May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Relative Quantitation of Major Basic Protein Species Found in the Human Tear Film
Author Affiliations & Notes
  • M. Senchyna
    Centre for Contact Lens Research,
    University of Waterloo School of Optometry, Waterloo, ON, Canada
  • E. Heikkila
    University of Waterloo School of Optometry, Waterloo, ON, Canada
  • J. Vehige
    Allergan Eye Care R&D, Irvine, CA
  • P. Simmons
    Allergan Eye Care R&D, Irvine, CA
  • L. Jones
    Centre for Contact Lens Research,
    University of Waterloo School of Optometry, Waterloo, ON, Canada
  • M. Bornstein
    University of Waterloo School of Optometry, Waterloo, ON, Canada
  • Footnotes
    Commercial Relationships  M. Senchyna, Allergan Eye Care R&D, Irvine, CA F; E. Heikkila, None; J. Vehige, Allergan Eye Care R&D, Irvine, CA E; P. Simmons, Allergan Eye Care R&D, Irvine, CA E; L. Jones, Allergan Eye Care R&D, Irvine, CA F; M. Bornstein, None.
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 4831. doi:
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      M. Senchyna, E. Heikkila, J. Vehige, P. Simmons, L. Jones, M. Bornstein; Relative Quantitation of Major Basic Protein Species Found in the Human Tear Film . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4831.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: The release of cationic proteins from mast cells and eosinophils plays a significant role in the pathophysiology of ocular allergies and potentially other chronic inflammatory diseases such as dry eye. Among the most common cationic proteins is Major Basic Protein (MBP), which exists in several forms, the two most common being the 13.8 kDa mature form (M–MBP) and the 25 kDa proform (proMBP). M–MBP is a known cellular toxin and possess proinflammatory activity where as proMBP is thought to mask these effects. Preliminary work in our laboratory suggested that both M–MBP and proMBP are expressed in the human tear film. The purpose of this work was to optimize a reliable and accurate method for the relative quantification of both M–MBP and proMBP in human tear film samples so as to gain insight into expression and potential clinical significance. Methods: Non–stimulated tear samples were collected using 5–µl microcapillary tubes from six normal female subjects (ages 28–35) on three separate occasions, spaced at two week intervals. Total protein in each tear sample was determined using a MicroBCA Assay (Pierce Biochemicals). SDS–PAGE and Western blotting was performed on a PhastSystemTM (Pharmacia), using 1.5 µg of tear protein loaded per lane on 10–15% gradient gels. MBP species were identified using a mouse anti–human MBP polyclonal antibody (Cymby Biotechnology) and peroxidase conjugated goat anti–mouse secondary antibody (Sigma–Aldrich). On each blot, 1.5 µg of a standard pool of tears was applied to serve as a semi–quantitative calibration standard. Immunoreactivity was visualized by incubating with ECL Plus chemiluminescent substrate (Pharmacia). Optical densities of the resulting bands were quantified from digitized images created with a Molecular® Dynamics StormTM 840 Imager using ImageQuantTM 5.1 software. Results: M–MBP and proMBP were identified in all tear film samples analyzed (n=18). The relative amount of M–MBP and proMBP did not vary significantly between collection times (p=0.21), and the ratio of M–MBP to proMBP also did not vary over time (p=0.34). Conclusion:In this pilot study, a semi–quantitative method demonstrated that both M–MBP and proMBP were ubiquitously expressed in the human tear film of normal subjects. These data suggest that M–MBP and/or proMBP may have a role in the ocular surface physiology of normal individuals, and warrant further investigation with more quantitative methods.

Keywords: cornea: tears/tear film/dry eye 
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