcomes. In other words, while patients in each treatment arm utilized different treatment modalities, the actual treatment received was, in principle, similar among the three groups (i.e., eyelid warming). Therefore, the patients from the three treatment arms were then grouped together to evaluate the longitudinal effects of eyelid warming per se on tear lipid profiles over the 2-week treatment period.

Table 4 Result of ANQVA analyses comparing changes in clnical indices with 12-week treatment amongst the three treatment arms

ANOVA Blephasteam- Eyegiene- Blephasteam- warm towel warm towel Eyegiene

ASchir I 0.20 0.19 0.41 0.83

ATBuT 0.39 0.36 0.70 0.80

Δ Ocular discomfort 0.07 0.68 0.31 0.06

Δ Light sensitivity 0.9 0.91 0.91 1 .00

Δ Blurred vision 0.77 0.98 0.87 0.77

ASummed global 0.59 0.85 0.88 0.56 symptom

APIugs 0.62 0.61 0.78 0.94

Δ Total corneal 0.49 0.57 1.00 0.53 staining

ATotal Yamaguchi's 0.21 0.18 0.55 0.68 score

Changes in dry eye clinical parameters before and after eyelid-warming treatment

Eyelid warming for 12 weeks resulted in appreciable alleviation of symptoms of ocular discomfort (P < 0.01 ) (Tables 5 and 6). The number of plugged orifices were significantly reduced (P < 0.01 ), and there was a noticeable improvement in TBuT (P< 0.10), which is in agreement with an earlier study demonstrating that the application of heat to the inner surface of the eyelids on a routine basis led to steady increases in both TBuT and the number of meibomian glands yielding liquid secretion over a 12-week treatment period (Korb and Blackie 2010). On the other hand, no significant changes were observed in Schir I after treatment, which was not surprising because the current MGD cohort did not have discernible lacrimal dysfunction to begin with, even at week 0 (i.e., mean Schir I > 5.5 mm) (Table 2). On another note, eyelid-warming treatment resulted in a reduction in ocular evaporation rate with marginal significance (P < 0.10).

Table 5 Changes in ocular symptoms and signs after routine eyelied-warminq treatment for 12 weeks

Week 0 Week 12 P

Symptoms

Ocular discomfort 35.7 1 3.9 c

30.7 ± 3.9

Light sensitivity 23.1 ± 5.3 18.7 ± 4.9 a

Blurred vision 25.9 ± 5.2 25.5 ± 5.6 NS

Summed global score 84.7 ± 10.7 74.9 ± 10.7 a

Signs

TButT 2.5 ± 0.3 4.0 ± 0.8 a

Schir I 9.8 ± 1 .5 9.6 ± 1.4 NS

Total corneal staining 3.5 ± 0.6 4.1 ± 0.7 NS

Total Yamaguchi score 5.1 + 0.6 6.5 + 0.4 c

Blocked glands 18.5 ± 2.0 13.9 ± 2.2 c

Ocular evaporation rate a

80.6 ± 3.2 71.6 ± 3.6

Values were presented as means ± standard errors. A total of 32 MGD patients successfully completed the entire course of treatment. NS, non significant ^a 0.05 < P < 0.10

f^c P < 0.05

C P < 0.01

d P < 0.001

Table 6 Changes in clinical parameters with eyelid warming treatment

Subject ASchir I ATBut AOcular ALight ABIurred ASummed Δ Plugs ATotal ATotal

(mm) (sees) discomfort sensitivity vision global Corneal Yamaguchi symptom staining score

A1 1 -0.3 0 0 0 0 12 2 0

A2 0 -0.6 0 0 0 0 -6 -2 0

A3 1 0.1 0 0 0 0 -12 1.5 2

A4 -8 23.8 -7.88 0 0 -7.88 -16 0 -4

A5 -13 0.1 0 0 0 0 -12 2.5 4

A6 -6 8 -12.4 -61.87 0 -74.27 -10 -4.5 0

A7 -5 -3 -3.15 0 0 -3.15 0 3 0

A8 -10 -0.9 -0.85 0 3 2.15 -6 -3 0

A9 -1 1 0 0 0 0 -6 -1.5 1

A10 -1 2.1 -14.19 0 -0.03 -14.22 -12 4 -1

B1 8 0.3 -3.77 -6.02 0 -9.79 0 -4 8

B2 -7 -0.1 -3.06 0 0 -3.06 21 3.5 -1

B3 10 -0.5 0 0 0 0 4 0 1

B4 4 0.4 -1.68 0 0 -1.68 -13 9.5 3

B5 1 2 -0.27 0 0 -0.27 -16 5 2

B6 5 -1 0 0 0 0 -5 3.5 2

B7 17 1.6 25.23 0 50.2 75.43 6 2 3

B8 -2 -7.5 -4.33 0 0 -4.33 -12 -1 3

B9 -3 -0.2 2.21 0 0 2.21 1 -2 0 B10 -4 5.2 - 8.44 -31.16 -36.6 -86.2 -15 0.5 3

C1 5 4.2 -12.2 -15.95 0 -28.15 -8 -1 -2

C2 0 0 0 0 0 0 -9 4 -1

C3 0 1.9 -5.36 0 0 -5.36 -1 1 3

C4 -2 -0.3 -5.98 10.51 5.08 9.61 -4 1 2

C5 -13 -0.9 -18.53 0 0 -18.53 10 4 7

C6 9 2.4 -4.54 -1.7 0 -6.24 0 1 8

C7 8 4.6 -4.01 -4.02 0 -8.03 -4 0 1

C8 -5 0.4 0 0 0 0 6 -8 2

C9 24 0.8 -11.39 -2.33 -8.91 -22.63 -9 -1 1

C10 -6 0.9 -0.12 -2.47 0 -2.59 -5 1 -1

C 1 -22 0.1 -31.42 -29.22 -24.13 -84.77 -9 -1 0

C12 10 2.1 -22.87 0 0 -22.87 -17 1 -3

A total of 32 MGD subjects were randomly assigned to one of the three treatment groups. Each subject underwent routine eyelid warming for a total period of 12 weeks using the respective eyelid warming modalities. Values were presented as changes with treatment (i.e. value at week 12 - value at week 0). A: warm towel; B: Blephasteam; C: EyeGiene

Changes in tear lipids before and after eyelid-warming treatment

Routine eyelid warming did not result in a discernible increase in the absolute amount of total lipids in the tear fluid (Figure 4), which is rather surprising considering the reductions in the number of plugged meibomian glands (see previous results section) following heat treatment. This could imply that the relief of meibomian plugs resulted in a restoration of normal lipid turnover, instead of an enhanced amount of lipids at the eyelid margin. In fact, these MGD patients did not have an absolute deficiency in total lipids to begin with because their mean molar concentration of total lipids in tears before treatment (-0.58 prnol ml^"1) (Figure 4A) was comparable to that of a healthy cohort asymptomatic for DES (~0.50 prnol ml^"1) as previously reported (Lam et a/., 2014b) his observation is in good agreement with this previous report that the quality of meibomian lipids, instead of the quantity, might be the actual cause of MGD-associated signs and symptoms of ocular discomfort (Lam et al., 2014b). However, stark changes in the compositions of tear lipids were observed after routine eyelid warming for 12 weeks.

The most striking changes after eyelid warming were observed in lysophospholipids (Figures 4B, 5). Molar fractions of major lysophospholipid classes, including lyso-plasmalogen phosphatidylethanolamines (LpPEs), lysophosphatidylcholines (LPCs), and lysophosphatidylinositols (LPIs), were reduced by almost half (P < 0.001 ) after treatment, while total lysophosphatidylethanolamine (LPE) also displayed a significant decrease (P < 0.05) (Figures 4B, 5). Remarkably, several individual species of LpPE, LPE, LPI, and LPC were also significantly reduced after treatment (Figure 6A-D), consistent with the overall trends observed for the respective lipid classes. Interestingly, the drastic reduction in total LpPE was paralleled by concomitant increases in total PE (Figure 4C) and several plasmalogen PE species that possessed a high degree of unsaturation (containing six or more double bonds) (Figure 7A). In addition, numerous diacyl PE, diacyl PC, and diacyl PI species were also significantly increased after the 12-week treatment period (Figure 7), which corresponded well with the observed reductions in their respective lysophospholipid counterparts. Moreover, similar to plasmalogen PE, various unsaturated PC, PS, and PI species containing two or more double bonds seem preferentially increased after eyelid warming. In addition, while total DAG (Figure 7D) did not change significantly after the treatment period, levels of individual DAG species containing PUFAs in their structures, namely DAG 16:1/22:5 (P < 0.05), DAG 18:0/20:4 (P < 0.01 ), and DAG 18:0/22:6 (P < 0.05), were almost halved following eyelid-warming treatment (Table 7). In addition, DAG 16:0/22:5 and DAG 16:0/22:6 were reduced by -30% following treatment with marginal significance (P < 0.10) (Table 7). The reductions in the various lysophospholipids and PUFA-containing DAG, together with the concomitant increases in PUFA-containing phospholipid species, indicated attenuation of PLA 2 and PLC activities, respectively, following eyelid-warming treatment. In particular, these phospholipases seem to preferentially target PUFAcontaining phospholipid species over their more saturated counterparts. Table 7 Fold change and p-value comparing the levels of individual DAG species at Week 0 and Week 12 of the study

DAG species Week O Week 12 Fold-change p-value

DAG14:1/16:1 5.71 E-07 4.77E-07 0.84 0.435

DAG14:0/16:0 1 .53E-06 1 .39E-06 0.91 0.761

DAG15:0/16:2 8.82E-08 5.59E-08 0.63 0.016

DAG18:2/14:0 3.80E-07 3.03E-07 0.80 0.365

DAG16:1/16:1 2.98E-06 2.71 E-06 0.91 0.774

DAG16:1/16:0 2.91 E-06 2.52E-06 0.87 0.579

DAG14:0/18:1 8.94E-07 8.64E-07 0.97 0.898

DAG16:0/16:0 1.07E-05 9.24E-06 0.86 0.327

DAG16:1/17:0 6.40E-07 5.62E-07 0.88 0.611

DAG18:1/15:0 9.69E-07 8.89E-07 0.92 0.734

DAG16:0/ 7:0 9.88E-07 8.75E-07 0.88 0.616

DAG16:0/18:2 1 .45E-05 1 .06E-05 0.74 0.086

DAG16:1/18:1 3.82E-06 3.59E-06 0.94 0.809

DAG16:0/18:1 1.39E-06 1.05E-06 0.76 0.101

DAG16:1/18:0 1.88E-05 1 .60E-05 0.85 0.302

DAG16:0/18:0 3.16E-06 2.57E-06 0.81 0.146

DAG18:1/17:0 9.92E-07 9.05E-07 0.91 0.547

DAG18:2/18:2 2.36E-06 1 .61 E-06 0.68 0.012

DAG18:2/18:1 8.24E-06 6.80E-06 0.82 0.205

DAG18:1/18:1 1.94E-05 1 .98E-05 1.02 0.890 DAG18:2/18:0 2.04E-05 1 .23E-05 0.60 0.008

DAG18:1/18:0 9.17E-06 6.47E-06 0.71 0.013

DAG16:1/22:5 2.84E-07 1.81 E-07 0.64 0.010

DAG16:0/22:6 2.1 1 E-06 1.47E-06 0.70 0.086

DAG16:0/22:5 8.83E-07 6.54E-07 0.74 0.096

DAG18: 1/20:3 9.55E-07 7.64E-07 0.80 0.184

DAG18:0/20:4 2.62E-05 1 .16E-05 0.44 0.001

DAG18:0/22:6 6.26E-06 3.25E-06 0.52 0.008

On another note, molar fractions of major tear film lipid amphiphiles, namely CS (P < 0.10) and OAHFA (P<0.001 ), were noticeably increased following heat treatment (Figure 8A, B). Also, the increases in total OAHFA and numerous OAHFA species, including OAHFA 18:1/26:1 , OAHFA 18: 1/30:2, and OAHFA 18:1 /34:1 , were significantly correlated (P < 0.05) with the reductions in ocular discomfort (Figure 8C), suggesting that the alleviation of ocular discomfort with treatment was associated with enhanced levels of OAHFA in the tear fluid.

Correlation between changes in the levels of tear lipid amphiphiles with reduction in ocular evaporation rate following heat treatment The increase in total OAHFA, as well as several other OAHFA species including OAHFA 18:1/25:0, OAHFA 18:1/28:1 , OAHFA 16:1/32:1 , OAHFA 18:1/30:1 , OAHFA 18:1/31 :0, OAHFA 18:2/32:1 , OAHFA 18:1/32:2, and OAHFA 18:1/34:2, was significantly (P < 0.05) correlated with the improvement in ocular evaporation rate following 2-week treatment (Figure 8D, Table 8). On the other hand, the reduction in total LpPE, as well as several other LpPE species including LpPE 16:0p, LpPE 18:1 p, LpPE 18:0p, and LpPE 20:1 p, was significantly correlated (P < 0.05) with the improvement in ocular evaporation rate (Figure 8E, Table 8). Similar trends were observed between changes in molar fractions of OAHFA and LpPE with improvement in corneal evaporation rate and scleral (conjunctival) evaporation rates (Table 8). These observations implied that while OAHFA might have a stabilizing effect on tear film structural integrity, elevated levels of LpPE might compromise tear fi Im stability, leading to an increase in evaporation rate.

Table 8 Results of correlation analyses between individual species of LPE, LpPE and OAHFA with ocular evaporation rate, corneal evaporation rate and scleral evaporation rate.

Lipid Ocular evaporation rate Corneal evaporation rate Scleral evaporation rate p-value r p-value r p-value r

LPE16:1 0.56 0. 1 0.51 0.12 0.51 0.12

LPE16:0 0.25 0.21 0.25 0.21 0.22 0.22

LPE18:2 0.03 0.38 0.02 0.41 0.03 0.40

LPE18:1 0.86 0.03 0.81 0.04 0.76 0.06

LPE18:0 0.08 0.32 0.07 0.33 0.06 0.34

LPE16:0p 0.04 0.37 0.04 0.36 0.03 0.38

LPE18:1p 0.04 0.37 0.04 0.37 0.03 0.39

LPE18:0p 0.03 0.39 0.03 0.40 0.02 0.40

LPE20:1p 0.02 0.40 0.02 0.40 0.02 0.42

LPE20:0p 0.09 0.31 0.08 0.32 0.08 0.31

OAHFA 18:1/24:1 0.82 0.04 0.81 0.04 0.76 0.06

OAHFA 18:1/24:0 0.85 0.04 0.94 0.01 0.80 0.05

OAHFA 18:1/25:0 0.01 -0.44 0.02 -0.42 0.02 -0.42

OAHFA 18:1/26:1 0.12 -0.28 0.15 -0.26 0.12 -0.28

OAHFA 18:1/26:0 0.51 -0.12 0.55 -0.11 0.42 -0.15

OAHFA 18:0/27:2 0.95 -0.01 0.95 -0.01 0.96 0.01

OAHFA 18:2/28:1 0.94 0.02 0.86 0.03 1.00 0.00

OAHFA 16:1/30:1 0.41 -0.15 0.46 -0.14 0.31 -0.18

OAHFA 18:1/28:1 0.02 -0.43 0.01 -0.43 0.01 -0.44

OAHFA 18:1/28:0 0.57 -0.10 0.62 -0.09 0.43 -0.14

OAHFA 16:1/31 :0 0.13 -0.28 0.09 -0.30 0.15 -0.26

OAHFA 16:1/32:2 0.21 -0.23 0.21 -0.23 0.18 -0.24

OAHFA 18:2/30:1 0.46 -0.13 0.38 -0.16 0.55 -0.11

OAHFA 18:1/30:2 0.29 -0.19 0.29 -0.19 0.25 -0.21

OAHFA 16:1/32:1 0.02 -0.40 0.04 -0.37 0.02 -0.43

OAHFA 18:1/30:1 0.01 -0.49 0.01 -0.44 0.00 -0.51

OAHFA 16:1/33:1 0.12 -0.28 0.12 -0.28 0.11 -0.29

OAHFA 18:2/31 :0 0.50 -0.12 0.47 -0.13 0.49 -0.13

OAHFA 18:1 /31 :1 0.11 -0.29 0.11 -0.29 0.12 -0.28

OAHFA 18:1/31 :0 0.05 -0.36 0.04 -0.36 0.04 -0.37

OAHFA 18:2/32:2 0.95 -0.01 0.93 -0.02 0.95 -0.01

OAHFA 16:1/34:2 0.80 -0.05 0.68 -0.08 0.91 -0.02

OAHFA 18:2/32:1 0.01 -0.48 I 0.01 I -0.48 0.01 -0.48 OAHFA 18:1/32 2 0.01 -0.45 0.01 -0.43 0.01 -0.47

OAHFA 16:1/34 1 0.27 0.20 0.24 0.21 0.29 0.19

OAHFA 18:1/32 1 0.06 -0.33 0.10 -0.30 0.05 -0.35

OAHFA 18:2/33 1 0.85 -0.03 0.88 -0.03 0.71 -0.07

OAHFA 18:1/33 1 0.78 -0.05 0.96 -0.01 0.63 -0.09

OAHFA 18:2/34 1 0.11 -0.29 0.12 -0.28 0.14 -0.27

OAHFA 18:1/34 2 0.02 -0.40 0.04 -0.37 0.02 -0.41

OAHFA 18:1/34 1 0.12 -0.28 0.20 -0.23 0.10 -0.29

Discussion

This is the very first study to investigate longitudinal changes in the tear lipid profiles of MGD patients over a 12-week treatment period. The longitudinal design allows detection of changes in tear lipid profiles corresponding to improvement in ocular symptoms in the same group of individuals over the treatment period, while minimizing confounding factors such as hormonal, genetic, and inter-individual variations inherent in cross-sectional case-control studies. Notably, drastic reductions in various classes of lysophospholipids and appreciable increases in amphiphilic lipids including OAHFA and CS were observed in the tears of patients following eyelid warming.

Involvement of ocular phospholipases in MGD pathogenesis

Group 11 A phospholipases A₂ (GIIAPLA₂s) are the most potent mammalian- secreted PLA₂s in terms of antibacterial efficiency against gram-positive bacteria and constitute part of the body's innate immunity as the first-line antimicrobial defense against invading microbes and pathogens (Nevalainen et al., 2008; Beers et al., 2002). The bactericidal properties of GIIAPLA₂ reside in the (1 ) high positive surface charge of the molecule and (2) its phospholipolytic enzymatic activity (Bayburt et al., 1993). In addition, GIIAPLA₂s possess higher binding affinity to anionic phospholipids such as PE compared with zwitterionic PC (the predominant phospholipid constituent of mammalian cell membranes) (Beers et al., 2002), therefore ensuring the preferential elimination of bacterial membranes over eukaryotic host tissues (Nevalainen et al., 2008).

As a consequence of their bactericidal properties, high levels of GIIAPLA₂ expression could be found at the various potential routes of pathogenic entry throughout the body, such as the corneal and intestinal mucosal epithelia. The presence of GIIAPLA₂ has been previously reported in the human main lacrimal glands and tears (Saari et al., 2001 ). In particular, the human tear fluid has been shown to contain one of the highest amounts of GIIAPLA₂ among other human secretions, with an estimated concentration of 54.5 ± 33.9 pg ml^"1 (Aho et al., 1996). The human lacrimal tissues have been found to contain two distinct acinar cell types each specialized for the production and secretion of lysozyme and GIIAPLA₂ , respectively (Saari et al., 2001 ). The cells expressing GIIAPLA₂ have been found in comparatively smaller numbers and localized mainly in central regions of the lobules in main and accessory lacrimal glands (Aho et al., 1996).

Aho et al., (2002) have demonstrated an almost 2-fold increase in GIIAPLA₂ levels in the tears of dry eye patients compared with age-matched healthy controls. The increase in GIIAPLA₂ might serve to compensate for the compromised antibacterial activity in the tear fluid, as a result of concomitant decreases in tear lysozyme and lactoferrin with DES, the major bactericidal proteins normally found in tear fluid (Seal et al., 1986). In addition, it has been found that the levels of tear GIIAPLA₂ were almost doubled in the tear fluid of chronic blepharitis patients compared with controls, and the authors have verified that the elevated GIIAPLA₂ in tears displayed preferential hydrolysis of PE over PC by an approximated ratio of 3:1 (Song et al., 1999).

In corroboration with earlier works on ocular phospholipases (Aho et al., 1999; Song et al., 1999), it was observed that the levels of major lysophospholipids in tears were reduced by almost half after the treatment period, suggesting that ocular PLA₂ activity was diminished following routine eyelid warming. The notable reductions in various lysophospholipids following eyelid-warming treatment, without application of other medications such as steroids or antibiotics, are reported for the first time in the current study. It may be that the diminished PLA₂ activity with eyelid warming might be attributed to the relief of intraglandular pressure within meibomian ductal systems via facilitating the melting and outflow of meibum from the obstructed orifices with heating. In obstructive MGD, the elevation in intraglandular pressure imposes considerable mechanical stress on the ductal and acinar epithelia of gland tissues, which could possibly activate mitogen-activated protein kinase activity leading to the downstream release of chemokines and cytokines, therefore eliciting the ocular inflammatory cascade, as has been previously demonstrated in conjunctival and corneal epithelial tissues (Luo et al., 2004; Li et al., 2007). The induction of extracellular PLA₂ secretion from various cell types under an inflammatory milieu has been documented (Landreville et al., 2004) Therefore, the release of PLA₂ from meibomian gland acinar and ductal epithelia as a result of elevated intraglandular pressure might represent a plausible mechanistic pathway for the enhanced levels of lysophospholipids observed in the tears of MGD patients. Consequentially, the relief of plugged orifices with eyelid warming cou!d remove the stimulus for the increased PLA₂ secretion, leading to normalization of lysophospholipids levels. Nonetheless, it remains to be determined whether the meibomian gland tissues could secrete PLA₂ in response to mechanical induction.

Alternatively, the mechanical stress could be communicated from the meibomian gland acinar epithelia to the corneal and conjunctival epithelial tissues, as well as the lacrimal gland epithelia, which collectively lead to an enhanced release of PLA₂. The expression and production of PI_A₂ in the cornea (Wang and Kolko 2010), conjunctiva, and lacrimal glands (Landreville et al., 2004) has been well documented. In fact, the ocular epithelial tissues could be regarded as a continuum, and the lacrimal gland and meibomian gland simply denote invaginations of this continuous epithelium (Obata 2002). Swartz and colleagues (Swartz et al., 2001 ) have previously shown that mechanical stress could be communicated between different cell types to bring about comprehensive matrix remodeling in the airway system. It remains to be elucidated, however, whether the mechanical stress imposed on meibomian epithelial tissues in obstructive MGD could be conveyed to lacrimal epithelia that subsequently lead to the production of aqueous tears with elevated levels of PLA₂, as has been observed in tear samples from dry eye (Aho et a/., 2002) and chronic blepharitis patients (Song et al., 1999).

Regardless of the precise mechanistic details, the increased release of PLA₂ enzymatic products (i.e., lysophospholipids and FFAs) to the ocular surface could substantially compromise tear film stability, in view of the detergent-like properties of the highly polar lysophospholipids (Makide et al. , 2009). Indeed, a significant correlation between reductions in the levels of LpPE with improvement in ocular (both corneal and scleral) evaporation rate was observed, indicating the detrimental effects that LpPE might exert on tear film structural integrity. In addition, the specific increases in highly unsaturated plasmalogen PE after the treatment period implied that such species might be preferentially targeted during MGD pathogenesis. Such highly unsaturated plasmalogen PE species contain PUFAs, such as arachidonic acid (FA20:4) and docosahexaenoic acid (FA22:6) at their sn-2 positions. Furthermore, the present data also indicated that PLC might exhibit the same trend as PLA₂ in selectively targeting PUFA-containing phospholipids to produce DAGs during MGD pathogenesis, as shown by the appreciable decreases in PUFA- containing DAGs such as DAG 18:0/20:4 and DAG 18:0/22:6 following heat treatment. Upon release, arachidonic acids could be converted to downstream inflammatory mediators including prostaglandins and leukotrienes that further fuel the sustained inflammatory cycle commonly observed in DES (Thakur et a/., 1998). Also, the presence of leukotriene B4 and platelet-activating factor has been reported in human tears (Thakur et a/., 1998). Accordingly, the reductions in these proinflammatory metabolite precursors with eyelid warming would be expected to alleviate symptoms of ocular discomfort.

Nonetheless, the consistent decreases in LPE, LpPE, LPC, and LPI with heat treatment suggest that PLA₂ other than GIIAPLA₂, which has a specific preference for PE over PC, might participate in MGD pathogenesis. Indeed, cytosolic PLA₂ with high selectivity for phospholipids that possess an sn-2 arachidonic fatty acyl group, which hydrolyze PE and PC with equal efficiency (Wang et a/., 2010), have also been found to be expressed in the corneal and conjunctival tissues (Wang et al., 20 0). Moreover, the contribution by bacterial phospholipases to the disease phenotype (i.e., elevated lysophospholipids) observed at the ocular surface should also be considered. The clearance of stagnant meibum would remove potential sources of nutrients for ocular commensals. Furthermore, increased lipid turnover would also promote the elimination of commensal bacteria, which are known to proliferate in obstructed gland ducts (McCulley and Dougherty 1986), via drainage through lacrimal puncta or onto peripheral eyelid skin. Therefore, a diminished level of commensal bacteria (and bacterial phospholipase activity) might also partially account for the observed reductions in lysophospholipids following eyelid- warming treatment in relieving meibomian plugs. Meibum-derived amphiphilic lipids in maintaining tear film stability

The relief of obstructed meibomian orifices would release the stagnated meibomian oils onto the eyelid margin, thereby facilitating lipid removal by bulk flow and increasing lipid turnover at the eyelid margins. In a previous study, it was postulated that the lipid anomalies associated with MGD and DES might have resulted primarily from diminished lipid turnover, instead of an absolute deficiency in lipids per se (Lam et al, 2014b), which is in accordance with the current observation that eyelid warming and disease symptom alleviation were not associated with enhanced absolute amounts of lipids in tears. The Increased lipid turnover would facilitate delivery of fresh meibomian and lacrimal secretions onto the ocular surface, while removing toxic contaminants and other metabolized products and reactive lipid species (Bron et al., 2004). Thus, the appreciable increases in amphiphilic lipids, such as CS and OAHFA, in the tears of MGD patients might be associated with increased delivery of fresh meibomian and lacrimal secretions onto eyelid margins following eyelid warming.

The higher levels of lipid amphiphiles brought about by eyelid warming would exert positive effects on tear film structural integrity and stability, as indicated by the increase in TBuT following treatment. Interestingly, significant correlations were found between the increases in total OAHFA, as well as numerous OAHFA species, with alleviation in the degree of ocular discomfort after treatment. In addition, increases in the levels of several OAHFA species were also significantly correlated with improvement in ocular evaporation rates after treatment. These implied that OAHFAs could serve as suitable indicators of treatment response and ocular symptom improvement. This finding is in good agreement with the observation that OAHFAs displayed consistent decreases with increasing DES severity (Lam et al., 201 1 ) and further strengthens the validity of OAHFAs as appropriate indicators of DES/MGD pathogenesis and severity.

Conclusion

The results show for the first time, a comprehensive tear lipid signature associated with alleviation of meibomian gland obstruction and ocular symptom improvement for a relatively homogenous cohort of MGD patients receiving eyelid-warming treatment over a period of 12 weeks. Remarkably, stark reductions in tear lysophospholipids and PUFA-containing DAG at the end of the treatment period were observed, which were associated with improvement in ocular evaporation rate. This observation points to the plausible involvement of ocular phospholipases in the pathogenesis of MGD and indicates that eyelid warming could itself serve to curtail excess phospholipase activity in MGD without external application of steroids, possibly by relieving intraglandular pressure and facilitating lipid turnover at the eyelid reservoir, in addition, the levels of numerous OAHFA species, deemed as critical amphiphilic components in maintaining tear film stability (Lam et al., 2014a, Butovich 2009), were significantly increased at the end of the treatment period. Importantly, the increase in OAHFA was associated with reductions in both the ocular evaporation rate and the degree of ocular discomfort. Thus, monitoring changes in the levels of tear fluid OAHFA could be effective indicators of MGD progression. Furthermore, the positive association between OAHFA and ocular symptom alleviation suggests that OAHFA might be suitable therapeutics for treatment of DES.

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Claims

1. A method for monitoring a subject suffering from meibomian gland dysfunction comprising comparing at least one lipid parameter of at least one tear sample from the subject at a first time point with the lipid parameter of at least one tear sample from the subject at a second time point.

2. The method according to claim 1 , wherein a change in the lipid parameter comprising:

(i) a decrease in the level of at least on elysophopholipid;

(ii) an increase in the level of at least one phospholipid;

(iii) a decrease in the level of at least one polyunsaturated fattyacid- containing diacylglycerol ; and/or

(iv) an increase in the level of cholesteryl sulphate and/or O-acy!-co hydroxy-fattyacid(OAHFA); in the tear sample from the subject at the second time point compared to the first time point is indicative that the subject has improved in symptoms of meibomian gland dysfunction at the second time point compared to the first time point. The method according to claim 1 , wherein a change in the lipid parameter comprising:

(v) an increase in the level of at least one lysophopholipid;

(vi) a decrease in the level of at least one phospholipid;

(viii) a decrease in the level of cholesteryl sulphate and/or O-acyl-ω- hydroxy-fatty acid(OAHFA); in the tear sample from the subject at the second time point compared to the first time point is indicative that the subject has deterioriated in symptoms of meibomian gland dysfunction at the second time point compared to the first timepoint.

The method according to claim 1 , wherein no significant change in: (ix )the level of at least one lysophopholipid;

(x) the level of at least one phospholipid;

(xi) the level of at least one polyunsaturated fattyacid-containing diacylglycerol; and/or (xii) the level of cholesteryl sulphate and/or O-acyl-co-hydroxy-fatty acid (OAHFA); in the tear sample from the subject at the second time point compared to the first time point is indicative that there is no change in the symptoms of meibomian gland dysfunction a the first and second time points.

The method according to any one of claims 2 to 4, wherein the

lysophospholipid comprises at least one lysophosphatidylcholine, at least one lysophosphatidylethanolamine, at least one lyso-plasmologen phosphatidylethanolamine , at least one lysophosphatidylinositol and/or at least one lysophosphatidylserine.

The method according to any one of claims 2 to 5, wherein the

phospholipid comprises at least one polyunsaturated f attya cid-co nta i n i ng phospholipid, at least one phosphatidylcholine, at least one

phosphatidylethanolamine, at least one plasmalogen

phosphatidylethanolamine, at least one phosphatidylinositol and/or at least one phosphatidylserine.

The method according to any one of claims 2 to 6, wherein the

phospholipid comprises total phosphatidylethanolamine.

The method according to any one of claims 2 to 7, wherein the

polyunsaturated fatty acid-containing diacylglycerol comprises DAG16:1/22:5; DAG18:0/20:4; DAG18:0/22:6; DAG16:0/22:5 or

DAG16:0/22:6.

[Wewouldappreciateiftheinventorcouldclarifywhatthesenumbersme anintermsofthepositionandno.ofdoublebondsintheDAGandwhichon eofthetwofattyacidsintheDAG?]

9. The method according to any one of claims 2 to 8, wherein theO-acyl-o hydroxy-fattyacid (OAHFA) comprises OAHFA 18:1/26:1 ; OAHFA

18:1/30:2, OAHFA 18:1/34:1 ; OAHFA 18:1/25:0, OAHFA 18:1/28:1 , OAHFA 16:1/32:1 , OAHFA 18:1 /30:1 ; OAHFA18:1/31 :0; OAHFA

18:2/32:1 ; OAHFA 18:1/32:2 or OAHFA18: 1/34:2.

[Wewouldagainappreciateiftheinventorcouldc!arifywhatthesenumbe rsmean?] 0. The method according to any one of claims 1 to 9, wherein the first time point is before and the second time point is after treatment for meibomian gland dysfunction.

1 1 . The method according to claim 10, wherein the treatment comprises warming therapy of the eyelid and/or steroid therapy.

12. A method for detecting if a subject is suffering from and/or at risk of meibomian gland dysfunction comprising comparing at least one lipid parameter of at least one tear sample from the subject with a control.