Upregulated cytokine genes were identified using Affymetrix gene chip analysis, and their 37 corresponding receptor genes were identified using the GO and GeneCards database

Upregulated cytokine genes were identified using Affymetrix gene chip analysis, and their 37 corresponding receptor genes were identified using the GO and GeneCards database. whereas two unique cytokine-receptor genes were upregulated in SCEs. Cytokine protein expression analysis showed that at least eight cytokines were upregulated in SLT-irradiated human Rabbit Polyclonal to USP32 CAOP tissues in situ/ex vivo. Conclusions This study has helped us identify a cytokine signaling pathway and to consider newly identified mechanisms regulating aqueous outflow that may lay the foundation for the future development of cytokine-based glaucoma therapies. values were calculated for each log2 intensity difference.14 These values were adjusted for multiple comparisons using the false discovery rate (FDR) method of Benjamini and Hochberg.15 The average log2 intensity difference between each experimental condition and its corresponding control was calculated by raising 2 to the power of the log2 intensity difference for each probe set. All calculations were made using the limma library of the R/Bioconductor software package16 in SAS 9.3 (SAS Institute, Inc., Cary, NC, USA). Upregulated Cytokine Genes. The top 50 upregulated probe sets for each of the six experimental comparisons were selected and combined to represent potentially upregulated genes. For genes with Tolrestat multiple probe sets, the particular probe with the largest fold value was selected for further analysis, resulting in 83 upregulated cytokine genes. Only cytokine genes with a calculated fold change 1.5 and a one-sided FDR-adjusted value less than 0.1 for at least one experimental condition qualified as upregulated genes. After excluding noncytokine genes (i.e., receptors, enzymes, cluster of differentiation proteins), 28 unique cytokine genes remained identified as upregulated based on the results of the six experimental Tolrestat comparisons. Downregulated Cytokine Genes. Using an approach parallel to that used above for detection of upregulated genes, the bottom 50 downregulated probe sets were selected to represent possibly downregulated genes. Only cytokine genes with a calculated fold value 0.5 and a one-sided FDR-adjusted value less than 0.1 for at least one experimental condition were considered downregulated genes. After excluding noncytokine genes from 39 candidates, 18 unique cytokine genes remained identified as downregulated. Affymetrix Cytokine-Receptor Gene Expression. Upregulated cytokine genes were identified using Affymetrix gene chip analysis, and their 37 corresponding receptor genes were identified using the GO and GeneCards database. Differentially expressed receptor genes, both upregulated and downregulated, were then selected by using the same methods as for identification of cytokine genes described above. Six unique cytokine-receptor genes were identified as differentially expressed (DE) in the six experimental comparisons evaluated. Cytokine Secretion. For in vitro experiments, cytokines secreted by cultured TMEs and SCEs were measured and FDR-adjusted Wilcoxon two-sample one-sided Tolrestat values for all those treatment-control comparisons were calculated for each cytokine. Box plots were constructed for comparison between laser-treated and untreated controls. For ex vivo experiments, the difference in the mean log2 intensity measurements between the treated media samples and the untreated media samples was calculated for each specimen and for each cytokine. Depending on the specific cytokine, between 5 and 22 specimens were available for analysis. To test for equality between laser-treated and control observations, the resultant specimen-specific paired means were analyzed using the Wilcoxon signed rank test for paired data, and the FDR-adjusted one-sided Wilcoxon value was obtained for each cytokine. A cytokine was considered upregulated when its protein expression exhibited a fold change 1.5 along with a one-sided value less than 0.1. Only cytokines that were identified as upregulated by gene expression analysis underwent testing for protein expression using specific antibodies. Thus, because TNF- was not upregulated by this criterion, it was not tested for protein expression using specific antibodies. Results Cytokine Gene Expression Upregulated Cytokine Genes. Table 1 shows a consistent asymmetry between TMEs and SCEs in their responses to each of three treatments. In each treatment, one cell type responded much more.