Practical issues concerning tear protein assays in dry eye
© D'Souza and Tong; licensee BioMed Central Ltd. 2014
Received: 28 June 2014
Accepted: 9 October 2014
Published: 13 November 2014
Dry eye is a common clinical condition diagnosed by cumulative evidence of symptoms and signs. Many new treatments in dry eye are either expensive, invasive, have potential for side effects, or are not easily accessible. In severe dry eye, the ideal modality of treatment to begin with is often not clear as specific molecular disturbances are not evident from just examination of clinical manifestations. Assessing the effects of ongoing treatment is not straight forward since there is lack of agreement between clinical signs and symptoms. There is a need to have more objective methods of selecting treatment for dry eye and monitoring the effect of treatment.
Recently, there are many new technologies applied to the discovery of tear biomarkers, for e.g., mass spectrometry based proteomics techniques and multiplex assays such as the bead-based sandwich indirect immunofluorescent assays. Tear proteins assays have even been made available as point-of-care devices. This review focuses on the evidence for the involvements of tear proteins in dry eye, possible changes in tear concentrations with therapy and the strength of evidence regarding dry eye pathology. Much remains to be done in terms of developing office-based assays and ascertaining their reliability, but current evidence suggests that tear proteins have a role in the clinical practice of dry eye.
KeywordsHuman Dry eye Review Tear Proteomics Biomarker
Diagnosis of dry eye
According to the International Dry Eye Workshop, “dry eye is a multifactorial disease of the tear and ocular surface that results in symptoms of discomfort, visual disturbance, and tear instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear and inflammation of the ocular surface” . Simply put, dry eye is a complex multifactorial disorder affected by pathological processes such as lacrimal gland inflammation , meibomian gland dysfunction , and tear hyperosmolarity .
The signs and symptoms of dry eye disease do not always correlate well even though both are important in the diagnosis and management of dry eye. There are a number of tests used in clinical practice for the diagnosis of dry eye including the traditional Schirmer test, tear break up time, rose bengal staining and the more advanced technologies like the tear osmolarity, meibography and tear film interferometry. Unfortunately the available diagnostic tests often do not correlate reliably with severity of the patient’s symptoms .
Recently, a panel of tear proteins has been found, which may be specific and sensitive for the detection of dry eye . A similar panel has been advocated for the diagnosis of primary Sjogren syndrome . The importance of this discovery lies in the fact that most of the diagnostic tests need to be used in combination to reliably diagnose dry eye. These tear proteins may thus prove invaluable to the practicing ophthalmologist. Disease specific signatures of tear proteins may demonstrate underlying disease pathways, and about 4-5% of proteins may be deranged in dry eye . Since tear fluid has various components secreted by different glands, the composition of tears reflects the health of different components of the ocular surface . For some time now, the level of secretory IgA in the tears was used as a measure of lacrimal function, and could be used as a marker of ocular surface inflammation. It fluctuates diurnally in normal people, with a higher value at noon time, and has also been found to be reduced in dry eye compared to controls .
What is the cause of increased cytokines in dry eye? It is hypothesised that in the dry eye, the increased concentration of cytokines is not due to evaporation, but rather attributed to the upregulation of inflammatory genes in the conjunctival epithelium. This results in the increased production of inflammatory cytokines in tears . The increase in tear cytokines was correlated to derangements in tear function tests such as osmolarity , suggesting that they reflect the underlying disease mechanisms.
In fact when a neural network was used to examine the tear proteins of dry eye patients and normal participants, multivariate analysis was able to discriminate the proteins from these two groups . Specific protein derangements in dry eye patients further point to clinical subgroups of patients such as meibomian gland dysfunction , preservative induced dry eye in glaucoma patients , aqueous deficient dry eye , or diabetes .
The symptoms of dry eye can sometimes be non-specific. Dry eye symptoms like chronic irritation, tearing and eye redness may be mimicked by other conditions like allergic conjunctivitis . Therefore, an objective test would be useful to distinguish dry eye from its mimickers. In fact, a decrease in tear break up time, which is a hallmark of certain types of dry eye disease, when present in isolation without the other signs of dry eye, may be a feature of allergic conjunctivitis , thus suggesting that it is unreliable to diagnose dry eye with only one single clinical test. In patients with chronic conjunctivitis, an elevated tear IgE may be a clue to underlying allergic conjunctivitis . Cytokines in dry eye involve mainly Th1 and Th17 subsets whereas in allergic eye disease, T helper type 2-related mechanisms are involved in the sensitization phase, but both T helper type 1 and type 2 cytokines are overexpressed in the active disease, contributing to the development of ocular inflammation .
Other conditions we need to differentiate from idiopathic dry eye are those in which dry eye is associated with an underlying systemic cause. It may be possible to have a higher index of suspicion for a systemic cause of dry eye based on the levels of specific tear glycoproteins in addition to relevant history and clinical findings. For example, conditions such as pemphigoid may affect the glycoproteins differently from age-related dry eye .
It is important to assess the suitability of a tear protein as a biomarker before analysis. In this article we consider a tear protein to be potentially suitable as a clinical biomarker if it fulfils the following three criteria in the literature available: Firstly, the tear proteins which have been studied in dry eye disease in humans and found to be consistently deranged with a clearly defined normal range may be considered to be more relevant for clinical use. Secondly, the tear proteins that have been shown to change with the severity of dry eye, especially in longitudinal studies or trials are given importance. Lastly, there should be some link between dry eye pathology and the function of the measured tear protein. Such a link can be provided by relevant in vivo experiments in animals or an intervention that produced a known biological effect such as inflammation in humans.
Evidence for assessing the suitability of tear proteins for clinical use
Class of tear protein
Potential clinical marker
Human tear levels show consistent dysregulation
Human tear levels linked to clinical signs/symptoms of dry eye
Change with treatment or severity of dry eye?
Biological function in dry eye pathology known?
Lipid binding protein
Available technologies and use in dry eye
Traditional separation of tear proteins was performed by electrophoresis , and identification and quantification by the enzyme linked immunosorbent (ELISA) method , but the recent advances in analytical technologies have provided ocular surface scientists with a host of other techniques for examination of tear proteins. The nano-scale sensitivity of some recent technologies enable the detection of proteins in tears from individuals rather than pooled samples ,. Some of the techniques described below can be used for relative or even absolute quantification if standards of known concentration are also available for calibration. One of the techniques of separating proteins rely on high performance liquid chromatography , or nano-electrospray liquid chromatography , where the size and charge of proteins provide the basis for their separation. The various techniques available are isobaric tagging using relative and absolute quantification (iTRAQ) technology, and the matrix assisted laser desorption ionisation (MALDI) time of flight (TOF) technique with mass spectrometry . A variant of this technique is the surface-enhanced laser desorption/ionization (SELDI) TOF which can also be coupled with mass spectrometry .
Two dimensional (2D) separation of tear proteins based on mass/charge followed by dye staining have also been performed for separation and later identification of tear proteins . In diabetic people, tear proteins have been evaluated using the 2D electrophoresis technology . Isoelectric focusing in combination with sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), an older technology, was able to detect glycation at specific residues of tear proteins such as lactoferrin and immunoglobulins .
Protein arrays have been used to detect proteins from tears of Sjogren syndrome patients . This involves the binding of tear proteins to a predetermined set of antibodies arranged in a fixed arrangement in a stationary platform. In some unique scenarios, for example, to compare the types of immunoglobulins in tears, a more traditional assay such as the Western or immunoblot assay can be performed . For office-based diagnosis, tear proteins can be separated in a very short time using a commercially available equipment, the Agilent Bioanalyser 2100, and analysed using the microfluidic based lab-on-chip technology . For the analysis of a dozen or more cytokines and chemokines in a few microliter of tears, it seems the best technology is the multiplex bead-based immunofluorescent sandwich assay, and the sensitivity for detection of some cytokines are in the order of several picogram/milliliter concentrations, surpassing even the minimum detection limits of mass spectrometry based methods .
There are some pitfalls to tear protein analysis such as: differences in methods of tear collection which can give different results . Tear collection may be done using Schirmer strips, capillary tubes, or even a special minisponge . A key consideration is the comfort and minimal stimulation of the patient during tear collection, avoiding reflex tearing, since reflex tears likely have different tear protein levels from basal tears. Tear concentrations of interleukin (IL)-1 alpha, precursor IL-1 beta, and IL-1 receptor antagonist (RA) may be altered in dry eye in the basal but not the reflex tear . The Schirmer test method has been shown to be reliable for the collection of tears for analysis of multiple cytokines , and such evaluation would be necessary as a prerequisite for new methods of tear collection proposed in the future.
The techniques of iTRAQ, MALDI TOF, SELDI TOF and 2D electrophoresis have shown to be useful for evaluation of tear proteins in a research setting but are unlikely to be used in clinics.
In summary, there is no universal methodology for handling or analysing tear proteins that is applicable to all clinics. Clinicians should consider the advantages and pitfalls of various methods should they want to apply these in their practice. Once applied, the same method should be continued for subsequent encounters and other patients.
A ‘traditional’ marker: Lysozyme
This protein, also known as muramidase or N-acetylmuramide glycanhydrolase is a glycoside hydrolase, which are enzymes that damage bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. Lysozymes are abundant in a number of secretions, such as tears, saliva, human milk, and mucus . Lysozyme is probably the earliest tear protein studied in conjunction with dry eye, and the level of tear lysozyme was noted to increase with age till 40 and decrease thereafter . It was found to be decreased in glaucoma patients with chronic medication induced dry eye . In a large study with 262 patients (78 patients with Sjogren syndrome), the concentrations of the lysozyme and lactoferrin protein in tear samples in Sjogren syndrome were determined . It was found that lysozyme was decreased in idiopathic dry eye and Sjogren syndrome compared to controls . However, a later study showed that lysozyme concentration did not differ between non-Sjogren dry eye, Sjogren syndrome or normal controls . Researchers also found the single protein lysozyme test to be insufficient for the diagnosis of dry eye . Nevertheless, lysozyme levels may be useful in specific contexts, for example, to check for adverse effects of beta-adrenergic receptor blocking drugs such as Practalol, which reduced tear lysozyme levels  or to detect dry eye from specific occupational exposures in coal mining . With so many existing and emerging technologies, it may be worthwhile to take a fresh look at the utility of lysozyme.
Lactoferrin, also known as lactotransferrin, is a non-haem iron-binding protein of the transferrin family. It is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as milk, saliva, tears and nasal secretions. Lactoferrin is one of the components of the immune system of the body. It has antimicrobial activity, is antiviral, antiparasitic, catalytic, immunomodulatory and anti-inflammatory .
It was found to be negatively correlated to Rose Bengal staining, indicating that reduced lactoferrin was a marker of ocular surface damage . However, in evaporative dry eye in the absence of epithelial defects, tear lactoferrin was also found to be reduced . In chronic hepatitis C patients, the cotton thread test of tear secretion was weakly correlated (r = 0.35) to the tear lactoferrin levels , indicating that these patients having significantly lower tear lactoferrin levels than control participants. The use of tear lactoferrin has been advocated for the diagnosis of primary Sjogren’s syndrome, where the test had a specificity of 95% and a sensitivity of 72% . These values were somewhat superior to using only the Schirmer I test for detection of this disease . Researchers have suggested a cut-off value of 1.1 mg/mL for tear lactoferrin so that the assay has optimal accuracy for the diagnosis of dry eye . Using this threshold, the test was able to detect dry eye with a sensitivity 79.4% and specificity of 78.3% . Using a convenient commercial lactoplate assay, the tear lactoferrin was measured in dry eye patients and was decreased relative to controls .
However, it has been found that lactoferrin changes either do not appear early enough for diagnosis of mild to moderate dry eye or that some cases of dry eye did not have the lacrimal dysfunction that this assay tested for . In any case, treatment of dry eye with punctal occlusion was associated with increased tear lactoferrin levels, suggesting that tear levels of this protein may be a measure of tear turnover . Recently a TearScan™ Lactoferrin Diagnostic Test Kit was made available commercially (Advanced Tear Diagnostics, Raleigh, NC). This device provides quantitative point-of-care tear concentration of lactoferrin.
The S-100A protein is a family of low molecular weight proteins characterized by two calcium binding sites of the helix-loop-helix conformation and at least 21 different types of S100 proteins are known. It is normally present in cells derived from the neural crest (Schwann cells, melanocytes and glial cells), chondrocytes, adipocytes, myoepithelial cells, macrophages, Langerhans cells, dendritic cells, and keratinocytes. S100 proteins are involved in the regulation of protein phosphorylation, transcription factors, calcium homeostasis, the dynamics of cytoskeleton constituents, enzyme activities, cell growth and differentiation, and the inflammatory response .
The S100 A8 and A9 proteins have been known to be pro-inflammatory . In a study using the isobaric tagging for relative and absolute quantification (iTRAQ) technology, we found the S100A8 and S100A9 proteins were among a panel of 6 upregulated proteins found in the tear of dry eye patients . The average ratio of S100A8 and S100A9 in dry eye versus control subjects was 1.82 (SD 1.41) and 1.92 (SD 1.48) respectively. In another study using a different SELDI-TOF technique, over-expression of S100A8 has also been reported .
Mucin and related molecules
The mucins (MUC) are a family of high molecular weight, hydrophilic, heavily glycosylated proteins, produced by epithelial tissues. The tear film on the ocular surface epithelia is maintained by the mucins on its surface as well as by membrane-associated mucins in the apical surface of the cell. They are secreted by goblet cells or other secretory cells and have a characteristic ability to form gels with the exception of the monomeric MUC7, hence they have various functions from lubrication to cell signalling to form chemical barriers. MUCs 1, 3A, 3B, 4, 12, 13, 15, 16, 17 and 20 are membrane associated and MUCs 2, 5 AC, 5B, 6, 7 and 19 have been classified as secreted mucins .
Using Schirmer strip samples, mean MUC5AC content in tears was found to be lower in the dry eye patients than in the age- and gender-matched healthy individuals . The levels of certain mucin molecules were associated with certain ocular surface states. For example, tear MUC5AC was reduced in symptomatic contact lens wearers, and MUC4 was correlated to the presence of temporal lid parallel conjunctival folds (conjunctivochalasis) and lid wiper epitheliopathy . The levels of mucins are not always depressed in dry eye. Sjogren syndrome patients exhibited increased soluble MUC16 in the tear compared to controls . The membrane-associated MUC16 and the mucin-associated T-antigen carbohydrate were associated with ocular surface epithelial protection .
Understanding of tear mucin regulation may produce insight into the mechanism of at least some types of dry eye . Phospholipid transfer protein, a protein that interacts with mucins, may also be relevant to the dry eye mechanism , but this has not been studied well enough to be used for clinical purposes. Since the regulation of mucin affects dry eye pathologic mechanisms, it gives greater credibility to the use of mucins as a marker for this disease.
Protease refers to a group of enzymes whose catalytic function is to hydrolyse (breakdown) peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyse various peptide bonds. Matrix metalloproteinases (MMPs) are a class of proteases belonging to the metzincin superfamily characterised by zinc cofactors, and they are the most important proteases found in tears ,.
These enzymes degrade extracellular matrix proteins, but can also process certain bioactive molecules. They are also associated with cleavage of cell surface receptors, the release of apoptotic ligands (like the FAS ligand), and chemokine/cytokine in/activation. MMPs also play a major role cell proliferation, migration (adhesion/dispersion), differentiation, apoptosis, and host defense . MMP-9, also known as 92 kDa type IV collagenase or gelatinase B, can be involved in the degradation of collagen IV present in the basement membrane and extracellular matrix.
In rosacea-associated meibomian gland disease or Sjogren’s syndrome, the tear activity of MMP9 was raised compared to controls . In another study, pro-MMP-9 levels were found to be significantly elevated in various ocular surface diseases: blepharitis (p = 0.013), allergic eye disease, dry eye and conjunctivochalasis (all p < 0.001) compared to controls . In a study involving 46 patients with newly diagnosed dry eye and 18 control participants, tear MMP-9 activity was assessed with an MMP-9 activity assay in 1 uL of basal tear fluid . The MMP-9 activity in the control group was 8.39 +/- 4.70 ng/mL and progressively higher levels of MMP-9 were found in the dry eye groups with the highest levels corresponding to the most severe dry eyes clinically . Although this is not a longitudinal study, it represents correlation of MMP-9 levels with clinical disease severity and suggests that it can be a clinical marker for monitoring patients.
In a study involving treatment of ocular surface disease, including dry eye, with a therapeutic contact lens, clinical improvement was observed and at the same time, tear MMP-9 was found to decrease by day 7 and further decrease to minimal levels by day 21 . These types of longitudinal results strengthen the validity of using the tear MMP-9 assay as a monitoring tool.
InflammaDry (RPSInc, Sarasota, FL, USA) is a rapid (10 minute) point of care diagnostic test for tear MMP-9, which gives a positive result if tear MMP-9 exceeds 40 ng/ml. This test can be easily administered by a nurse or technician. It has a sensitivity of 87% and specificity of 92% (RPS clinical study: protocol number 09-001, version no 2.4) when compared to a combined diagnostic criteria of dry eye tests (OSDI > =13, Schirmer II < 10 mm, TBUT < 10 sec and staining > =1). This disposable test kit is based on direct sampling of tears from the inferior fornix using microfiltration and capturing of MMP-9 between specific monoclonal and polyclonal antibodies, and does not require specialised equipment. However, in its current form, the InflammaDry does not yield the actual MMP-9 concentration .
Experimentally, dry eye models in animals have demonstrated the importance of tear MMPs in the induction of ocular surface damage in dry eyes , and have linked these to inflammatory signaling , thereby strengthening the biological basis for clinical use of MMPs. In experimental dry eye, the raised MMP-9 can also be detected in the cornea and lacrimal tissues, implying that tear concentrations were not raised purely due to evaporation . In primary lacrimal gland cultures, pro-MMPs were secreted into the culture medium, suggesting that the lacrimal gland may be a source of MMPs .
Lipocalins are a group of extracellular low molecular weight proteins , which use multiple recognition properties including ligand binding to small hydrophobic molecules, macro-molecular complexes and binding to specific cell surface receptors. Tear lipocalin is a major protein in tears, which binds a variety of lipophilic molecules. It can also bind to macromolecules like lactoferrin and lysozyme and has a variety of functions in tears, including anti-inflammatory activity, binding and release of lipids , regulation of tear viscosity, endonuclease inactivation of viral DNA and used as a biomarker for dry eye .
In dry eye disease, downregulation of lipophilin-1 and lipocalin-1 have been found . In fact, the tear lipocalin was even lower in Sjogren’s syndrome patients compared to non-Sjogren’s dry eye patients . In a treatment trial involving dry eye volunteers, improvement of clinical signs of dry eye with an increase in the stability of tear film was found in conjunction with an increase in the tear lipocalin levels . One risk factor for dry eye is contact lens wear, and dry eye sufferers also tend to have intolerance to contact lens. Surprisingly, people who are intolerant of contact lens wear demonstrated higher tear lipocalin levels compared to people who tolerated contact lens , suggesting that tear lipocalin alone should not be used for the diagnosis of dry eye. To the best of our knowledge, there has been no longitudinal clinical study on levels of lipocalin to-date.
Experimentally, rabbit levels of tear lipocalin were hormone dependent. Ovariectomy in rabbits decreased tear lipocalin and in contrast, administration of estrogen or male androgen dihydrotestosterone (DHT) increased the levels . These studies point out that hormones act upstream of the production of lipocalin. Interestingly, addition of exogenous sex steroids resulted in the binding of these steroids to the tear lipocalin . Since levels of hormones influence the human dry eye, these findings provide some evidence linking lipocalins to the biology of dry eye. Unfortunately, there has been no evidence of tear lipocalin deficiencies in animal models of dry eye so far.
The interleukins (IL), a group of cytokines that were first seen to be expressed by leukocytes, have an important role in the adaptive immune response in that they are required for the propagation of inflammation . Recently, a panel of four key inflammatory cytokines (IL-1β, IL-6, IFN-γ, and TNF-α) were found to be highly reproducible and reliable when determined in tear samples as little as 4-10 μL. Standard operating instructions for tear collection, shipping, storage and processing were recommended .
Tear ILs, in particular IL-1β has been shown to increase in aqueous deficiency dry eye .
In patients with meibomian gland dysfunction MGD and those with Sjogren’s syndrome, compared with normal participants, the concentrations of tear IL-1α and mature IL-1β were increased, and precursor IL-1β was decreased . Experimentally, the production of IL-1β in tears has been linked to inflammation ,,,,,.
Tear IL-6 has been found to be elevated in Sjogren’s and non-Sjogren syndrome dry eye patients ,. In another study, significant elevation of tear IL-6 has also been observed after 2 weeks of soft contact lens wear. Since the tears break up time and expression of MUC5AC was also decreased over this time, the study suggests that increased tear IL-6 may be an indicator of worsening epithelial and mucoid function . Interestingly, one of the polymorphisms in the IL-6 gene was associated with dry eye disease . This not only suggests a genetic predisposition of dry eye for certain populations, but also supports the use of IL-6 as a marker of dry eye in susceptible people.
The tear concentrations of IL-17 in patients with filamentary keratitis, graft versus host disease, autoimmune keratitis, Sjogren’s syndrome, dry eyes, MGD and Steven Johnson syndrome were significantly higher than in normal study participants . This relationship of increased tear IL-17 was only observed in patients with systemic autoimmune disease but not in those whose inflammation is restricted to the eye. Interestingly, serum IL-17 levels were correlated to fluorescein staining scores, but this study did not investigate tear IL-17 levels .
Tumour necrosis factor-α (TNF-α) and Interferon-γ (IFN-γ)
These are both acute response cytokines that enhance cellular immune responses . Although tear TNF-α levels were higher in dry eye than controls , there was no significant correlation between these levels and the dry eye clinical tests . There was also no difference between the tear TNF-α levels between Sjogren’s and non-Sjogren’s dry eye patients . The pro-inflammatory role of tear TNF-α however, has been verified in experimental models ,,,,. The main drawback in the use of tear cytokines as markers is that none of these have been measured longitudinally in clinical studies to-date.
Tear levels of IFN-γ were elevated in Sjogren’s syndrome compared to controls . Similarly, the tear levels of IFN-γ in cystic fibrosis, a systemic disease associated with dry eye, were significantly higher than those in non-cystic fibrosis controls ,. As in the case of TNF-α, the pro-inflammatory role of IFN-γ has been verified in experimental murine dry eye ,.
Chemokines and interleukin 8 (CXCL8)
The tear chemokines which have been implicated in dry eye are CX3CL1 , CXCL10 , CCL4/MIP-1beta ,, CCL3/MIP-1alpha ,,, CCL5/RANTES , CXCL9, -10, and -11 . The tear levels of CXCL9, -10 and -11 were 1,148 +/- 1,088, 24,338 +/- 8,706, and 853 +/- 334 pg/mL, in dry eye, and only 272 +/- 269, 18,149 +/- 5,266, and 486 +/- 175 pg/mL in controls respectively . Hyperosmolarity induced the production of Monocyte chemotactic protein ( MCP)-1 in experimental (epithelial cell culture) settings, providing a link between tear chemokines and dry eye pathology . Nevertheless, there is currently no evidence that chemokines are reduced after treatment of dry eye or that measurement of chemokines is specific and sensitive for the detection of dry eye.
The tear levels of proinflammatory cytokine IL-8 was found to be higher in Sjogren’s syndrome compared to controls . The levels were also higher in dry eye with and without MGD compared to normal controls . In experimental dry eye, IL-8 was also found to be upregulated in the lacrimal gland ,. Ocular pain levels were correlated to tear IL-8 .
Growth factors and wound healing molecules
A number of growth factors are produced by the lacrimal gland/ocular surface and may provide trophic effects for ocular surface epithelium. Intuitively, the levels of tear growth factors may reflect the extent of trophic support that is vital for epithelial health .
Nerve growth factor and related proteins
It is logical to assess proteins related to nerve endings in dry eye, since dry eye may result from an interruption of neural reflex at the afferent nerve endings. The tear nerve growth factor (NGF) levels were higher in dry eye patients compared to age- and gender-matched healthy control participants. In these patients, prednisolone treatment for 28 days resulted in a decrease in tear NGF levels, which occurred together with clinical improvement of dry eyes . In another study, tear levels of NGF were increased in dry eye patients whereas related peptides: Calcitonic Gene Related Peptide (CGRP) and Neuropeptide (NP) Y concentrations were decreased compared to healthy participants. Furthermore, the level of tear NGF was correlated with clinical severity of dry eye while CGRP and NPY levels were inversely correlated to these clinical parameters . Tear NGF/total tear protein ratio was increased in photorefractive keratectomy and laser in situ keratomileusis, and the early post-operative levels were also correlated with tear function 6 months later . NGF was elevated in tears of contact lens wearers with dry eye, and the levels were associated with a decrease in the nerve plexus density in the cornea , supporting the theory that dry eye results from a decrease in the afferent part of the lacrimal loop.
The tear concentration of another peptide, substance P, was also associated with dry eye, and was elevated after excimer corneal surgery compared to pre-surgical levels .
Is there any evidence of functional mechanisms involving neural peptides? Experimentally, NGF has been shown to induce goblet differentiation and increase in MUC5AC both in human conjunctival epithelial cells exposed to increasing NGF concentrations and confirmed in primary cultures . Dry eye was induced in rats by subcutaneous scopolamine treatment and aqueous tear production, tear clearance, fluorescein corneal staining, and tear break-up time were evaluated. The NGF mimetic was able to induce an improvement of dry eye test parameters and glycoprotein secretion . The use of supplementary NGF for dry eye therapy is however, hard to justify if the tear level of this protein is already higher than normal to start with. Further studies are required to determine why the NGF levels are elevated in dry eye, and perhaps the use of NGF as a biomarker should be delayed until substantial longitudinal studies of NGF levels are available.
Other growth factors
Epidermal growth factor (EGF) is a low molecular weight polypeptide that acts by binding with high affinity to the epidermal growth factor receptor (EGFR) on the cell surface. Stimulation of the intrinsic protein-tyrosine kinase activity of the receptor ultimately leads to DNA synthesis and cellular proliferation, differentiation, and survival . The concentration of tear EGF was significantly decreased in non-Sjogren dry eye, Sjogren syndrome, and Steven Johnson syndrome patients compared with controls . On the other hand, higher levels of tear EGF has been linked to subepithelial fibrosis in dry eye . In animal models, addition of EGF has been linked to longer tear break up times and lower fluorescein staining scores, establishing a functional effect of this growth factor in the ocular surface .
Transforming growth factor (TGF) is a peptide known to be involved in inflammatory and fibrotic pathways. TGF-β1 is the prototypic member of the transforming growth factor superfamily and it elicits diverse cellular responses like proliferation, induction and regulation depending on cell type, state of differentiation and culture conditions . In dry eye, tear TGF-β bioactivity, as assessed by a cell based assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in controls (4129.3 +/- 1342.9 pg/mL). The level of TGF-β bioactivity was highest in those with Sjogren syndrome, compared to controls and non-Sjogren dry eye . The biological role of TGF-β has also been investigated in experimental dry eye settings ,.
Corneal neovascularisation may be observed in very advanced dry eye and ocular surface failure . Vascular Endothelial Growth Factor (VEGF) is a signal protein that stimulates vasculogenesis and angiogenesis, cell survival, migration and differentiation. When VEGF is overexpressed, it can contribute to neovascularisation . Tear VEGF levels have been found to be raised in dry eye compared to controls in a study comparing evaporative dry eye to normal subjects. Epidermal growth factor (EGF), fractalkine/CX3CL1, IL-1-receptor antagonist (RA), IL-8/CXCL8, interferon inducible protein (IP)-10/CXCL10 were found elevated along with VEGF in 94%–100% of samples . Despite the promise of using growth factors as tear biomarkers, there has yet been no longitudinal study that evaluated the tear levels of these proteins.
Miscellaneous tear proteins and future directions
There are some interesting proteins that have not been evaluated as thoroughly as those mentioned above, but future research will show their usefulness or otherwise in clinical scenarios. The phospholipase (PL)A2-IIa concentration was found to be lower in patients with ocular rosacea (31.0+/-18.4 μg/ml) and in patients who had dry eye (25.8+/-15.1 μg/ml), compared to normal controls . The activity of serum PLA2-IIa was significantly increased in tears from dry eye diseased patients compared to those from normal subjects. In addition, serum PLA2-IIa stimulated the production of prostaglandin E(2) in ocular surface epithelial cell cultures, linking this tear protein to inflammation . We have previously reported the involvement of other proteins (e.g., prolactin-induced protein, enolase and orosomucoid) in dry eye . These have not been as well characterized as the other tear proteins above. Lacritin is a tear protein that is reduced in dry eye. Because it protects against cells from stress, replacement of lacritin has been advocated as a form of treatment in dry eye. Furthermore, an ELISA assay has been developed to quantify lacritin levels .
Specific molecules such as B cell activating factor may indicate severity of inflammation in Sjogren syndrome . Similarly, tear aquaporin 5 was increased in the tears of Sjogren syndrome, indicating the level of lacrimal damage . Increased levels of anti-Ro or anti-La may also be detected in tears of Sjogren syndrome , although the role of this assay is currently not clear. The tear levels of pro-apoptotic proteins such as sFas have been measured in patients with dry eye associated with cystic fibrosis , and it is possible that subject to further studies, this marker may have some clinical application.
In addition, the tear levels of albumin may also serve as a marker of inflammation. In one study the levels of albumin were found to be increased in glaucoma patients with dry eye . Some proteins are poorly understood and there is no clear strategy that can ‘restore’ this to normal. For example, we found the scaffolding protein 14-3-3 to be uniquely upregulated in glaucoma patients with dry eye who used chronic medications but not in other dry eye patients . It may be possible to use such markers to monitor these patients’ progress even if the biological link is unclear, but more research such as longitudinal studies would be required to address this.
A great difficulty in diagnostic science is the wide variation of techniques used in analysis. The differences in techniques can result in the variation of the ‘normal’ range of specific tear proteins . The extent of variation of tear proteins during processes such as fasting should also be further investigated . Particular care should be paid to the conditions during tear collection: close eye tears and open eye tears differ in the fibronectin concentrations . It may be necessary for an international panel to set up universal standards for the testing of specific tear proteins, or testing of patients for specific purposes, such as for the selection of treatment modality in dry eye.
Much remains to be done in terms of developing clinic based assays and ascertaining their reliability. Technologies that rapidly produce results and take up minimal amount of space are highly promising, such as the one using a microfluidics chip mentioned above .
Although this review focuses on proteins, tear lipids mediators in the tear may also have a major role in inflammation, and are potentially useful biomarkers. For example, the omega-3 fatty acid metabolic pathways produce both pro-inflammatory lipids (leukotrienes) as well as anti-inflammatory lipids (e.g., the 18R-, 17R- and 18S- resolvins). The resolvins (Rv)E1, neuroprotectin D1 and RvD1 dampen a range of immune responses including T cell responses, cytokine production, and endothelial adhesion. In fact, the RvE1 analog Rx-10045 has completed phase II testing for treatment of dry eye (clinicaltrials.gov NCT01675570). Assuming that diagnostic technology of lipid detection overcomes certain practical obstacles, tear lipids may be useful biomarkers for guiding therapies.
We propose that when a patient presents with dry eye symptoms, the history and the clinical examination will alert to any systemic diseases that may be linked to dry eye, for example, rheumatoid arthritis or Sjogren’s syndrome. In recalcitrant cases of dry eye, tear proteins may be useful for selection of treatment or following up the patient for response to treatment. For example, increased tear MMPs may suggest the more prolonged use of MMP inhibitors like the doxycyclines. In conclusion, clinicians are reminded that tear protein science is an evolving area of research and with increasing knowledge on the pathogenesis of specific types of dry eyes, more appropriate biomarkers may replace these mentioned in this article.
Retrieval of articles
The NCBI Pubmed database was searched for the keywords “Tear Proteins” and “Dry eye” in any field. This yielded 471 articles, and these have been manually curated to 131 relevant articles by excluding those that do not support the criteria in Table 1. Seven articles were added to introduce the proteins in each section.
Grant Support: National Medical Research Council grants, NMRC/CSA/045/2012.
- The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007).Ocul Surf 2007, 5:75–92.,Google Scholar
- Barabino S, Dana MR: Dry eye syndromes. Chem Immunol Allergy. 2007, 92: 176-184.PubMedView ArticleGoogle Scholar
- Bron AJ: Eyelid secretions and the prevention and production of disease. Eye (Lond). 1988, 2 (Pt 2): 164-171.View ArticleGoogle Scholar
- Smith J, Nichols KK, Baldwin EK: Current patterns in the use of diagnostic tests in dry eye evaluation. Cornea. 2008, 27: 656-662.PubMedGoogle Scholar
- Zhou L, Beuerman RW, Chan CM, Zhao SZ, Li XR, Yang H, Tong L, Liu S, Stern ME, Tan D: Identification of tear fluid biomarkers in dry eye syndrome using iTRAQ quantitative proteomics. J Proteome Res. 2009, 8: 4889-4905.PubMedView ArticleGoogle Scholar
- Tomosugi N, Kitagawa K, Takahashi N, Sugai S, Ishikawa I: Diagnostic potential of tear proteomic patterns in Sjogren’s syndrome. J Proteome Res. 2005, 4: 820-825.PubMedView ArticleGoogle Scholar
- Laurie GW, Olsakovsky LA, Conway BP, McKown RL, Kitagawa K, Nichols JJ: Dry eye and designer ophthalmics. Optom Vis Sci. 2008, 85: 643-652.PubMed CentralPubMedView ArticleGoogle Scholar
- Ohashi Y, Dogru M, Tsubota K: Laboratory findings in tear fluid analysis. Clin Chim Acta. 2006, 369: 17-28.PubMedView ArticleGoogle Scholar
- Hoshino M, Shoji J, Inada N, Sawa M, Kato H: Clinical evaluation of a measurement method for secretory IgA in tears. Nihon Ganka Gakkai Zasshi. 2006, 110: 276-281.PubMedGoogle Scholar
- Massingale ML, Li X, Vallabhajosyula M, Chen D, Wei Y, Asbell PA: Analysis of inflammatory cytokines in the tears of dry eye patients. Cornea. 2009, 28: 1023-1027.PubMedView ArticleGoogle Scholar
- VanDerMeid KR, Su SP, Ward KW, Zhang JZ: Correlation of tear inflammatory cytokines and matrix metalloproteinases with four dry eye diagnostic tests. Invest Ophthalmol Vis Sci. 2012, 53: 1512-1518.PubMedView ArticleGoogle Scholar
- Grus FH, Augustin AJ: Analysis of tear protein patterns by a neural network as a diagnostical tool for the detection of dry eyes. Electrophoresis. 1999, 20: 875-880.PubMedView ArticleGoogle Scholar
- Tong L, Zhou L, Beuerman RW, Zhao SZ, Li XR: Association of tear proteins with Meibomian gland disease and dry eye symptoms. Br J Ophthalmol. 2011, 95: 848-852.PubMedView ArticleGoogle Scholar
- Wong TT, Zhou L, Li J, Tong L, Zhao SZ, Li XR, Yu SJ, Koh SK, Beuerman RW: Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication. Invest Ophthalmol Vis Sci. 2011, 52: 7385-7391.PubMedView ArticleGoogle Scholar
- Grus FH, Augustin AJ, Evangelou NG, Toth-Sagi K: Analysis of tear-protein patterns as a diagnostic tool for the detection of dry eyes. Eur J Ophthalmol. 1998, 8: 90-97.PubMedGoogle Scholar
- Fujishima H, Toda I, Shimazaki J, Tsubota K: Allergic conjunctivitis and dry eye. Br J Ophthalmol. 1996, 80: 994-997.PubMed CentralPubMedView ArticleGoogle Scholar
- Toda I, Shimazaki J, Tsubota K: Dry eye with only decreased tear break-up time is sometimes associated with allergic conjunctivitis. Ophthalmology. 1995, 102: 302-309.PubMedView ArticleGoogle Scholar
- Baudouin C, Bourcier T, Brignole F, Bertel F, Moldovan M, Goldschild M, Goguel A: Correlation between tear IgE levels and HLA-DR expression by conjunctival cells in allergic and nonallergic chronic conjunctivitis. Graefes Arch Clin Exp Ophthalmol. 2000, 238: 900-904.PubMedView ArticleGoogle Scholar
- Moriyama M, Hayashida JN, Toyoshima T, Ohyama Y, Shinozaki S, Tanaka A, Maehara T, Nakamura S: Cytokine/chemokine profiles contribute to understanding the pathogenesis and diagnosis of primary Sjogren’s syndrome. Clin Exp Immunol. 2012, 169: 17-26.PubMed CentralPubMedView ArticleGoogle Scholar
- Berta A, Torok M: Tear glycoprotein determinations in the diagnosis and differential diagnosis of dry eyes. Scand J Rheumatol Suppl. 1986, 61: 228-233.PubMedGoogle Scholar
- Versura P, Frigato M, Mule R, Malavolta N, Campos EC: A proposal of new ocular items in Sjogren’s syndrome classification criteria. Clin Exp Rheumatol. 2006, 24: 567-572.PubMedGoogle Scholar
- Da Dalt S, Moncada A, Priori R, Valesini G, Pivetti-Pezzi P: The lactoferrin tear test in the diagnosis of Sjogren’s syndrome. Eur J Ophthalmol. 1996, 6: 284-286.PubMedGoogle Scholar
- Fukuda M, Wang HF: Dry eye and closed eye tears. Cornea. 2000, 19: S44-S48.PubMedView ArticleGoogle Scholar
- Wang HF, Fukuda M, Shimomura Y: Diagnosis of dry eye. Semin Ophthalmol. 2005, 20: 53-62.PubMedView ArticleGoogle Scholar
- Hu FR, Wang TH, Lin LL, Ko LS: Tear lactoferrin in keratoconjunctivitis sicca. Taiwan Yi Xue Hui Za Zhi. 1989, 88: 422-425.PubMedGoogle Scholar
- Danjo Y, Lee M, Horimoto K, Hamano T: Ocular surface damage and tear lactoferrin in dry eye syndrome. Acta Ophthalmol (Copenh). 1994, 72: 433-437.View ArticleGoogle Scholar
- Abe T, Nakajima A, Matsunaga M, Sakuragi S, Komatsu M: Decreased tear lactoferrin concentration in patients with chronic hepatitis C. Br J Ophthalmol. 1999, 83: 684-687.PubMed CentralPubMedView ArticleGoogle Scholar
- Reese V, Youngbar PR: The effect of punctal occlusion on tear lactoferrin in aqueous deficient dry eye patients. Adv Exp Med Biol. 2002, 506: 1269-1271.PubMedView ArticleGoogle Scholar
- Fujihara T, Nagano T, Nakamura M, Shirasawa E: Lactoferrin suppresses loss of corneal epithelial integrity in a rabbit short-term dry eye model. J Ocul Pharmacol Ther. 1998, 14: 99-107.PubMedView ArticleGoogle Scholar
- Solomon A, Dursun D, Liu Z, Xie Y, Macri A, Pflugfelder SC: Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci. 2001, 42: 2283-2292.PubMedGoogle Scholar
- Acera A, Rocha G, Vecino E, Lema I, Duran JA: Inflammatory markers in the tears of patients with ocular surface disease. Ophthalmic Res. 2008, 40: 315-321.PubMedView ArticleGoogle Scholar
- Chotikavanich S, De Paiva CS, De Li Q, Chen JJ, Bian F, Farley WJ, Pflugfelder SC: Production and activity of matrix metalloproteinase-9 on the ocular surface increase in dysfunctional tear syndrome. Invest Ophthalmol Vis Sci. 2009, 50: 3203-3209.PubMed CentralPubMedView ArticleGoogle Scholar
- Hadassah J, Bhuvaneshwari N, Rao U, Sehgal PK: Evaluation of succinylated collagen bandage lenses in corneal healing by the expression of matrix metalloproteinases (MMP-2 and MMP-9) in tear fluid. Ophthalmic Res. 2009, 42: 64-72.PubMedView ArticleGoogle Scholar
- Corrales RM, Stern ME, De Paiva CS, Welch J, Li DQ, Pflugfelder SC: Desiccating stress stimulates expression of matrix metalloproteinases by the corneal epithelium. Invest Ophthalmol Vis Sci. 2006, 47: 3293-3302.PubMedView ArticleGoogle Scholar
- De Paiva CS, Corrales RM, Villarreal AL, Farley WJ, Li DQ, Stern ME, Pflugfelder SC: Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye. Exp Eye Res. 2006, 83: 526-535.PubMedView ArticleGoogle Scholar
- Seo MJ, Kim JM, Lee MJ, Sohn YS, Kang KK, Yoo M: The therapeutic effect of DA-6034 on ocular inflammation via suppression of MMP-9 and inflammatory cytokines and activation of the MAPK signaling pathway in an experimental dry eye model. Curr Eye Res. 2010, 35: 165-175.PubMedView ArticleGoogle Scholar
- Nagelhout TJ, Gamache DA, Roberts L, Brady MT, Yanni JM: Preservation of tear film integrity and inhibition of corneal injury by dexamethasone in a rabbit model of lacrimal gland inflammation-induced dry eye. J Ocul Pharmacol Ther. 2005, 21: 139-148.PubMedView ArticleGoogle Scholar
- Zylberberg C, Seamon V, Ponomareva O, Vellala K, Deighan M, Azzarolo AM: Estrogen up-regulation of metalloproteinase-2 and -9 expression in rabbit lacrimal glands. Exp Eye Res. 2007, 84: 960-972.PubMedView ArticleGoogle Scholar
- Luo L, Li DQ, Doshi A, Farley W, Corrales RM, Pflugfelder SC: Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci. 2004, 45: 4293-4301.PubMedView ArticleGoogle Scholar
- Luo L, Li DQ, Corrales RM, Pflugfelder SC: Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface. Eye Contact Lens. 2005, 31: 186-193.PubMedView ArticleGoogle Scholar
- Song XJ, Li DQ, Farley W, Luo LH, Heuckeroth RO, Milbrandt J, Pflugfelder SC: Neurturin-deficient mice develop dry eye and keratoconjunctivitis sicca. Invest Ophthalmol Vis Sci. 2003, 44: 4223-4229.PubMedView ArticleGoogle Scholar
- Pflugfelder SC, Farley W, Luo L, Chen LZ, De Paiva CS, Olmos LC, Li DQ, Fini ME: Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye. Am J Pathol. 2005, 166: 61-71.PubMed CentralPubMedView ArticleGoogle Scholar
- Boukes RJ, Boonstra A, Breebaart AC, Reits D, Glasius E, Luyendyk L, Kijlstra A: Analysis of human tear protein profiles using high performance liquid chromatography (HPLC). Doc Ophthalmol. 1987, 67: 105-113.PubMedView ArticleGoogle Scholar
- Nielsen NV, Prause JU, Eriksen JS: Lysozyme, alfa-1-antitrypsin and serum albumin in tear fluid of timolol treated glaucoma patients with and without symptoms of dry eyes. Acta Ophthalmol (Copenh). 1981, 59: 503-509.View ArticleGoogle Scholar
- Caffery B, Joyce E, Boone A, Slomovic A, Simpson T, Jones L, Senchyna M: Tear lipocalin and lysozyme in Sjogren and non-Sjogren dry eye. Optom Vis Sci. 2008, 85: 661-667.PubMedView ArticleGoogle Scholar
- Zhao H, Jumblatt JE, Wood TO, Jumblatt MM: Quantification of MUC5AC protein in human tears. Cornea. 2001, 20: 873-877.PubMedView ArticleGoogle Scholar
- Caffery B, Joyce E, Heynen ML, Jones L, Ritter R, Gamache DA, Senchyna M: MUC16 expression in Sjogren’s syndrome, KCS, and control subjects. Mol Vis. 2008, 14: 2547-2555.PubMed CentralPubMedGoogle Scholar
- Berry M, Pult H, Purslow C, Murphy PJ: Mucins and ocular signs in symptomatic and asymptomatic contact lens wear. Optom Vis Sci. 2008, 85: E930-E938.PubMedView ArticleGoogle Scholar
- Gipson IK, Spurr-Michaud SJ, Senchyna M, Ritter R, Schaumberg D: Comparison of mucin levels at the ocular surface of postmenopausal women with and without a history of dry eye. Cornea. 2011, 30: 1346-1352.PubMed CentralPubMedView ArticleGoogle Scholar
- Nakamura Y, Yokoi N, Tokushige H, Kinoshita S: Sialic Acid in human tear fluid decreases in dry eye. Jpn J Ophthalmol. 2004, 48: 519-523.PubMedView ArticleGoogle Scholar
- Versura P, Bavelloni A, Grillini M, Fresina M, Campos EC: Diagnostic performance of a tear protein panel in early dry eye. Mol Vis. 2013, 19: 1247-1257.PubMed CentralPubMedGoogle Scholar
- Schoenwald RD, Vidvauns S, Wurster DE, Barfknecht CF: Tear film stability of protein extracts from dry eye patients administered a sigma agonist. J Ocul Pharmacol Ther. 1997, 13: 151-161.PubMedView ArticleGoogle Scholar
- Glasgow BJ, Gasymov OK, Abduragimov AR, Engle JJ, Casey RC: Tear lipocalin captures exogenous lipid from abnormal corneal surfaces. Invest Ophthalmol Vis Sci. 2010, 51: 1981-1987.PubMed CentralPubMedView ArticleGoogle Scholar
- Seamon V, Vellala K, Zylberberg C, Ponamareva O, Azzarolo AM: Sex hormone regulation of tear lipocalin in the rabbit lacrimal gland. Exp Eye Res. 2008, 87: 184-190.PubMedView ArticleGoogle Scholar
- Crow JM, Nelson JD, Remington SG: Human lipocalin-1 association with 3H-testosterone and 3H-estradiol. Curr Eye Res. 2009, 34: 1042-1049.PubMedView ArticleGoogle Scholar
- Yoon KC, Jeong IY, Park YG, Yang SY: Interleukin-6 and tumor necrosis factor-alpha levels in tears of patients with dry eye syndrome. Cornea. 2007, 26: 431-437.PubMedView ArticleGoogle Scholar
- Lam H, Bleiden L, De Paiva CS, Farley W, Stern ME, Pflugfelder SC: Tear cytokine profiles in dysfunctional tear syndrome. Am J Ophthalmol. 2009, 147: 198-205. e191.PubMed CentralPubMedView ArticleGoogle Scholar
- Dogru M, Ward SK, Wakamatsu T, Ibrahim O, Schnider C, Kojima T, Matsumoto Y, Ogawa J, Shimazaki J, Tsubota K: The effects of 2 week senofilcon-A silicone hydrogel contact lens daily wear on tear functions and ocular surface health status. Cont Lens Anterior Eye. 2011, 34: 77-82.PubMedView ArticleGoogle Scholar
- Corrales RM, Villarreal A, Farley W, Stern ME, Li DQ, Pflugfelder SC: Strain-related cytokine profiles on the murine ocular surface in response to desiccating stress. Cornea. 2007, 26: 579-584.PubMedGoogle Scholar
- Narayanan S, Corrales RM, Farley W, McDermott AM, Pflugfelder SC: Interleukin-1 receptor-1-deficient mice show attenuated production of ocular surface inflammatory cytokines in experimental dry eye. Cornea. 2008, 27: 811-817.PubMedView ArticleGoogle Scholar
- Cavet ME, Harrington KL, Ward KW, Zhang JZ: Mapracorat, a novel selective glucocorticoid receptor agonist, inhibits hyperosmolar-induced cytokine release and MAPK pathways in human corneal epithelial cells. Mol Vis. 2010, 16: 1791-1800.PubMed CentralPubMedGoogle Scholar
- Zywalewska-Gorna N, Mrugacz M, Bakunowicz-Lazarczyk A: The evaluation of chosen cytokines in induction of ocular changes in Sjogren’s syndrome of dry eye. Klin Oczna. 2007, 109: 435-437.PubMedGoogle Scholar
- Mrugacz M, Kaczmarski M, Bakunowicz-Lazarczyk A, Zelazowska B, Wysocka J, Minarowska A: IL-8 and IFN-gamma in tear fluid of patients with cystic fibrosis. J Interferon Cytokine Res. 2006, 26: 71-75.PubMedView ArticleGoogle Scholar
- Enriquez-de-Salamanca A, Castellanos E, Stern ME, Fernandez I, Carreno E, Garcia-Vazquez C, Herreras JM, Calonge M: Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease. Mol Vis. 2010, 16: 862-873.PubMed CentralPubMedGoogle Scholar
- Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, Wirthlin A, Pfeiffer N: SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci. 2005, 46: 863-876.PubMedView ArticleGoogle Scholar
- Roth J, Vogl T, Sorg C, Sunderkotter C: Phagocyte-specific S100 proteins: a novel group of proinflammatory molecules. Trends Immunol. 2003, 24: 155-158.PubMedView ArticleGoogle Scholar
- Qiu X, Gong L, Sun X, Guo J, Chodara AM: Efficacy of acupuncture and identification of tear protein expression changes using iTRAQ quantitative proteomics in rabbits. Curr Eye Res. 2011, 36: 886-894.PubMedView ArticleGoogle Scholar
- Ohashi Y, Ishida R, Kojima T, Goto E, Matsumoto Y, Watanabe K, Ishida N, Nakata K, Takeuchi T, Tsubota K: Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol. 2003, 136: 291-299.PubMedView ArticleGoogle Scholar
- Rao K, Farley WJ, Pflugfelder SC: Association between high tear epidermal growth factor levels and corneal subepithelial fibrosis in dry eye conditions. Invest Ophthalmol Vis Sci. 2010, 51: 844-849.PubMed CentralPubMedView ArticleGoogle Scholar
- Xiao X, He H, Lin Z, Luo P, Zhou T, Zhou Y, Liu Z: Therapeutic effects of epidermal growth factor on benzalkonium chloride-induced dry eye in a mouse model. Invest Ophthalmol Vis Sci. 2012, 53: 191-197.PubMedView ArticleGoogle Scholar
- Lambiase A, Micera A, Sacchetti M, Cortes M, Mantelli F, Bonini S: Alterations of tear neuromediators in dry eye disease. Arch Ophthalmol. 2011, 129: 981-986.PubMedView ArticleGoogle Scholar
- Liu Q, McDermott AM, Miller WL: Elevated nerve growth factor in dry eye associated with established contact lens wear. Eye Contact Lens. 2009, 35: 232-237.PubMedView ArticleGoogle Scholar
- Lee HK, Ryu IH, Seo KY, Hong S, Kim HC, Kim EK: Topical 0.1% prednisolone lowers nerve growth factor expression in keratoconjunctivitis sicca patients. Ophthalmology. 2006, 113: 198-205.PubMedView ArticleGoogle Scholar
- Lee HK, Lee KS, Kim HC, Lee SH, Kim EK: Nerve growth factor concentration and implications in photorefractive keratectomy vs laser in situ keratomileusis. Am J Ophthalmol. 2005, 139: 965-971.PubMedView ArticleGoogle Scholar
- Lambiase A, Micera A, Pellegrini G, Merlo D, Rama P, De Luca M, Bonini S: In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression. Invest Ophthalmol Vis Sci. 2009, 50: 4622-4630.PubMedView ArticleGoogle Scholar
- Jain P, Li R, Lama T, Saragovi HU, Cumberlidge G, Meerovitch K: An NGF mimetic, MIM-D3, stimulates conjunctival cell glycoconjugate secretion and demonstrates therapeutic efficacy in a rat model of dry eye. Exp Eye Res. 2011, 93: 503-512.PubMedView ArticleGoogle Scholar
- Batellier L, Rea A, Chaumeil C, Scat Y: Protein evaluation of tears: different biological parameters and their respective value. J Fr Ophtalmol. 1996, 19: 520-526.PubMedGoogle Scholar
- Mackie IA, Seal DV: Diagnostic implications of tear protein profiles. Br J Ophthalmol. 1984, 68: 321-324.PubMed CentralPubMedView ArticleGoogle Scholar
- Jacob JT, Ham B: Compositional profiling and biomarker identification of the tear film. Ocul Surf. 2008, 6: 175-185.PubMedView ArticleGoogle Scholar
- Wollensak G, Mur E, Mayr A, Baier G, Gottinger W, Stoffler G: Effective methods for the investigation of human tear film proteins and lipids. Graefes Arch Clin Exp Ophthalmol. 1990, 228: 78-82.PubMedView ArticleGoogle Scholar
- Grus FH, Augustin AJ: High performance liquid chromatography analysis of tear protein patterns in diabetic and non-diabetic dry-eye patients. Eur J Ophthalmol. 2001, 11: 19-24.PubMedGoogle Scholar
- Nichols JJ, Green-Church KB: Mass spectrometry-based proteomic analyses in contact lens-related dry eye. Cornea. 2009, 28: 1109-1117.PubMedView ArticleGoogle Scholar
- Cappadona S, Nanni P, Benevento M, Levander F, Versura P, Roda A, Cerutti S, Pattini L: Improved label-free LC-MS analysis by wavelet-based noise rejection. J Biomed Biotechnol. 2010, 2010: 131505-PubMed CentralPubMedView ArticleGoogle Scholar
- Grus FH, Sabuncuo P, Augustin AJ: Analysis of tear protein patterns of dry-eye patients using fluorescent staining dyes and two-dimensional quantification algorithms. Electrophoresis. 2001, 22: 1845-1850.PubMedView ArticleGoogle Scholar
- Herber S, Grus FH, Sabuncuo P, Augustin AJ: Two-dimensional analysis of tear protein patterns of diabetic patients. Electrophoresis. 2001, 22: 1838-1844.PubMedView ArticleGoogle Scholar
- Reitz C, Breipohl W, Augustin A, Bours J: Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C. Graefes Arch Clin Exp Ophthalmol. 1998, 236: 894-899.PubMedView ArticleGoogle Scholar
- Li S, Sack R, Vijmasi T, Sathe S, Beaton A, Quigley D, Gallup M, McNamara NA: Antibody protein array analysis of the tear film cytokines. Optom Vis Sci. 2008, 85: 653-660.PubMed CentralPubMedView ArticleGoogle Scholar
- Grus FH, Dick B, Augustin AJ, Pfeiffer N: Analysis of the antibody repertoire in tears of dry-eye patients. Ophthalmologica. 2001, 215: 430-434.PubMedView ArticleGoogle Scholar
- Schmut O, Horwath-Winter J, Zenker A, Trummer G: The effect of sample treatment on separation profiles of tear fluid proteins: qualitative and semi-quantitative protein determination by an automated analysis system. Graefes Arch Clin Exp Ophthalmol. 2002, 240: 900-905.PubMedView ArticleGoogle Scholar
- Carreno E, Enriquez-de-Salamanca A, Teson M, Garcia-Vazquez C, Stern ME, Whitcup SM, Calonge M: Cytokine and chemokine levels in tears from healthy subjects. Acta Ophthalmol. 2010, 88: e250-e258.PubMedView ArticleGoogle Scholar
- Saijyothi AV, Angayarkanni N, Syama C, Utpal T, Shweta A, Bhaskar S, Geetha IK, Vinay PS, Thennarasu M, Sivakumar RM, Prema P: Two dimensional electrophoretic analysis of human tears: collection method in dry eye syndrome. Electrophoresis. 2010, 31: 3420-3427.PubMedView ArticleGoogle Scholar
- Lopez-Cisternas J, Castillo-Diaz J, Traipe-Castro L, Lopez-Solis RO: Use of polyurethane minisponges to collect human tear fluid. Cornea. 2006, 25: 312-318.PubMedView ArticleGoogle Scholar
- VanDerMeid KR, Su SP, Krenzer KL, Ward KW, Zhang JZ: A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex. Mol Vis. 2011, 17: 1056-1063.PubMed CentralPubMedGoogle Scholar
- Ronen D, Eylan E, Romano A, Stein R, Modan M: A spectrophotometric method for quantitative determination of lysozyme in human tears: description and evaluation of the method and screening of 60 healthy subjects. Invest Ophthalmol. 1975, 14: 479-484.PubMedGoogle Scholar
- Mackie IA, Seal DV: Confirmatory tests for the dry eye of Sjogren’s syndrome. Scand J Rheumatol Suppl. 1986, 61: 220-223.PubMedGoogle Scholar
- Mackie IA, Seal DV, Pescod JM: Beta-adrenergic receptor blocking drugs: tear lysozyme and immunological screening for adverse reaction. Br J Ophthalmol. 1977, 61: 354-359.PubMed CentralPubMedView ArticleGoogle Scholar
- Sun Z, Hong J, Liu Z, Jin X, Gu C: Coal dust contiguity-induced changes in the concentration of TNF-alpha and NF-kappa B p65 on the ocular surface. Ocul Immunol Inflamm. 2009, 17: 76-82.PubMedView ArticleGoogle Scholar
- Gonzalez-Chavez SA, Arevalo-Gallegos S, Rascon-Cruz Q: Lactoferrin: structure, function and applications. Int J Antimicrob Agents. 2009, 33 (301): e301-e308.Google Scholar
- Versura P, Nanni P, Bavelloni A, Blalock WL, Piazzi M, Roda A, Campos EC: Tear proteomics in evaporative dry eye disease. Eye (Lond). 2010, 24: 1396-1402.View ArticleGoogle Scholar
- Yolton DP, Mende S, Harper A, Softing A: Association of dry eye signs and symptoms with tear lactoferrin concentration. J Am Optom Assoc. 1991, 62: 217-223.PubMedGoogle Scholar
- Marenholz I, Heizmann CW, Fritz G: S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochem Biophys Res Commun. 2004, 322: 1111-1122.PubMedView ArticleGoogle Scholar
- Spurr-Michaud S, Argueso P, Gipson I: Assay of mucins in human tear fluid. Exp Eye Res. 2007, 84: 939-950.PubMed CentralPubMedView ArticleGoogle Scholar
- Argueso P, Tisdale A, Spurr-Michaud S, Sumiyoshi M, Gipson IK: Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye. Invest Ophthalmol Vis Sci. 2006, 47: 113-119.PubMed CentralPubMedView ArticleGoogle Scholar
- Setala NL, Holopainen JM, Metso J, Yohannes G, Hiidenhovi J, Andersson LC, Eriksson O, Robciuc A, Jauhiainen M: Interaction of phospholipid transfer protein with human tear fluid mucins. J Lipid Res. 2010, 51: 3126-3134.PubMed CentralPubMedView ArticleGoogle Scholar
- De Souza GA, Godoy LM, Mann M: Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol. 2006, 7: R72-PubMed CentralPubMedView ArticleGoogle Scholar
- Van Lint P, Libert C: Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation. J Leukoc Biol. 2007, 82: 1375-1381.PubMedView ArticleGoogle Scholar
- Kaufman HE: The practical detection of MMP-9 diagnoses ocular surface disease and may help prevent its complications. Cornea. 2013, 32: 211-216.PubMedView ArticleGoogle Scholar
- Dartt DA: Tear lipocalin: structure and function. Ocul Surf. 2011, 9: 126-138.PubMed CentralPubMedView ArticleGoogle Scholar
- Flower DR: The lipocalin protein family: structure and function. Biochem J. 1996, 318 (Pt 1): 1-14.PubMed CentralPubMedView ArticleGoogle Scholar
- Glasson M, Stapleton F, Willcox M: Lipid, lipase and lipocalin differences between tolerant and intolerant contact lens wearers. Curr Eye Res. 2002, 25: 227-235.PubMedView ArticleGoogle Scholar
- Ben Menachem-Zidon O, Avital A, Ben-Menahem Y, Goshen I, Kreisel T, Shmueli EM, Segal M, Ben Hur T, Yirmiya R: Astrocytes support hippocampal-dependent memory and long-term potentiation via interleukin-1 signaling. Brain Behav Immun. 2011, 25: 1008-1016.PubMedView ArticleGoogle Scholar
- Wei Y, Gadaria-Rathod N, Epstein S, Asbell P: Tear cytokine profile as a noninvasive biomarker of inflammation for ocular surface diseases: standard operating procedures. Invest Ophthalmol Vis Sci. 2013, 54: 8327-8336.PubMed CentralPubMedView ArticleGoogle Scholar
- Boehm N, Riechardt AI, Wiegand M, Pfeiffer N, Grus FH: Proinflammatory cytokine profiling of tears from dry eye patients by means of antibody microarrays. Invest Ophthalmol Vis Sci. 2011, 52: 7725-7730.PubMedView ArticleGoogle Scholar
- Zoukhri D, Macari E, Kublin CL: A single injection of interleukin-1 induces reversible aqueous-tear deficiency, lacrimal gland inflammation, and acinar and ductal cell proliferation. Exp Eye Res. 2007, 84: 894-904.PubMed CentralPubMedView ArticleGoogle Scholar
- Xiao W, Wu Y, Zhang J, Ye W, Xu GT: Selecting highly sensitive non-obese diabetic mice for improving the study of Sjogren’s syndrome. Graefes Arch Clin Exp Ophthalmol. 2009, 247: 59-66.PubMedView ArticleGoogle Scholar
- Polanska V, Sery O, Fojtik Z, Hlinomazova Z: The presence of dry eye syndrome and corneal complications in patients with rheumatoid arthritis and its association with -174 gene polymorphism for interleukin 6. Cesk Slov Oftalmol. 2008, 64: 77-80.PubMedGoogle Scholar
- Kang MH, Kim MK, Lee HJ, Lee HI, Wee WR, Lee JH: Interleukin-17 in various ocular surface inflammatory diseases. J Korean Med Sci. 2011, 26: 938-944.PubMed CentralPubMedView ArticleGoogle Scholar
- Oh JY, Kim MK, Choi HJ, Ko JH, Kang EJ, Lee HJ, Wee WR, Lee JH: Investigating the relationship between serum interleukin-17 levels and systemic immune-mediated disease in patients with dry eye syndrome. Korean J Ophthalmol. 2011, 25: 73-76.PubMed CentralPubMedView ArticleGoogle Scholar
- Albertsmeyer AC, Kakkassery V, Spurr-Michaud S, Beeks O, Gipson IK: Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells. Exp Eye Res. 2010, 90: 444-451.PubMed CentralPubMedView ArticleGoogle Scholar
- Trousdale MD, Zhu Z, Stevenson D, Schechter JE, Ritter T, Mircheff AK: Expression of TNF inhibitor gene in the lacrimal gland promotes recovery of tear production and tear stability and reduced immunopathology in rabbits with induced autoimmune dacryoadenitis. J Autoimmune Dis. 2005, 2: 6-PubMed CentralPubMedView ArticleGoogle Scholar
- Siemasko KF, Gao J, Calder VL, Hanna R, Calonge M, Pflugfelder SC, Niederkorn JY, Stern ME: In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation. Invest Ophthalmol Vis Sci. 2008, 49: 5434-5440.PubMedView ArticleGoogle Scholar
- Mrugacz M, Zelazowska B, Bakunowicz-Lazarczyk A, Wysocka J, Minarowska A: IFN-gamma in tear fluid in patients with cystic fibrosis. Klin Oczna. 2005, 107: 287-288.PubMedGoogle Scholar
- De Paiva CS, Villarreal AL, Corrales RM, Rahman HT, Chang VY, Farley WJ, Stern ME, Niederkorn JY, Li DQ, Pflugfelder SC: Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-gamma. Invest Ophthalmol Vis Sci. 2007, 48: 2553-2560.PubMedView ArticleGoogle Scholar
- Mrugacz M: CCL4/MIP-1beta levels in tear fluid and serum of patients with cystic fibrosis. J Interferon Cytokine Res. 2010, 30: 509-512.PubMedView ArticleGoogle Scholar
- Malesinski R, Bakunowicz-Lazarczyk A, Wysocka J: The role of chemokines CCL3/ MIP-1 alfa and CCL4/ MIP-1 beta in pathogenesis of dry eye syndrome. Klin Oczna. 2008, 110: 277-279.PubMedGoogle Scholar
- Mrugacz M, Zelazowska B, Bakunowicz-Lazarczyk A, Kaczmarski M, Wysocka J: Elevated tear fluid levels of MIP-1alpha in patients with cystic fibrosis. J Interferon Cytokine Res. 2007, 27: 491-495.PubMedView ArticleGoogle Scholar
- Yoon KC, Park CS, You IC, Choi HJ, Lee KH, Im SK, Park HY, Pflugfelder SC: Expression of CXCL9, -10, -11, and CXCR3 in the tear film and ocular surface of patients with dry eye syndrome. Invest Ophthalmol Vis Sci. 2010, 51: 643-650.PubMed CentralPubMedView ArticleGoogle Scholar
- Klenkler B, Sheardown H, Jones L: Growth factors in the tear film: role in tissue maintenance, wound healing, and ocular pathology. Ocul Surf. 2007, 5: 228-239.PubMedView ArticleGoogle Scholar
- Varnell RJ, Freeman JY, Maitchouk D, Beuerman RW, Gebhardt BM: Detection of substance P in human tears by laser desorption mass spectrometry and immunoassay. Curr Eye Res. 1997, 16: 960-963.PubMedView ArticleGoogle Scholar
- Herbst RS: Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys. 2004, 59: 21-26.PubMedView ArticleGoogle Scholar
- Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, Doetschman T: Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature. 1992, 359: 693-699.PubMed CentralPubMedView ArticleGoogle Scholar
- Zheng X, De Paiva CS, Rao K, Li DQ, Farley WJ, Stern M, Pflugfelder SC: Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay. Cornea. 2010, 29: 1048-1054.PubMed CentralPubMedView ArticleGoogle Scholar
- Petsoglou C, Balaggan KS, Dart JK, Bunce C, Xing W, Ali RR, Tuft SJ: Subconjunctival bevacizumab induces regression of corneal neovascularisation: a pilot randomised placebo-controlled double-masked trial. Br J Ophthalmol. 2013, 97: 28-32.PubMedView ArticleGoogle Scholar
- Kut C, Mac Gabhann F, Popel AS: Where is VEGF in the body? A meta-analysis of VEGF distribution in cancer. Br J Cancer. 2007, 97: 978-985.PubMed CentralPubMedView ArticleGoogle Scholar
- Kari O, Aho VV, Peltonen S, Saari JM, Kari M, Maatta M, Collan Y, Saari KM: Group IIA phospholipase A(2) concentration of tears in patients with ocular rosacea. Acta Ophthalmol Scand. 2005, 83: 483-486.PubMedView ArticleGoogle Scholar
- Still KM, Soyars CL, Velez F, Bower KS, Sia RK, Ryan DS, Seifert K, Laurie GW, McKown RL: Development of quantitative sandwich ELISAs for lacritin and the lacritin-c splice variant in human tears. Invest Ophthalmol Vis Sci. 2012, 53 (ARVO E-Abstract): 4233-Google Scholar
- Yavuz S, Asfuroglu E, Bicakcigil M, Toker E: Hydroxychloroquine improves dry eye symptoms of patients with primary Sjogren’s syndrome. Rheumatol Int. 2011, 31: 1045-1049.PubMedView ArticleGoogle Scholar
- Toker E, Yavuz S, Direskeneli H: Anti-Ro/SSA and anti-La/SSB autoantibodies in the tear fluid of patients with Sjogren’s syndrome. Br J Ophthalmol. 2004, 88: 384-387.PubMed CentralPubMedView ArticleGoogle Scholar
- Mrugacz M, Zelazowska-Rutkowska B, Bakunowicz-Lazarczyk A, Wysocka J, Kaczmarski M: The role of apoptosis in induction ocular changes in patients with cystic fibrosis. Klin Oczna. 2007, 109: 22-24.PubMedGoogle Scholar
- Ng V, Cho P, To C: Tear proteins of normal young Hong Kong Chinese. Graefes Arch Clin Exp Ophthalmol. 2000, 238: 738-745.PubMedView ArticleGoogle Scholar
- Sariri R, Varasteh A, Sajedi RH: Effect of Ramadan fasting on tear proteins. Acta Med (Hradec Kralove). 2010, 53: 147-151.Google Scholar
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