Document Detail


Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.
MedLine Citation:
PMID:  20539212     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
PURPOSE: To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye.
METHODS: Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay.
RESULTS: The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active.
CONCLUSIONS: The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.
Authors:
Xiaofen Zheng; Cintia S De Paiva; Kavita Rao; De-Quan Li; William J Farley; Michael Stern; Stephen C Pflugfelder
Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cornea     Volume:  29     ISSN:  1536-4798     ISO Abbreviation:  Cornea     Publication Date:  2010 Sep 
Date Detail:
Created Date:  2010-08-23     Completed Date:  2010-11-10     Revised Date:  2013-04-15    
Medline Journal Info:
Nlm Unique ID:  8216186     Medline TA:  Cornea     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1048-54     Citation Subset:  IM    
Affiliation:
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, USA.
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MeSH Terms
Descriptor/Qualifier:
Adult
Aged
Animals
Biological Assay / methods
Bromodeoxyuridine
Cell Division
Cell Line
Cell Proliferation
DNA / biosynthesis
Dry Eye Syndromes / metabolism*
Enzyme-Linked Immunosorbent Assay
Epithelial Cells / metabolism
Humans
Lung / cytology
Middle Aged
Tears / metabolism*
Transforming Growth Factor beta1 / metabolism*
Transforming Growth Factor beta2 / metabolism*
Grant Support
ID/Acronym/Agency:
EY11915/EY/NEI NIH HHS; FY06 PR064719/PR/OCPHP CDC HHS; R01 EY011915-13/EY/NEI NIH HHS
Chemical
Reg. No./Substance:
0/Transforming Growth Factor beta1; 0/Transforming Growth Factor beta2; 59-14-3/Bromodeoxyuridine; 9007-49-2/DNA
Comments/Corrections

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