Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods.

PURPOSE: Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups. METHODS: Tears were collected from Schirmer strips of the right and left eyes of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR), and 20 DE subjects and stored at -80 °C. Tear proteins were eluted and precipitated using ammonium bicarbonate and acetone. The right and left eye samples were combined for each subject. Following the Bradford protein quantitation method, 10 µg of total protein was used for each of the two analyses, Quantibody® Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Magnetic Bead Kit (Millipore). The assays were run using the GenePix® 4000B Scanner (Molecular Devices) or the Luminex MagPix® plate reader (Luminex), respectively. The data were then compared between the two instruments and the three subject groups. RESULTS: Of the 40 proteins on the Quantibody® microarray, seven had average expression levels above the lower limit of detection: ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF-RI. Significant differences in expression levels (p<0.05) were detected between the CL and DE groups for MCSF, TIMP-1, and TNF R1, between the NOR and DE groups for ICAM-1, and between the CL and NOR groups for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2, and TNF-R1 when using the Student t test. Of the 13 proteins tested with Luminex, IL-1ß, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level, and these were most often detected using the Luminex assay compared to the Quantibody® microarray. Contrarily, IL-2, IL-12, IL-13, INF-g, and GM-CSF were detected more frequently using the Quantibody® microarray than the Luminex assay. Significant differences in expression levels (p<0.05) were only detected between the CL and DE groups for IL-7 and IL-8 and between the CL and NOR subjects for IL-8. CONCLUSIONS: In addition to detecting more significant differences between the subject groups, the Quantibody® microarray detected more inflammatory cytokines in total within the range of detection than the Luminex assay. Differences were also noted in the types of cytokines each assay could detect from the limited protein samples. Both methods offer advantages and disadvantages; therefore, these factors should be considered when determining the appropriate assay for analyzing tear protein samples.

Differential Profiling of T-Cell Cytokines as Measured by Protein Microarray Across Dry Eye Subgroups.

PURPOSE: The purpose of this study is to explore the role of the adaptive immune system in patients with aqueous-deficient dry eye (ADDE) and lipid-deficient dry eye (LDDE). METHODS: Patients (n = 29) with moderate to severe dry eye (dry eye workshop [DEWS] severity grading scheme) were enrolled in a cross-sectional study and classified as ADDE (Schirmer < 10), LDDE (abnormal meibum), combined (meeting both criteria), or generic (meeting neither criterion). Tears were collected by Schirmer strips, and samples for both eyes were pooled for each subject. Thirty micrograms of total protein was used in a normalized volume for microarray analysis (Quantibody Human Inflammation Array 3; RayBiotech). Six markers of TH1 cells (interferon [IFN]γ, interleukin [IL]-2), TH2 cells (IL-4, IL-5, IL-13), and TH17 cells (IL-17) were assessed. RESULTS: ADDE demonstrated the highest total cytokine concentration, followed by the LDDE, combined, and generic groups. IFNγ and IL-2 were detectable in all subgroups. IL-4, -5, and -13 were detectable in ADDE and LDDE, but only IL-13 was detected in both the combined and generic groups. IL-17 was present in the ADDE, LDDE, and combined groups. CONCLUSIONS: TH1 cells seem to be involved in all forms of dry eye. ADDE and LDDE seem to be mediated by TH1, TH2, and TH17 cells. The combined-mechanism group is mediated by TH1 and TH17 cells, and generic dry eye seems to be mediated by TH1 cells only. ADDE has the greatest overall T-cell-mediated pathophysiology compared with the other subgroups, which is consistent with previous reports of improved efficacy with antiinflammatory therapy in these patients.

Large-scale gene trapping in C57BL/6N mouse embryonic stem cells.

We report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.

PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation.

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.