Aqueous Macrophages Contribute to Conserved CCL2 and CXCL10 Gradients in Uveitis.

Purpose: Uveitis is a heterogenous group of inflammatory eye disease for which current cytokine-targeted immune therapies are effective for only a subset of patients. We hypothesized that despite pathophysiologic nuances that differentiate individual disease states, all forms of eye inflammation might share common mechanisms for immune cell recruitment. Identifying these mechanisms is critical for developing novel, broadly acting therapeutic strategies. Design: Experimental study. Subjects: Biospecimens from patients with active or inactive uveitis and healthy controls. Methods: Protein concentration and single cell gene expression were assessed in aqueous fluid biopsies and plasma samples from deidentified patients with uveitis or healthy controls. Main Outcome Measures: The concentration of 31 inflammatory proteins was measured in all aqueous samples, as well as plasma samples from patients with active uveitis. Chemokine and cytokine ligand and receptor expression were assessed in individual cell types from aqueous biopsies obtained from patients with active uveitis. Results: We identified 6 chemokines that were both elevated in active uveitis compared with controls and enriched in aqueous compared with plasma during active uveitis (C-C motif chemokine ligand [CCL]2, C-X-C motif chemokine ligand [CXCL]10, CXCL9, CXCL8, CCL3, and CCL14), forming potential gradients for migration of immune cells from the blood to the eye. Of these, CCL2 and CXCL10 were consistently enriched in the aqueous of all patients in our cohort, as well as in a larger cohort of patients from a previously published study. These data suggest that CCL2 and CXCL10 are key mediators in immune cell migration to the eye during uveitis. Next, single cell RNA sequencing suggested that macrophages contribute to aqueous enrichment of CCL2 and CXCL10 during human uveitis. Finally, using chemokine ligand and receptor expression mapping, we identified a broad signaling network for macrophage-derived CCL2 and CXCL10 in human uveitis. Conclusions: These data suggest that ocular macrophages may play a central role, via CCL2 and CXCL10 production, in recruiting inflammatory cells to the eye in patients with uveitis. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT)--relationship to newly identified mitochondrial pyruvate carrier proteins.

Thiazolidinedione (TZD) insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44) and BRP44 Like (BRP44L), which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT) cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13)C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT) and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA.

Cytosine-methylation changes are stable and thought to be among the earliest events in tumorigenesis. Theoretically, DNA carrying tumor-specifying methylation patterns escape the tumors and may be found circulating in the sera from cancer patients, thus providing the basis for development of noninvasive clinical tests for early cancer detection. Indeed, using methylation-specific PCR-based techniques, several groups reported the detection of tumor-associated methylated DNA in the sera from cancer patients with varying clinical success. However, by design, such analytical approaches allow assessment of the presence of molecules with only one methylation pattern, leaving the bigger picture unexplored. The limited knowledge about circulating DNA methylation patterns hinders the efficient development of clinical methylation tests and testing platforms. Here, we report the results of a comprehensive methylation pattern analysis from breast cancer clinical tissues and sera obtained using massively parallel bisulphite pyrosequencing. The four loci studied were recently discovered by our group, and demonstrated to be powerful epigenetic biomarkers of breast cancer. The detailed analysis of more than 700,000 DNA fragments derived from more than 50 individuals (cancer and cancer-free) revealed an unappreciated complexity of genomic cytosine-methylation patterns in both tissue derived and circulating DNAs. Both tumor and cancer-free tissues (as well as sera) contained molecules with nearly every conceivable cytosine-methylation pattern at each locus. Tumor samples displayed more variation in methylation level than normal samples. Importantly, by establishing the methylation landscape within circulating DNA, this study has better defined the development challenges facing DNA methylation-based cancer-detection tests.

Identification of novel high-frequency DNA methylation changes in breast cancer.

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

Gene expression in pharyngeal arch 1 during human embryonic development.

Craniofacial abnormalities are one of the most common birth defects in humans, but little is known about the human genes that control these important developmental processes. To identify relevant genes, we analyzed transcription profiles of human pharyngeal arch 1 (PA1), a conserved embryonic structure that develops into the palate and jaw. Using microdissected, normal human craniofacial structures, we constructed 12 SAGE (serial analysis of gene expression) libraries and sequenced 606 532 tags. We also performed Affymetrix microarray analysis on 25 craniofacial targets. Our data revealed not only genes "enriched" or differentially expressed in PA1 during fourth and fifth week of human development, but also 6927 genes newly identified to be expressed in human PA1. Many of these genes are involved in biosynthetic processes and have binding function and catalytic activity. We compared expression profiles of human genes with those of mouse homologs to look for genes more specific to human craniofacial development and found 766 genes expressed in human PA1, but not in mouse PA1. We also identified 1408 genes that were expressed in mouse as well as human PA1 and could be useful in creating mouse models for human conditions. We confirmed conservation of some human PA1 expression patterns in mouse embryonic samples with whole mount in situ hybridization and real-time RT-PCR. This comprehensive approach to expression profiling gives insights into the early development of the craniofacial region and provides markers for developmental structures and candidate genes, including SET and CCT3, for diseases such as orofacial clefting and micrognathia.