News

    DRY-EYE and OCULAR-SURFACE DISEASES

     

    Prof. Dr. Dr. F. Grus PhD MD
    Dr. N. Perumal, PhD
    Sichang Gu, Master of Ophthalmology

     

    Biochemical analysis of tear fluid for the non-invasive follow-up, diagnosis, and search for disease-associated biomarkers

    The dry-eye syndrome is a very common disease in ophthalmology. In the last years the knowledge about the pathogenesis of the disease could be improved. However, the development of new potential treatments are hampered by the fact that there are no objective criteria available to prove the success or failure of the treatment in an objective manner. Tear fluid is easy to obtain and thus we raised the question if it could be possible to use it for non-invasive diagnosis and follow-up. In several previous studies we investigated the tear protein patterns by means of electrophoretical separations and subsequent multivariate statistics. The analysis of tear proteins is furthermore promising, because in the last year many proteomics approaches could demonstrate pathogenic proteins. The search for pathogenic proteins might be useful, because even changes in m-RNA-expressions are only weakly correlated (r<0.3) with the proteins which can be found in the cell and which exert their pathologic functions. Using our technologies, we could reveal differences in the tear protein patterns of patients suffering from dry-eye and healthy volunteers. Those differences in complex profiles can be assessed quantitatively using our algorithms. Further studies could show that it is possible to quantify if a pathologic tear protein profile will become more similar during a treatment to those patterns of healthy subjects. This result would be expected to see if a causative treatment really works. In dry-eye disease our approach could close an important gap to measure the success of treatments not only in terms of some unreliable subjective symptoms that rely widely on the compliance of patients but in a more objective manner. Furthermore, the search for disease-associated biomarkers in tear fluid can help to understand more about the pathogenesis of diseases. In recent studies we could in cooperation with Proteognostics (Switzerland) establish a very fast and easy to use approach for the evaluation of tear proteins: the use of proteinchips based on mass-spectrometry (s. News Section). This method has a very high mass accuracy and sensitivity in comparison to electrophoretical techniques and allows for a very fast and reproducible analysis. The result of such a complex protein tear profiling can be obtained within 1-2 hours. The next challenge could be the setup of test-strips, where antibodies against those biomarkers recognize the proteins and can be easily detected.

    To learn more about the pathogenic importance of the changes levels of tear protein expressions we find in our studies, we performed a two-dimensional liquid chromatography combined with tandem mass spectrometry (MS-MS). Using this technique, we were able to successfully identify more than 500 proteins in tear fluid. Studies to investigate these proteins are still ongoing.

    Dry-Eye Syndrome:

    In dry-eye disease, we could demonstrate in a study with more than 600 patients that there are significantly different changes in the tear protein pattern of patients suffering from sicca-syndrome and healthy subjects. Furthermore, the analysis could show which molecular regions are the most different between all clinical groups. By means of artificial neural networks we could use successfully the tear protein patterns for diagnosis and follow-up of the dry-eye disease. The quantitative changes in tear profiles correlate signicantly better with the subjective sympomts of patients (as a measure of the severity of the disease) than the Schirmer’s test (BST basal secretory test) [18, 16, 15, 12, 10, 4].

    Diabetes mellitus and diabetic retinopathy:

    Is is well known that diabetic patients develop a dry-eye syndrome more frequently compared to otherwise healthy subjects. That’s why we decided to analyze the tears of diabetic patients which are additionally suffering from dry-eye syndrome. We found very specific changes in the protein patterns that could only be found in diabetic tears. These changes in protein expressions correlate significantly with the duration and the severity of the diabetic retinopathy and the diabetes itself. In diabetic retinopathy more prognostic markers are urgently needed. Current studies have to show, if the proteins found can be used as prognostic and diagnostic biomarkers of the disease and if those biomarkers can be found maybe prior to the manifestation of the disease. First results of this study are very promising. The specific proteins found in previous studies can not be explained solely by glycosylation. Figure 1 demonstrate a group map of a two-dimensional electrophoretical separation of tear fluids of diabetic patients (blue) and healthy subjects (red). The dramatic increase in protein spots of diabetic patients can easily be detected in some specific regions [13, 5, 7].

    The proteins promise to be specific for this disease and can be reproducible detected.

    Autoimmune components in dry-eye disease:

    In the last years it is widely discussed that the dry-eye disease has some underlying immunologic and autoimmune components. Based on those topics a new treatment using topical applied cyclosporine A could be established. However, if there is an underlying autoimmune component in dry-eye disease, some autoantibodies might be detectable in tear fluid. Unfortunately, the analysis of autoantibodies is hampered by the fact that complex autoantibody repertoires can also be found in healthy subjects. In international cooperations we could establish an approach to isolate those antibody-antigen reactions in the sera of patients from the immunologic noise that could be disease associated. Using this approach, we could demonstrate the existence and different patterns of autoantibodies in Tourette syndrome, sydenham chorea, endocrine orbitopathy, and myopathies [22, 20, 21, 19, 17, 8, 14, 2, 3]. It was the aim of this study to apply this approach to the analysis of autoantibodies in the tear fluid of patients suffering from dry-eye syndrome. In this study, we could show a very different pattern of autoantibodies against ocular antigens in the tear fluid of patients suffering from dry-eye syndrome. Again, the non-invasive analysis of autoantibodies and proteins in tears could find potential hints for underlying autoimmune processes in dry-eye syndrome. T Figure 2 demonstrates the intensity of antigen-autoantibody-reactions (y-axis, IgA-antibodies) against ocular antigens for each molecular weight region (x-axis) in the tear fluid of dry-eye patients (DRY) and healthy controls (CTRL).

    LASIK and PRK:

    After refractive surgery most of patients will develop a severe dry-eye syndrome for an average of 6 months. It is discussed that this dry-eye syndrome develops due to an neurotrophic cornea. However, this might not be the only reason for dry-eye following LASIK or PRK. In this paper, we wanted to investigate, if patients will develop changes in their tear fluid protein pattern following LASIK or PRK, as we know that from previous studies with “idiopathic” dry-eye patients. Therefore, we analyzed tear fluid from patients undergoing refractive surgery (LASIK or PRK in different studies) just before surgery, 4 days, 2 weeks, and 4 weeks following the surgical procedure. The tear proteins were separated using electrophoretical techniques and were subsequently analyzed by means of multivariate procedures according to our standard protocols. The LASIK study was performed in our department. The PRK study was done in cooperation with the university of Budapest, Hungary. We could demonstrate that the surgery is followed by specific changes in the tear fluid patterns. There is an increase of tear fluid protein spots that increases furthermore with the time after the surgery. In parallel, just shortly after the surgery we found in both PRK and LASIK a strong decrease in the main proteins just like lysozyme and lipocalin which was not observed in any of our preceeding studies with idiopathic dry-eye syndrome. In most cases, the protein concentrations of these main proteins will recover to normal values within the next weeks. Further studies have to show, what will happen to these changes following longer time periods after surgery. However, the results demonstrate that the changes in the environment of the ocular surface following LASIK or PRK are much more complex as they could be explained only by a consequence of a disruption of corneal innervation due to the LASIK procedure. Figure 3 shows the number of protein spots before surgery (A), 4 days, 2 weeks, and 4 weeks following the procedure.

    ProteinChips in the analysis of tear film.

    The analysis of tear film proteins by means of electrophoresis and the subsequent multivariate profiling is a very complex and time consuming process. Therefore, we attempted to use proteinchip techniques that are based on mass spectrometry. We could show for the first time that it is possible to analyze tear proteins using proteinchip approaches. Figure 4 shows such a proteinchip setup and some protein profiles . The advantages of this method are a very high sensitivity, the small amount of sample (1-3 µl), and the speed of the procedure (results in 1-2 h), which is very helpful in screening experiments. Our results from previous studies using electrophoretical techniques, which could show significant changes in the tear protein patterns of dry-eye patients, could be confirmed using proteinchips. In comparison to electrophoresis, another big advantage in using those proteinchips is that one can mostly identify the biomarkers by onchip procedures. In our pilotstudy, the cystatin S expression could be identified to be significantly altered in dry-eye patients.

     

    Publications

    • Boerger M, Funke S, Leha A, Roser AE, Wuestemann AK, Maass F, Bahr M, Grus F, Lingor P (2019) Proteomic analysis of tear fluid reveals disease-specific patterns in patients with Parkinson's disease - A pilot study. Parkinsonism Relat Disord 63:3-9.
    • Manicam C, Perumal N, Wasielica-Poslednik J, Ngongkole YC, Tschabunin A, Sievers M, Lisch W, Pfeiffer N, Grus FH, Gericke A (2018) Proteomics Unravels the Regulatory Mechanisms in Human Tears Following Acute Renouncement of Contact Lens Use: A Comparison between Hard and Soft Lenses. Sci Rep 8:11526.
    • Perumal N, Manicam C, Steinicke M, Funke S, Pfeiffer N, Grus FH (2017) Characterization of the human aqueous humour proteome: A comparison of the genders. PLoS One 12:e0172481.
    • Sullivan DA, Rocha EM, Aragona P, Clayton JA, Ding J, Golebiowski B, Hampel U, McDermott AM, Schaumberg DA, Srinivasan S, Versura P, Willcox MDP (2017) TFOS DEWS II Sex, Gender, and Hormones Report. Ocul Surf 15:284-333.
    • Funke S, Beck S, Lorenz K, Kotterer M, Wolters D, Perumal N, Pfeiffer N, Grus FH (2016) Analysis of the effects of preservative-free tafluprost on the tear proteome. Am J Transl Res 8:4025-4039.
    • Perumal N, Funke S, Pfeiffer N, Grus FH (2016) Proteomics analysis of human tears from aqueous-deficient and evaporative dry eye patients. Sci Rep 6:29629.
    • Perumal N, Funke S, Wolters D, Pfeiffer N, Grus FH (2015) Characterization of human reflex tear proteome reveals high expression of lacrimal proline-rich protein 4 (PRR4). Proteomics 15:3370-3381.
    • Perumal N, Funke S, Pfeiffer N, Grus FH (2014) Characterization of lacrimal proline-rich protein 4 (PRR4) in human tear proteome. Proteomics 14:1698-1709.
    • Boehm N, Funke S, Wiegand M, Wehrwein N, Pfeiffer N, Grus FH (2013) Alterations in the tear proteome of dry eye patients--a matter of the clinical phenotype. Invest Ophthalmol Vis Sci 54:2385-2392.
    • Bell K, Buksinska E, Pfeiffer N, Grus FH (2012) Comparison of the effects of different lens-cleaning solutions on the protein profiles of human conjunctival cells. Graefes Arch Clin Exp Ophthalmol 250:1627-1636.
    • Bohm D, Keller K, Pieter J, Boehm N, Wolters D, Siggelkow W, Lebrecht A, Schmidt M, Kolbl H, Pfeiffer N, Grus FH (2012) Comparison of tear protein levels in breast cancer patients and healthy controls using a de novo proteomic approach. Oncol Rep 28:429-438.
    • Funke S, Azimi D, Wolters D, Grus FH, Pfeiffer N (2012) Longitudinal analysis of taurine induced effects on the tear proteome of contact lens wearers and dry eye patients using a RP-RP-Capillary-HPLC-MALDI TOF/TOF MS approach. J Proteomics 75:3177-3190.
    • Boehm N, Riechardt AI, Wiegand M, Pfeiffer N, Grus FH (2011) Proinflammatory cytokine profiling of tears from dry eye patients by means of antibody microarrays. Invest Ophthalmol Vis Sci 52:7725-7730.
    • Grus FH, Joachim SC, Pfeiffer N (2007) Proteomics in ocular fluids. Proteomics Clin Appl 1:876-888.
    • Grus FH, Kramann C, Bozkurt N, Wiegel N, Bruns K, Lackner N, Pfeiffer N (2005a) Effects of multipurpose contact lens solutions on the protein composition of the tear film. Cont Lens Anterior Eye 28:103-112.
    • Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, Wirthlin A, Pfeiffer N (2005b) SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci 46:863-876.
    • Grus FH, Sabuncuo P, Augustin A, Pfeiffer N (2002) Effect of smoking on tear proteins. Graefes Arch Clin Exp Ophthalmol 240:889-892.

     

    References

    1. Fust A, Veres A, Kiszel P, Nagy ZZ, Cervenak L, Csakany B, Maka E, Suveges I, Grus FH: Changes in tear protein pattern after photorefractive keratectomy. Eur J Ophthalmol 2003, 13(6):525-531.
    2. Grus FH, Joachim SC, Pfeiffer N: Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry. Proteomics 2003, 3(6):957-961.
    3. Singer HS, Loiselle CR, Lee O, Garvey MA, Grus FH: Anti-basal ganglia antibody abnormalities in Sydenham chorea. J Neuroimmunol 2003, 136(1-2):154-161.
    4. Grus FH, Sabuncuo P, Herber S, Augustin AJ: Analysis of tear protein patterns for the diagnosis of dry eye. Adv Exp Med Biol 2002, 506(Pt B):1213-1216.
    5. Grus FH, Sabuncuo P, Dick HB, Augustin AJ, Pfeiffer N: Changes in the tear proteins of diabetic patients. BMC Ophthalmol 2002, 2(1):4.
    6. Grus FH, Sabuncuo P, Augustin A, Pfeiffer N: Effect of smoking on tear proteins. Graefes Arch Clin Exp Ophthalmol 2002, 240(11):889-892.
    7. Herber S, Grus FH, Sabuncuo P, Augustin AJ: Changes in the tear protein patterns of diabetic patients using two-dimensional electrophoresis. Adv Exp Med Biol 2002, 506(Pt A):623-626.
    8. Dziewas R, Kis B, Grus FH, Zimmermann CW: Antibody pattern analysis in the Guillain-Barre syndrome and pathologic controls. J Neuroimmunol 2001, 119(2):287-296.
    9. Grus FH, Dick B, Augustin AJ, Pfeiffer N: Analysis of the antibody repertoire in tears of dry-eye patients. Ophthalmologica 2001, 215(6):430-434.
    10. 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(9):1845-1850.
    11. Grus FH, Sabuncuo P, Augustin AJ: [Quantitative analysis of tear protein profile for soft contact lenses--a clinical study]. Klin Monatsbl Augenheilkd 2001, 218(4):239-242.
    12. 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(1):19-24.
    13. Herber S, Grus FH, Sabuncuo P, Augustin AJ: Two-dimensional analysis of tear protein patterns of diabetic patients. Electrophoresis 2001, 22(9):1838-1844.
    14. Wendlandt JT, Grus FH, Hansen BH, Singer HS: Striatal antibodies in children with Tourette's syndrome: multivariate discriminant analysis of IgG repertoires. J Neuroimmunol 2001, 119(1):106-113.
    15. Grus FH, Augustin AJ: [Protein analysis methods in diagnosis of sicca syndrome]. Ophthalmologe 2000, 97(1):54-61.
    16. 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(4-5):875-880.
    17. Grus FH, Augustin AJ, Toth-Sagi K: Diagnostic classification of autoantibody repertoires in endocrine ophthalmopathy using an artificial neural network. Ocul Immunol Inflamm 1998, 6(1):43-50.
    18. 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(2):90-97.
    19. Grus FH, Augustin AJ: Analysis of the IgG autoantibody repertoire in endocrine ophthalmopathy using the MegaBlot technique. Curr Eye Res 1998, 17(6):636-641.
    20. Grus F, Augustin A, Toth-Sagi K, Koch F: Detection and Evaluation of the IgG Autoantibody Repertoire against Human skeletal-muscle antigens in Graves' Disease. Advances in Therapy 1997, 14(1):8-20.
    21. Grus FH, Zimmermann CW: Identification and classification of autoantibody repertoires (Western blots) with a pattern recognition algorithm by an artificial neural network. Electrophoresis 1997, 18(7):1120-1125.
    22. Zimmermann CW, Grus FH, Dux R: Multivariate statistical comparison of autoantibody-repertoires (western blots) by discriminant analysis. Electrophoresis 1995, 16(6):941-947.