Senior Honors Projects, 2010-current

Date of Award

Spring 2014

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Integrated Science and Technology

Advisor(s)

Robert L. McKown

Kyle Seifert

Ron Raab

Abstract

Purpose: Human breast milk and tears share a number of common proteins including lysozyme, lactoferrin, albumin, secretory immunoglobulins, and mucins. Human breast milk has been used as self-medication for conjunctivitis and has been recently reported to improve corneal healing in an animal model system. Lacritin is prosecretory, mitogenic and when cleaved, antimicrobial. Topical application promotes sustained basal tearing in rabbits. This study aims to determine if lacritin can be detected in human breast milk. Methods: Milk fat from human breast milk was removed by centrifugation and samples were subjected to DEAE and Hydrophobic Interaction Chromatography (HIC) to separate proteins in the sample. The samples were analyzed by SDS-PAGE and Western blot using polyclonal anti- lacritin antibodies. Blots included recombinant lacritin, human tear samples, and human milk samples. In addition, a colony forming unit (CFU) antimicrobial assay was performed against Escherichia coli. Results: Protein bands between 18-25 kDa on the Western blots for milk, tears, and recombinant lacritin were detected using N-terminal antibodies. C-terminal specific lacritin antibodies produced distinct primary bands between 18-20 kDa for tears and recombinant lacritin. A band at 75 kDa on the blot was detected for milk corresponding to a previously reported cross-linked lacritin complex. DEAE purification was unsuccessful, and HIC partially purified lacritin. Milk diluted 0.25X with PBS had 37% antimicrobial activity in the Colony Forming Unit (CFU) assay. Conclusions: Anti-lacritin antibodies that detect recombinant lacritin and human tear lacritin also detect a protein in human milk after HIC purification with similar electrophoretic mobility suggesting that lacritin or lacritin-like proteins are expressed in human breast milk. These proteins are present with antimicrobial properties.

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