Metastatic progression and gene expression between breast cancer cell lines from African American and Caucasian women

African American (AA) women have a lower overall incidence of breast cancer than do Caucasian (CAU) women, but a higher overall mortality. Little is known as to why the incidence of breast cancer is lower yet mortality is higher in AA women. Many studies speculate that this is only a socio-economical problem. This investigation suggests the possibility that molecular mechanisms contribute to the increased mortality of AA women with breast cancer. This study investigates the expression of 14 genes which have been shown to play a role in cancer metastasis. Cell lines derived from AA and CAU patients were analyzed to demonstrate alterations in the transcription of genes known to be involved in cancer and the metastatic process. Total RNA was isolated from cell lines and analyzed by RT-PCR analysis. Differential expression of the 14 targeted genes between a spectrum model (6 breast cancer cell lines and 2 non-cancer breast cell lines) and a metastasis model (12 metastatic breast cancer cell lines) were demonstrated. Additionally, an in vitro comparison of the expression established differences in 5 of the 14 biomarker genes between African American and Caucasian breast cell lines. Results from this study indicates that altered expression of the genes Atp1b1, CARD 10, KLF4, Spint2, and Acly may play a role in the aggressive phenotype seen in breast cancer in African American women

Development of a Multiplex Real-Time PCR Assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test

In Vitro Characterization of Inflammatory Biomarkers across Species

There are currently no validated animal models or suitable biomarkers with which to ascertain the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in equine, bovine or ovine species during conditions of endotoxemia. This has resulted in approval of only one NSAID, flunixin meglumine, for controlling inflammation due to endotoxemia in bovine and equine animals, while none are approved in ovine animals for this claim. This study aims to investigate biomarkers with which to test efficacy of NSAIDs in these species. To this end, the effects of Escherichia coli lipopolysaccharide (LPS) induced inflammation on gene expression were investigated. Whole blood from each species was cultured and stimulated with LPS, after which RNA was extracted at various times. RNA was analyzed via quantitative RT-PCR (qRT-PCR) to determine differential expression of biomarkers. Results indicated up-regulation of cluster of differentiation 1 (CD1) gene in bovine and serum amyloid A (SAA) gene in ovine cultures. Down-regulation of cluster of differentiation 4 (CD4) gene and Caspase 1 was seen in bovine, and of CD1 in equine cultures. This work demonstrates that LPS stimulation alters expression of these genes in these species. These genes may be useful biomarkers for inflammation which could serve as markers for NSAID efficacy