Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter-Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method.

BACKGROUND: It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task. METHODS: A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions. RESULTS: The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ∼1.5 and 0.9 Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products. CONCLUSIONS: Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.

Pax-8-PPAR-γ fusion protein in thyroid carcinoma.

Thyroid carcinoma is the most common endocrine malignancy, and its incidence is continuing to increase. Most thyroid carcinomas contain one of several known driver mutations, such as the Val600Glu substitution in B-Raf, Ras mutations, RET gene fusions, or PAX8-PPARG gene fusions. The PAX8-PPARG gene fusion results in the production of a Pax-8-PPAR-γ fusion protein (PPFP), which is found in approximately one-third of follicular thyroid carcinomas, as well as some follicular-variant papillary thyroid carcinomas. In vitro and in vivo evidence indicates that PPFP is an oncoprotein. Although specific mechanisms of action remain to be defined, PPFP is considered to act as a dominant-negative inhibitor of wild-type PPAR-γ and/or as a unique transcriptional activator of subsets of PPAR-γ-responsive and Pax-8-responsive genes. Detection of the fusion transcript in thyroid nodule biopsy specimens can aid clinical decision-making when cytological findings are indeterminate. The PPAR-γ agonist pioglitazone is highly therapeutic in a transgenic mouse model of PPFP-positive thyroid carcinoma, suggesting that PPAR-γ agonists might be beneficial in patients with PPFP-positive thyroid carcinomas.

Immunotoxicology: fifty years of global scientific progress.

The Immunotoxicology Specialty Section of the Society of Toxicology (SOT) celebrated the 50(th) Anniversary of the SOT by constructing a poster to highlight the milestones of Immunotoxicology during that half-century period. This poster was assembled by an ad hoc committee and intertwines in words, citations, graphics, and photographs our attempts to capture a timeline reference of the development and progressive movement of immunotoxicology across the globe. This poster was displayed during the 50(th) Annual SOT Meeting in Washington DC in March, 2011. The poster can be accessed by any Reader at the SOT Website via the link http://www.toxicology.org/AI/MEET/AM2011/posters_rcsigss.asp#imss. We dedicate this poster to all of the founders and the scientists that followed them who have made the discipline of Immunotoxicology what it is today.

15-Deoxy-Δ¹²,¹4-prostaglandin J2-glycerol, a putative metabolite of 2-arachidonyl glycerol and a peroxisome proliferator-activated receptor γ ligand, modulates nuclear factor of activated T cells.

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative released on demand from membrane precursors. 2-AG-mediated suppression of interleukin (IL)-2 depends on cyclooxygenase 2 (COX-2) metabolism and peroxisome proliferator-activated receptor γ (PPARγ) activation. 15-Deoxy-Δ¹²,¹4-prostaglandin J2-glycerol ester (15d-PGJ2-G), a putative COX-2 metabolite of 2-AG, acts as a PPARγ ligand and produces IL-2 suppression in activated Jurkat T cells, in part, by decreasing nuclear factor of activated T cells (NFAT) transcriptional activity. The objective of the present studies was to investigate the mechanism by which 15d-PGJ2-G modulates NFAT activity to suppress IL-2. 15d-PGJ2-G treatment decreased phorbol 12-myristate 13-acetate (PMA)/calcium ionophore (I0)-induced NFAT DNA binding to the human IL-2 promoter and nuclear NFAT2 accumulation. It is noteworthy that 15d-PGJ2-G treatment increased active nuclear HDM2 (human homolog of the oncoprotein and E3 ubiquitin ligase murine double minute 2) expression, whereas there was no change in the expression of glycogen synthase kinase 3ß, both of which regulate NFAT. 15d-PGJ2-G and other PPARγ agonists, such as rosiglitazone and ciglitazone, decreased PMA/I0-mediated elevation in intracellular calcium concentration ([Ca²âº](i)) in activated Jurkat cells. We were surprised to find that the PPARγ antagonists 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907) and 2-chloro-5-nitrobenzanilide (GW9662) also decreased the PMA/I0-mediated elevation in [Ca²âº](i) in activated T cells. In addition, the presence of T0070907 plus 15d-PGJ2-G produced an additive decrease in PMA/I0-mediated elevation of [Ca²âº](i), suggesting that the 15d-PGJ2-G effects on calcium might be either PPARγ-independent or -dependent on occupation of the PPARγ ligand binding domain. Collectively, our findings suggest that 15d-PGJ2-G increases active nuclear HDM2, which could lead to a decrease in NFAT2 and IL-2 suppression.

15-Deoxy-delta12,14-prostaglandin J2-glycerol ester, a putative metabolite of 2-arachidonyl glycerol, activates peroxisome proliferator activated receptor gamma.

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative capable of suppressing interleukin (IL)-2 production by activated T cells. 2-AG-mediated IL-2 suppression is dependent on cyclooxygenase-2 (COX-2) metabolism and peroxisome proliferator activated receptor γ (PPARγ) activation. The objective of the present studies was to examine whether 15-deoxy-Δ(12,14)-PGJ(2)-glycerol ester (15d-PGJ(2)-G), a putative metabolite of 2-AG, can mimic the actions of 2-AG on IL-2 regulation through PPARγ activation. 15d-PGJ(2)-G bound PPARγ-ligand binding domain in a PPARγ competitive binding assay. 15d-PGJ(2)-G treatment activated PPARγ in a reporter assay, and PPARγ activation was attenuated when a PPARγ antagonist, 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907), was present. 15d-PGJ(2)-G treatment suppressed IL-2 production by activated Jurkat cells, which was partially attenuated when pretreated with T0070907. Moreover, IL-2 suppression was pronounced when 15d-PGJ(2)-G was present 30 min before or after T-cell activation. Concordant with IL-2 suppression, 15d-PGJ(2)-G treatment decreased nuclear factor of activated T cells (NFAT) transcriptional activity in transiently transfected Jurkat cells. It is noteworthy that T0070907 alone markedly increased NFAT reporter activity, suggesting the existence of endogenous PPARγ activation and modulation of NFAT. Because COX-2 metabolism of 2-AG is important for IL-2 suppression, the effect of 2-AG on COX-2 and PPARγ mRNA expression was investigated. 2-AG treatment decreased the up-regulation of COX-2 mRNA after T-cell activation, which suggests negative feedback limiting COX-2-mediated metabolism of 2-AG. PPARγ mRNA expression was increased upon activation, and 2-AG treatment produced a modest decrease in PPARγ mRNA expression. Collectively, our findings suggest that 15d-PGJ(2)-G activates PPARγ to decrease NFAT transcriptional activity and IL-2 expression in activated T cells.

A COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol, mediates suppression of IL-2 secretion in activated Jurkat T cells.

Previous studies from this laboratory have demonstrated that a COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol (2-AG), inhibits IL-2 secretion in activated T cells through PPARgamma activation independent of the cannabinoid receptors, CB1/CB2. Because numerous cyclooxygenase (COX) products have been shown to activate PPARgamma, the primary purpose of the present studies was to determine the role of COX metabolism in the inhibition of IL-2 secretion by 2-AG. Pretreatment with nonselective and COX-2-specific inhibitors completely abrogated 2-AG-mediated suppression of IL-2 secretion. In contrast, pretreatment with COX-1-specific inhibitors had no effect upon 2-AG-mediated inhibition of IL-2 secretion. Interestingly, the current studies also demonstrate that while the potency of 2-AG is comparable between human Jurkat T cells and murine splenocytes, anandamide (AEA) is markedly more potent in suppressing IL-2 production in Jurkat T cells compared to murine splenocytes. Additionally, the present studies also demonstrate that COX-2 protein is readily detectable in resting Jurkat T cells, which is in contrast to resting murine splenocytes in which COX-2 protein is virtually undetectable. Furthermore, COX-2 protein and mRNA levels are significantly increased over basal levels by 2h following activation of Jurkat cells, whereas increases in COX-2 protein in murine splenocytes are not observed until 4h after cellular activation. These studies suggest that the potency of AEA in the suppression of IL-2 secretion may correlate with COX-2 protein levels in different T cell models. The present studies are also significant in that they demonstrate 2-AG-mediated inhibition of IL-2 secretion is dependent upon COX-2 metabolism.

Lipid peroxidation and cyclooxygenase enzyme inhibitory activities of acidic aqueous extracts of some dietary supplements.

The botanical supplement market is growing at a fast pace with more and more people resorting to them for maintaining good health. Echinacea, garlic, ginkgo, ginseng, Siberian ginseng, grape seed extract, kava kava, saw palmetto and St John's wort are some of the popular supplements used for a variety of health benefits. These supplements are associated with various product claims, which suggest that they possess cyclooxygenase (COX) enzyme and lipid s inhibitory activities. COX enzymes are found to be at elevated levels in inflamed and cancerous cells. To test some of the product claims, selected supplements were analysed for their ability to inhibit COX-1 and -2 enzymes and lipid peroxidation in vitro. The supplements were extracted with acidified water (pH 2) at 37 degrees C to simulate the gastric environment. The supplements tested demonstrated varying degrees of COX enzyme inhibition (5-85% for COX-1 and 13-28% for COX-2). Interestingly, extracts of garlic (Meijer), ginkgo (Solaray), ginseng (Nature's Way), Siberian ginseng (GNC, Nutrilite, Solaray, Natrol), kava kava (GNC, Sundown, Solaray) and St John's wort (Nutrilite) selectively inhibited COX-2 enzyme. These supplements also inhibited lipid peroxidation in vitro (5-99%). The results indicated that the consumption of these botanical supplements studied possess health benefits.

Evaluation of metal and microbial contamination in botanical supplements.

The sale of botanical dietary supplements in the United States is on the rise. However, limited studies have been conducted on the safety of these supplements. There are reports on the presence of undesired metals in some of the botanical dietary supplements. In this study, echinacea, garlic, ginkgo, ginseng, grape seed extract, kava kava, saw palmetto, and St. John's wort supplements manufactured by Nature's Way, Meijer, GNC, Nutrilite, Solaray, Sundown and Natrol, have been analyzed for lead, mercury, cadmium, arsenic, uranium, chromium, vanadium, copper, zinc, molybdenum, palladium, tin, antimony, thallium, and tungsten using inductively coupled plasma mass spectrometry. All samples were devoid of mercury contamination. Results indicated that the botanical supplements analyzed did not contain unacceptable concentrations of these metals. These supplements were also evaluated for microbial contamination, and most samples analyzed showed the presence of bacteria or fungi or both. Microbes were not counted nor were microbial counts determined in these samples.